Image-Pro®

Version 10 for Windows®

Users Guide

Media Cybernetics
401 N. Washington St. Suite 350
Rockville, MD 20850
+1-301-495-3305, FAX +1-240-328-6193
+1-800-263-2088 (North America only)
http://www.mediacy.com
COPYRIGHT NOTICE
Copyright, 2012 - 2017 Media Cybernetics, Inc.
All Rights Reserved
TRADEMARK ACKNOWLEDGMENTS
Image-Pro and Image-Pro with 3D Module are registered trademarks of Media Cyber-
netics, Inc..
All trademarks in this document are trademarks or registered trademarks of their respect-
ive companies.
License Agreement
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interest in such trademarks is granted hereunder.
Table of Contents

1
Preface xvii
Product Features xviii
Acquire and Open Images xviii
Automate and Customize xviii
Read and Write Image Data xix
Process and Analyze xix
Measure and Count xix
3D Image Viewing and Rendering xx
Report and Share xx
Conventions Used in this Guide xxi
A Note about this User Guide xxi
Contacting Technical Support xxii
Chapter 1: Installing Image-Pro 1
Package Contents 2
System Requirements for Image-Pro 10 3
Supported Operating Systems: 3
Minimum Requirements 3
Recommended Requirements 3
High Performance Requirements 4
System Requirements for Image-Pro with 3D Module 5
Rule of Thumb: 5
Minimum Requirements (for small datasets only, performance will be low) 5
Table of Contents

Recommended Requirements 5
High Performance Requirements 6
Hardware Priorities 7
Graphics Card 7
System Memory 7
Hard Drives 8
CPU 8
Installing Image-Pro 9
Activating Your Image-Pro License 16
Verifying your Registration 19
Windows Security Alert 21
Product Versions and Updates 22
Updating Your Product 23
Verifying Updates 26
Help and About Image-Pro 29
Upgrading Image-Pro 35
Upgrading the Installed Software 36
Upgrading Your Software 36
How to Return your Original Image-Pro License 38
Upgrading your License 39
Configuring License Use 40
Sending Feedback 42
Chapter 2: Getting Started 44
The Image-Pro Application Window 45
Floating Panels 45
Workspace Tabs 46
Viewing Tooltips 46

ii
 Table of Contents

Tools and Layouts 47

Using the Multiple Document Interface (MDI) 48
The Image Context Menu 53
Viewing Image Document Information 55
The Welcome Screen 56
The Image-Pro Ribbon 59
Different Types of Content 60
The File Menu 61
Opening an Image 63
Opening an Image Sequence 65
Opening an Image Set 67
Saving an Image 74
Save 74
Save All 75
Using Quick Save 75
Save As 77
Reload 79
Printing an Image 80
Quick Access Toolbar 82
Options Buttons 83
Close or Hide Dialogs for Extra Space 83
The Image-Pro Status Bar 84
Tabs and Ribbons 87
Capture tab 87
Adjust Tab 88
Process Tab 88
Select Tab 88

iii
Table of Contents

Count/Size Tab 88
Measure Tab 89
Review Tab 89
View Tab 89
Image Tab 89
Optional Tabs 90
Automate Tab 90
Apps Tab 90
Custom Tab 90
Extras Tab 91
Context-Sensitive Tabs 91
3D View Tab 91
3D Measure Tab 91
3D Animation Tab 92
Chapter 4: Viewing Intensity Measurements 94
Image Histogram, Line Profile, and Saturation Warning 95
Viewing an Image Histogram 96
Advanced Display 99
Viewing a Line Profile 103
Line Profile Tools 104
Background Tools 108
To Add a Line Profile to the Current Collection 108
Edge Detection Tools 110
Using the Saturation Warning 119
Chapter 5: Working with Images 121
121
Viewing Optical Characteristics 122

iv
 Table of Contents

Using Spatial Calibrations 125

Pixels per Unit 125
Aspect Ratio 125
System and Reference Calibration 126
Creating a Spatial Calibration 126
Quick Calibration Method 127
Auto-Calibration 128
Calibrating by Aspect Ratio 129
Calibrating by Feature 133
Predictive Calibration 134
Saving Calibrations 136
Locking Calibrations 137
Importing and Exporting Calibrations 138
Applying a Calibration Marker to the Active Image 139
Using Intensity Calibration 142
Step Tablet Wizard 145
The Image Tab 146
Viewing a Surface Plot 146
Draw Mode 148
Resolution 151
Style 151
Geometry 153
Output 154
Using Gallery View 155
Gallery Ribbon 156
Using the Slicer Viewer 159
The Slicer Viewer Ribbon 160

v
Table of Contents

Using the 3D Viewer 161

The 3D Viewer Ribbon 162
Bitmap Analysis 166
Zooming Images 170
Zooming by Using the Zoom Button 170
Zooming with Local Zoom 171
Zooming by Using the Mouse Wheel 172
Zooming by Using the Scale Bar 172
Panning Images 172
Panning Using the Pan Button 173
Panning in the Image Strip Using Image Navigator 173
Enhancing an Image 174
Resizing an Image 174
Rotating an Image 175
Using the Canvas 177
Converting Images 179
Using Color Group Commands 181
Dye/Tint Images 181
Using Color Composite 182
Pseudo-Coloring an Image 186
Adjusting Color Channels 188
Changing the Display Range 189
Adjusting Brightness, Contrast, and Gamma 190
Resetting the Image 191
Making Enhancements Permanent 191
Using the Process Tab Features 193
Merge Files 193

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 Table of Contents

Sub-sampling 194
Aligning Images 195
Using Extended Depth of Field 203
Tiling and Stitching 209
Comparing Images 218
Image Math 220
Correcting the Background 224
Applying Filters 226
Advanced Filters 227
Using the Grid Overlay 229
Filtering with Fast Fourier Transforms 232
Viewing the Image Spectrum 233
Image Sequences 237
Using the Active Data Overlay 242
Customizing the Data Overlay 244
Using Live Extended Depth of Field 247
Using Live Tiling 251
Creating an Audit Trail 256
Audit Trail Options 257
Audit Trail Tools 258
Very Large Images 259
Large Image Tools 260
Image-Pro Experiments 263
Managing Mixed Content Experiments 263
Working with Image Sets 266
Open Set from File 266
Image Set Controls 272

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Table of Contents

Chapter 6: Using Regions of Interest 276

The Select Tab 277
What is an ROI? 278
Defining an ROI 279
Features Manager 281
To Add an ROI to the Current Collection 282
To Apply an ROI to the Active Image 283
To Save an ROI Collection 284
To Load an ROI Collection 284
Placing Multiple ROIs on an Image 285
Arranging ROIs 287
Moving an ROI 287
Resizing an ROI 287
Using XOR Pixels 287
Rotating an ROI 289
Deleting an ROI 291
Setting the Appearance of ROIs 293
Changing the Appearance of an ROI 293
Drawing Properties 294
Label Properties 294
Setting the Default Appearance for ROIs 295
Using the Magic Wand 296
Polygon Nudge Tool 299
Using Overlays 301
Chapter 7: Using Annotations 302
The Select Tab 303
Creating an Annotation 304

viii
 Table of Contents

Creating a Graphical Annotation 304

308
Annotation Shapes 308
Creating a Text Annotation 309
Inserting a Bitmap Annotation 311
Arranging Annotations 312
Moving an Annotation 312
Rotating an Annotation 312
Resizing/Reshaping an Annotation 313
Grouping Annotations 313
Ordering Annotations 314
Deleting an Annotation 314
Setting the Appearance of Annotations 316
Changing the Appearance of an Annotation 316
Drawing Properties 317
Label Properties 318
Additional Options 318
Setting the Default Appearance for Annotations 319
Saving Annotations 320
Chapter 8: Measuring, Counting, and Tracking 322
Overview of the Measurement Feature 323
Data Measurements 324
Edit Ranges 326
Measurement Options 326
Manual Measurement Tools 329
Selection Tools 329
Direct Measurements 330

ix
Table of Contents

Edge Measurements 333

Best-fit measurements 335
Relative Measurements 336
Distance between objects 337
Incremental Distance 339
Setting the Default Appearance for Measurement Labels 343
Using the Auto-Trace feature 346
Manual Tagging 349
Viewing Measurement Information 351
Viewing a Data Table 351
Viewing a Data Histogram 355
Viewing an Object Window 356
Colocalization 358
Colocalization Panel 361
Object-Based Colocalization Analysis 365
Object segmentation using Parent Image 367
Object segmentation using colocalization channels 368
Manual object drawing 368
L*a*b* Color measurements and Color Correction 370
Counting Objects in Images 380
Displaying Individual Objects 385
Count Options 389
Thresholding 391
Auto-Threshold 394
Segmentation Bias 395
Grow or Shrink Object 396
Open or Close Object 397

x
 Table of Contents

Smart Segmentation 399

Select and Count 400
Smart Segmentation Training 401
Mask and Overlay 402
Recipe Options 403
Recipe Summary 408
Classifying Objects 410
Single Variable Classification 410
Auto Classification 411
Learning Classification 413
How to View a Histogram of Classified objects. 415
Finding Shapes 417
Lines 417
Circles 418
Irregular Shapes 420
Creating a Voronoi Map 422
Tracking Objects 424
Manual Tracking 424
Automatic Tracking 431
Intensity Tracking 433
Select Tools 434
Intensity Measurements 435
Intensity Tracking Options 436
Intensity Tracking Data Views 436
Features Manager 441
Converting Features 443
Data Collector 445

xi
Table of Contents

Data Types 446

Data Windows 451
Data Table 452
Viewing a Data Table 453
Data Blocks 456
Data Graph 457
Data Histogram 458
Scatterplot 459
Chapter 9: Customizing the User Interface and Automating Processes 461
Using the Quick Access Toolbar 462
To Add a Shortcut to the Quick Access Toolbar 462
Customizing the Image Strip 465
Viewing Tooltips 468
Activating and Closing 469
Using the Multiple Document Interface (MDI) 471
Floating Panels 473
Tools and Layouts 475
Creating a Custom Tab 477
Rename and Make your Custom Tab the First Tab 478
Hide or Show Tabs 480
Showing or Hiding Groups 482
Expanding and Collapsing 482
Image-Pro in Japanese 484
Working with Macros 485
Project Explorer 485
Project Workbench 486
The Macro Script Ribbon 488

xii
 Table of Contents

The Macro Edit Ribbon 490

Project Properties 491
Project Workbench Options 492
All about Apps 494
Loading an App 494
Running Apps 496
Using the Project Explorer 499
Creating a New Project, Module or App 501
Adding a New Project 501
Adding a New Module 502
Adding a New App 503
Designing Apps 505
Saving Your Project 508
Packaging a Project 510
Licensing and Encryption 511
Project License Generator 514
Batch Processing 516
Selecting Macros 517
Select Images 519
Process the Batch 521
Grouping and Sorting 521
Advanced Batch Processing Options 522
Grouping Macros 524
Task Management 524
Task Scheduling Options 527
General 528
Task Manager 528

xiii
Table of Contents

Advanced Options 528

Chapter 10: Generating and Printing Reports 530
Creating and Printing Reports 531
Report Tools 534
Reporting in the Project Workbench 535
The Report Tools in the Project Workbench 536
Project Workbench 537
Reports as App/Project Components 538
The Report Data and Report Fields panels 538
Chapter 11: Managing Multiple Users 542
Managing Identities 543
Password Protection 545
Chapter 12: Using the 3D Features 548
The Image-Pro with 3D Module Workspace 549
The 3D View Ribbon 551
Navigate Group 552
Voxel Size Group 553
Decoration Group 554
The View Style Group 556
Composition Group 564
Geometry Group 566
Volume of Interest Group 568
The 3D Measure Ribbon 572
Navigate Group 572
Count Group 572
Volume Measurements Group 576
Holes and Negative Volumes 582

xiv
 Table of Contents

3D Measurement Type Options 582

3D Options Panel 584
Appearance options 584
Measurements Group 586
Active Tool Group 586
Relative Measurements Group 588
Guided Segmentation 590
Collections Sub-Panel 592
2D Segmentation Sub-panel 593
Z-Propagation 597
Isosurface 599
View Group 602
3D Options 605
Add Isosurface 608
Add Colocalization 613
Add Ortho Slices 615
Add Oblique Slices 616
Add External Objects 618
The 3D Slicer Ribbon 619
The 3D Animation Ribbon 623
Timeline Group 623
Animation Path Group 624
Frames Group 626
Playback Group 627
Options Group 628
Output Group 630
Navigate Group 631

xv
Table of Contents

Adding a New Animation 631

Appendix A 633
AVI (*.AVI) 633
BMP (*.BMP, *.DIB, *.RLE) 633
JPEG (*.JPG, *.JPEG, *.JP2, *.JPF) 633
Flex Image Transport System (*. FTS, *.FIT, *.FITS) 633
Thermo Fisher Experiments (*.MCE) 633
Media Cybernetics Sets (*.MCS) 634
MPEG-4 (*.MP4, *.MOV, *.M4V) 634
Portable Internet Graphics (*.PNG) 634
Picture (*.PCT, .PICT) 634
Sequence (*.SEQ) 634
TIFF (*.TIF, *.TIFF) 634
Hitachi File Formats 636
Appendix B 637
Installation Questions 638
Application Questions 640
Index 641

xvi
Preface
Before You Begin
Welcome to the Image-Pro® User’s Guide. This product is designed to run under the
Microsoft® Windows® 7.0 Professional or Ultimate (service pack 1) (32- and 64-bit), Win-
dows® 8.0 or 8.1 Enterprise or Professional (32- and 64-bit), and Windows® 10 Enterprise
or Professional (32- and 64-bit).

xvii
Preface 1

Product Features
Image-Pro 9.3 is a comprehensive application for image acquisition and measurement for
industrial and physical scientists. It has been designed to automate and simplify routine
image capture, inspection, and measurement tasks.
With Image-Pro, you can.....

Acquire and Open Images

l See when Live Preview is running with the new Live Preview Pane.
l Capture HDR images.
l Easily acquire single frame images and time-lapse experiments.
l Acquire and analyze high dynamic range (HDR) images.
l Stream multi-gigabyte movies directly to your hard drive.
l Use Live Tiling and Live Extended Depth of Field (EDF) to acquire large, beau-
tifully focused images without an automated stage or z motor.
l Use the crosshair grid to align images while capturing.
l Compare live images with previously acquired images using the Image Compare
workspace.

Automate and Customize

l Show and hide tabs or groups to display only the tools you need.
l Create and display mini-programs (Apps) and macros to automate procedures that
you use frequently on the new Apps tab.
l Save and load your preferred workspace layout, and share your layout with others.
l Review your macros in an intuitive graphical macro editor or use the more
advanced code-based editing tools to test, edit and debug your scripts.
l Download Image-Promacros and Apps from the App Center website.
l Customize the user interface, so that you can set up Image-Pro in the most effi-
cient way for your work, including dockable tool bars and the ability to show or

xviii
 Product Features

hide different toolbars and buttons. You can also reset your interface to “factory”
or “default” settings if you don’t like what you’ve set up and want to start over.
l Never lose your work with Image-Pro’s AutoSave and AutoRecover.

Read and Write Image Data

l Read and write image data in all the standard image file formats including, TIFF,
JPEG, AVI, and many others.
l Open and view multi-resolution image files, including Leica .scn, Aperio .svs and
BigTiff, and extract out images to analyze.

Process and Analyze

l Use Extended Depth of Field (EDF) test strips to preview EDF settings options to
save time and ensure best results when creating in-focus images from a z-stack.
l Analyze colocalized channels. Perform parent/child analysis, analyze within spe-
cific regions and more.
l Perform image enhancement using powerful color and contrast filters, including
morphology, field flattening, background subtraction, Fast Fourier Transforms
(FFT), and other spatial and geometric operations.
l Align, tile, or stitch multiple images together.
l Extract features with spatial tools that isolate a Region of Interest (ROI) from the
rest of the image, or with segmentation tools that extract features by color or
intensity value.
l Use 1-2-3 Batch Processing to apply your image processing steps to multiple
opened images or folders of images.
l View and analyze very large images (64-bit systems only).

Measure and Count

l Adjust the size and shape of irregular ROIs and Measurements with the Polygon
Nudge Tool.
l Use the Grow and Shrink tools to adjust the size of your counted objects .
l Use the Close and Merge tools to combine neighboring objects manually when
using automatic segmentation and counting tools.

xix
Preface 1

l Automatically measure incremental distances between two irregular edges to

quantify changes in surface size and shape.
l Trace and count objects manually or automatically. Measure object attributes
such as: area, angle, perimeter, diameter, roundness and aspect ratio. Calibrate
your spatial scale to any unit of measure.
l Use Smart Segmentation to easily identify and segment challenging images with
faintly-colored objects, textured objects and uneven backgrounds.
l View collected data numerically, statistically or in graphic form (histogram and
scattergram). Save the measurement data to disk.
l Sort and classify your measurement data according to your predefined criteria.
Color code your objects by class.
l Manually or automatically follow objects as they move over time using Object
Tracking.
l Track changes in intensity over time.

3D Image Viewing and Rendering

l View 3D images from a variety of angles and views.

Report and Share

l Save images with all measurement overlays intact for future analysis.
l Apply annotations, labels, and overlays to your images.
l Export measurements and data graphs to Microsoft Excel™.
l Export all opened images into Microsoft PowerPoint™.
l Generate Adobe PDF™ reports that can include images, measurement data, text,
and pictures.

xx
 Conventions Used in this Guide

Conventions Used in this Guide

The Image-Pro User’s Guide uses the following documentation conventions:

A Note about this User Guide

This guide was designed to be a comprehensive overview ofImage-Pro and Image-Pro
with 3D Module. For that reason, some of the tab names, colors, screens, and images fea-
tured in this manual may be slightly different from those in your product.
For example, if you are using the versions of this product that do not include the ability to
capture images, you will see the Home tab instead of the Capture tab. Also, if you are
using Image-Pro with 3D Module 9.3, you will some of the tabs labeled as
2D Measure or 2D Count/Size to distinguish them from similar features in the
Image-Pro with 3D Module toolset.

xxi
Chapter 1

Contacting Technical Support

Media Cybernetics provides technical support to registered users. We can be reached by
phone, fax, or e-mail, as follows:

Telephone: 301- 495-3305, 800-263-2088 (North America only)

FAX: 301- 495-5964
E-mail: [email protected]
Web Page: http://support.mediacy.com

Operating Hours (Eastern Time)

Mon-Fri: 9:00 am - 5:00 pm
Sat-Sun: Closed
Holidays: Closed

Support for all registered users includes access to the latest information and downloads
regarding updates, hot-fixes, and service packs. Technical support for all users is free for
the first 30 days.
Support for users who have a valid Image-Pro maintenance agreement includes the fol-
lowing:
l Continuing access to the latest updates and fixes.
l Access to the Image Analysis Forum Discussion Group. Image-Pro Support Spe-
cialists and users from around the world participate and grow this Knowledge Base. It
includes valuable information concerning the use and support of Image-Pro. To access
this forum, click on the Image Analysis link on the Help menu:

xxii
 Contacting Technical Support

l Access to Image-Pro Support Specialists. If you cannot find a solution in the Forum
you can submit your problem to our Image-Pro Support Specialists. They will attempt
to answer you as quickly as possible.

Before you contact Thermo Fisher's Technical Support for assistance, please be prepared to
provide the following information:
l Your registration information: the product serial number, your name, phone number,
organization name, mailing address, and e-mail address. The serial numbers are dis-
played on the About Image-Pro screen (open from the Help menu):

l The software version number and system information.

l A description of the problem, and any relevant information regarding prior technical
support assistance.

xxiii
Chapter 1 : Installing Image-Pro

Installing Image-Pro

This chapter provides an overview of installation requirements and step-by-step instruc-

tions for installing and activating Image-Pro/Image-Pro with 3D Module.

1
Chapter 1

Package Contents
Before installing Image-Pro, please check that you have received everything in your
Image-Pro package:
l Image-Pro USB Installation Drive
l Image-Pro USB Licensing Dongle (if applicable)

2
 System Requirements for Image-Pro 10

System Requirements for Image-Pro 10

To install and work with Image-Pro you will need the following equipment and software:

Supported Operating Systems:

l Windows® 7 Professional and Ultimate (SP 1) – 32-bit and 64-bit
l Windows® 8.0 or 8.1 Enterprise and Professional – 32-bit and 64-bit
l Windows® 10 Enterprise and Professional – 32-bit and 64-bit

Minimum Requirements
l Dual core processor
l 4 GB of RAM or more
l 5 GB or more of available hard-disk space
l 1024 x 768 display with a 32-bit video card
l DVD/CD-ROM drive
l Microsoft .NET 4.0 Framework (automatically installed by application)
l USB port required for hard licenses and offline license activation
l Internet connection required for online services.

Note that Image-Pro will not install on computers running Windows® XP or Win-
dows® Vista™. Nor will it run under Windows® N.

Recommended Requirements
l OS: Windows 7, 8.1 or 10, 32-bit or 64-bit
l Processor: 2.8 GHz Intel quad-core 64-bit processor (Core i7 Series) or better
l RAM: 16 GB memory or higher
l Free Disk Space: Multiple High Speed SATA Hard Disks or SSDs; 8 GB free on
installation drive + free space for images (500+GB)

3
Chapter 1

l Graphics Card: nVIDIA GeForce GTX Cards with 4GB graphics memory & Open
GL 4.2 or higher
l DVD-ROM drive
l USB port required for hard licenses and offline license activation
l Internet Explorer version 9 or higher
l Internet connection required for online services*

High Performance Requirements

l OS: Windows 7, 8.1 or 10, 64-bit
l Processor: 3.0 GHz Intel quad-core 64-bit processor (Core i7 Series) or better
l RAM: 48 GB memory or higher
l Free Disk Space: Multiple High Speed SATA Hard Disks or SSDs configured in
RAID 5; 16 GB free on installation drive + free space for images (1 TB+)
l Graphics Card: nVIDIA GeForce GTX Cards with 4GB graphics memory & Open
GL 4.2 or higher
l DVD-ROM drive
l USB port required for hard licenses and offline license activation
l Internet Explorer version 9 or higher
l Internet connection required for online services*

4
 System Requirements for Image-Pro with 3D Module

System Requirements for Image-Pro with 3D Module

Manufacturers change system specifications regularly so it is difficult to describe the exact
configuration that is requiredowever there are some important guidelines to consider before
purchasing or upgrading your computer hardware for use with Image-Pro with 3D Module.

Rule of Thumb:
In short… More S Better. More GPU (Graphics Processing Unit) cores, more system RAM,
more graphics RAM, more CPU cores, more speed for 3D viewing rates.
Image-Pro with 3D Module has no limits on the number of objects, the size of images, the
number of images, or the number of frames. Everything depends on memory and speed. For
users the best possible experience and product performance review the requirements below
and follow-up with a review of the hardware priorities to understand how the whole sys-
tem plays a part in the application’s performance.
To install and work with Image-Pro with 3D Module you will need the following equip-
ment and software:

Minimum Requirements (for small datasets only, performance will be low)

l OS: Windows 7, 8.1 or 10, 32-bit or 64-bit
l Processor: 2.4 GHz CPU Intel dual-core processor or better
l RAM: 4 GB memory
l Free Disk Space: 4 GB on installation drive + free space for images (20+GB)
l Graphics Card: nVIDIA GeForce GTX 560 (1GB & Open GL 4.2 or higher)
l DVD-ROM drive
l USB port required for hard licenses and offline license activation
l Internet Explorer version 9 or higher
l Internet connection required for online services*.

Recommended Requirements
l OS: Windows 7, 8.1 or 10, 64-bit

5
Chapter 1

l Processor: 2.8 GHz CPU Intel quad-core processor or better

l RAM: 16 GB memory or higher
l Free Disk Space: Multiple High Speed SATA Hard Disks or SSDs; 8 GB free on
installation drive + free space for images (20+GB)
l Graphics Card: nVIDIA GeForce GTX 580, 760 ( 2GB & Open GL 4.2 or higher)
or AMD Radeon R9 290 (4.0 GB & Open GL 4.3 or higher)
l DVD-ROM drive
l USB port required for hard licenses and offline license activation
l Internet Explorer version 9 or higher
l Internet connection required for online services*.

High Performance Requirements

l OS: Windows 7, 8.1 or 10, 64-bit
l Processor: 3.0 GHz Intel quad-core processor or better
l RAM: 48 GB memory or higher
l Free Disk Space: Multiple High Speed SATA Hard Disks or SSDs configured in
RAID 5; 16 GB free on installation drive + free space for images (1 TB+)
l Graphics Card: nVIDIA GeForce GTX 680 (4GB & Open GL 4.2 or higher) or
AMD Radeon R9 290X (4GB and Open GL 4.3)
l DVD-ROM drive
l USB port required for hard licenses and offline license activation
l Internet Explorer version 9 or higher
l Internet connection required for online services*.
*This product may allow you to access certain features that are hosted online ("online ser-
vices"), provided you have a high-speed Internet connection. The online services include
but are not limited to, automatic updates, support links, access to video tutorials, and more.

6
 Hardware Priorities

Hardware Priorities

Graphics Card
Image-Pro with 3D Module is a 3D graphics-intensive product that requires a very capable
graphics adapter. For this reason, purchase the most powerful graphics card possible
because the single most important determinant of 3D viewing performance is the graphics
card.
When choosing a graphics card for optimal visualization performance Open GL 4.2 is
recommended along with at least 1GB of graphics memory, but ideally 2GBpreferable.
Securing the best possible graphics card is not buying the most expensive one, as many of
these cards are designed for unique rendering applications, such as the nVidia Quadro
series. These cards will not necessarily provide an Image-Pro with 3D Module user with
the best price to performance ratio. NVidia GeForce GTX and AMD Radeon HD and ATI
Radeon X cards typically are designed for video gaming, but deliver excellent performance
for 3D image analysis as well. When looking into nVidia cards, be aware of their series of
cards such as the 500, 600, and 700 have high and low endsmean a GTX 650 could be
potentially worse than a GTX 580 because the first number denotes a series and not the
level of performance. In this case the second number of the model explains how they
match up.
If you are working with very large images or sets, then it is important to focus on the
second priority, as much as you do the graphics card for visualization. Our suggestions
regarding memory are in the next section.
l Users are discouraged from multi-video card (Crossfire and SLI) solutions for now,
as this technology is known to decrease performance compared to single graphics
card solutions.

System Memory
System memory is the second most important factor for users who need to inspect large
data sets in particular, large volume data with direct volume rendering. This is why Image-
Pro with 3D Module is a 64-bit ONLY software product (32-bit operating systems are lim-
ited to a maximum amount of about 3.2 GB of system memory. It is important to note that
you may need much more memory than the actual size of the data you want to work with
since some processing may require several times the memory required by the original data
set.

7
Chapter 1

For instance, if you want to load a 4GB data set in memory, you may need up to 16 or
20GB of additional memory for the intermediate results of your processing. More com-
monly you will need 2 or 3 times the memory footprint for data being processed.

Hard Drives
When working with large files that contain either a long sequence or many dimensions of
data, reading from the disk can slow down your productivity.
Option 1 is a standard hard drive (e.g. 7200rpm SATA disk) that can only send data to
your application at a continuous rate of about 60MB/second. That is the theoretical limit;
your actual experience is likely to be closer to 40MB/second.
Option 2 is to configure multiple hard drives (3 or more) in a RAID5 configuration but
please note that RAID configurations may require substantially more system admin-
istration.
Option 3 is to use solid state drives (SSD) instead of standard hard drives (HDD) in either
a single-drive or RAID configuration. SSD’s are becoming more popular as the prices con-
tinue to drop. The SSD can perform better than a HDD, generally having file read-
ing/writing at speeds of 200-550 MB/second. The price of solid state drives is still higher
and the capacity is not typically equivalent, but the performance can certainly be worth
the investment.

CPU
The Image-Pro with 3D Module platform is designed to support multiple cores/processors
using state-of-the-art background processing. This feature is on by default and enables the
software to execute tasks in parallel. Since Image-Pro with 3D Module combines the best
of 3D and 2D processing and analysis, additional CPU cores and/or processor speed will
enhance the responsiveness of the Graphical User Interface (GUI), data tables, 2D outlines,
and large image filters. If you are using the 3D rendering and measurements, they will be
mostly exploiting GPU power, and little of Image-Pro’s visualization processes are strongly
affected by CPU performance. Some image processing routines are CPU-limited, so a better
CPU can benefit performance on those routines.

8
 Installing Image-Pro

Installing Image-Pro
To install Image-Pro, follow the steps below. Please close any other applications before
starting the installation process. The setup screen will indicate which version of Image-Pro
you are installing.
1. Insert the Image-Pro product into the correct drive.
If the program does not start automatically, you can install Image-Pro using the Start
button on your desktop. Choose Run and then use the Browse button to find the
autorun.exe file on the CD. Double-click autorun.exe to begin the installation process.

Image-Pro requires the Microsoft *.NET framework. If this is not installed on your
computer, the application will install it automatically.
If you have restricted user access control, you may see a warning asking if you want
the program to make changes to your system. Click Yes.
You will see the Installation Start screen. This screen has the following buttons:
l Install Image-Pro
l Documentation
l Install Demo Images
l Capture Support.

2. Click Install Image-Pro.

You will see a dialog extracting the Image-Pro *.MSI file. If you have restricted
user access control, you may see a warning message asking if you want the *.MSI
file to make changes to your system. Click Yes.

Image-Pro requires the Microsoft *.NET framework. If this is not installed on your
computer, the application will install it automatically.
Please wait.
In a few moments, you will see the Image-Pro Setup screen.
3. Click Next.
You will see the Image-Pro License Agreement.

9
Chapter 1

4. Check the Accept box, and click Next.

The Select Destination Folder location dialog box appears. This screen asks you for
the destination for the Image-Pro program files and folders, and for who can access
this installation.

10
 Installing Image-Pro

5. Click Change if you want to use a different location.

6. When the path is correct, click Next to continue.
The next screen verifies the destination folder for the Image-Pro data files:

11
Chapter 1

7. Confirm that the destination is correct. Click Change if you want to use a different loc-
ation.
8. When the path is correct, click Next to continue.
The next screen indicates that your computer is ready to install
Image-Pro/Image-Pro with 3D Module.

12
 Installing Image-Pro

9. Click the Install button to begin the installation. This screen gives you a chance to
verify your Image-Pro location and settings.
The Installing dialog box appears. The setup program will begin copying and
installing files.

13
Chapter 1

The final installation screen indicates that Image-Pro is now installed.

14
 Installing Image-Pro

10. Click Finish to conclude the installation process.

Note: During the installation process, you may see the following warning message:

This warning can be ignored safely, and installation of your product will complete suc-
cessfully.

15
Chapter 1

Activating Your Image-Pro License

USB license holders must activate their licenses after installing the software and plugging
in the hardware key.
Activating the Image-Pro Program
Before you can use Image-Pro, a valid license file must be obtained from the Thermo
Fisher Web site. A 14-day trial license is included for evaluation purposes.
1. If you have an Image-Pro hardware key, attach it to a USB port now.
2. When you first startImage-Pro, you will see the following dialog:

3. Click Activate Now to activate your license.

This will load the License Activation Wizard which will guide you through the steps
of activating your software.

16
 Activating Your Image-Pro License

If your PC has Internet access

1. Confirm your license type and USB dongle.
2. Enter the Unlocking Code(s) that was sent with the product.
3. Provide standard registration information.
4. Once your information is received, all your licenses will be installed on
your machine.
If your PC does not have Internet access
1. If you choose Activate Now, and your PC is not connected to the internet,
the License Activation Wizard will bring you to the following prompt after
the dongle selection:

2. Get your license file(s).

a. If your dongle is not activated or you don’t have access to the original
computer, you can download your license(s) by visiting http://www.-
mediacy.com/activate using the provided shortcut with your unlocking
code(s).

17
Chapter 1

b. If you have an already activated dongle you can obtain the license(s)
using the License Activation Wizard to export them from the original
computer.
3. In both cases, you can import the license(s) on the second page of the
License Activation Wizard.

18
 Verifying your Registration

Verifying your Registration

1. (Optional) Verify your registration by going to the Help menu and choosing About
Image-Pro:

The About page should show your (non-evaluation) license key:

19
Chapter 1

This page will indicate if and when your license expires. It also provides information
about your maintenance package, if any

20
 Windows Security Alert

Windows Security Alert

Depending on your Windows Firewall settings, you may see an alert when you start
Image-Pro for the first time. This alert indicates that Windows may have blocked some
aspects of the application.
Your software is checking on whether updates are available, and no personal information
is being sent. You will want to check the "Domain networks, such as a workplace
network" option, and then click Allow access.

21
Chapter 1

Product Versions and Updates

Most of the time, updates to Image-Pro happen automatically. If you have an Internet con-
nection, you can set your application software to check for updates when you start
the product. You must have an internet connection to update your product automatically.
For information about upgrading your product and license to Image-Pro 9.3, please see the
information about upgrading later in this chapter.

22
 Updating Your Product

Updating Your Product

Image-Pro uses the Auto-Update feature to ensure that you are running the most current ver-
sion of the software. Auto-Update is a small, unobtrusive feature that does not collect any
personal information from you.
In order for the process to work, you must have an Internet connection available.
Select the Automatic Update Check option in the Application page of the Options dialog
box, as shown here:

Checking this box ensures that software update checks will happen automatically.
When an update is available, your application will offer to download the available
updates. The downloads run in the background, and you may continue to use Image-
Pro while they run. You can decide later when you want to install the downloaded
updates. More information about Application Options appears in Chapter 2.

23
Chapter 1

If Automatic Update Check is not selected, follow the steps below to look for updates
to your software installation.
1. Click the small drop-down arrow at the upper right-hand corner of your application.

2. ClickCheck For Updates. If your product is current, you will see a message saying
that you are running the most current version and no update is necessary.

3. If an update is found, you will see the Update Available dialog:

4. To install the update now, click Yes. To install the update later, click No.
If you click Yes the application closes (and prompts to save documents as appro-
priate), and the update is applied once the application has exited. If you click No, the
update is installed after the end of the current session.
When the update process is complete, you may see a message, or a page of release
notes pertaining to this upgrade.

24
 Updating Your Product

You may see a message reminding you to restart your computer before using
Image-Pro. If you do, please restart you computer before using Image-Pro.
If you do not have an Internet connection, please contact Media Cybernetics’ technical
support for assistance.

25
Chapter 1

Verifying Updates
You can find out which updates of the application software have been applied by visiting
the Windows Control Panel:
1. Go to Programs:

2. Select View Installed Updates:

26
 Verifying Updates

You will see which programs and updates have been installed on your machine.

3. Once you have verified the update and version number, close the Window Control
Panel dialog.
You can view the About box to verify the current software version and see additional
information about your Image-Pro system.

27
Chapter 1

28
 Help and About Image-Pro

Help and About Image-Pro

The Help menu, User Guide, Support Center, About Image-Pro, Check for Updates, and
other useful features of Image-Pro can be accessed by clicking the drop-down arrow next

to the question mark in the upper right-hand corner of the application :

Once the drop-down menu appears, clicking anywhere on the menu item or its icon in the
Help menu items will take you to that feature of Image-Pro.
Clicking the Help question mark icon or H will open the first page of the in-product help
file:

29
Chapter 1

The Table of Contents, Index, and Search tabs in the left-side panel will help you navigate
through the Help files.
l Clicking the Image-Pro Users Guide link or G opens the first page of the Users Guide
in PDF. There are links and bookmarks to help you find the information you need in
the User’s Guide.
l Click the Image Analysis Forum or F sends you to the image analysis forum dis-
cussion group. This group includes links for you to send feedback, report a problem,
or make a feature request.
l Clicking Welcome to Image-Pro or W opens the Welcome screen. Note that if you do
not have an internet connection, the Welcome screen will display only offline content.
Tutorials, news, and forum posts will be available only if you have an internet con-
nection.
l Click Look for Updates or U to see if a new version of Image-Pro is available, as
described earlier in this chapter.
l Click SupportCenter or S to open a support session with a Technical Support spe-
cialist:
l Click About Image-Proor A to see important information about your Image-Prosystem.
The About page displays the version, copyright, license, and maintenance information:

30
 Help and About Image-Pro

The License page displays the current license agreement.

31
Chapter 1

The Credits page contains credits and acknowledgements:

The System Information page displays information about your system:

32
 Help and About Image-Pro

You can expand the system information folders to see the details of your installation. You
may need this information when working with Technical Support.
The next screen shows the system information for a rental software license.

Finally, a Licenses tab is included in the About dialog to help some users configure their
license use, see Configuring License Use.

33
Chapter 1

34
 Upgrading Image-Pro

Upgrading Image-Pro
There are two aspects to any upgrade:
l Upgrading the installed software from the previous product or version to the Image-
Pro 10 software, and
l Upgrading the license that authorizes Image-Pro to run.
You will want to review the next section, "Upgrading the Installed Software" on the next
page, regardless of what type of upgrade you are doing.
If you are upgrading from a previous version of Image-Pro, then you will want to review
the section "Upgrading your License" on page 39.
l If you are running a licensed and registered copy of Image-Pro 9, this will be a
paid upgrade to version 10, and it will not be available via Auto-Update.

35
Chapter 1

Upgrading the Installed Software

There are several ways that you can upgrade the software installed on your computer to
Image-Pro:
l You may download the Image-Pro evaluation software and install it from the
Thermo Fisher website.
l If you have ordered an upgrade for Image-Pro, you will receive a USB stick that
will install the software.
Regardless of how you obtain the Image-Pro installer, and regardless of which product and
version you are upgrading from, Image-Pro can be installed alongside your other software.
This allows you to evaluate Image-Pro for your purposes. When you are done with the
older software and completely migrated to Image-Pro, then you can uninstall the older soft-
ware from the Control Panel.

Upgrading Your Software

If you are running a licensed and registered copy of Image-Pro you will be offered free
upgrades via Auto-Update. If you have Auto-Update enabled, you will be notified of the
version upgrade when you start Image-Pro, and can download and install it as convenient.
If you do not have Auto-Update enabled, you can follow the steps below.
1. Click the small drop-down arrow at the upper right-hand corner of your application

.
2. Choose Look For Updates. You will see the Update Available dialog:

3. Click Yes to continue.

Note that if you are upgrading to Image-Pro 10 from Image-Pro 9, you are required
to update your product license as well as the software. The new version includes a

36
 Upgrading Your Software

14-day temporary license. Follow the instructions regarding the License Upgrade Wiz-
ard later in this chapter to obtain a permanent license.

37
Chapter 1

How to Return your Original Image-Pro License

In order to receive a new Image-Pro license, you MUST return your original Image-
Prolicense dongle. If you do not return your dongle, you will be invoiced for the dif-
ference. Please contact Thermo Fisher's Customer Service for information about receiving a
Return Materials Authorization (RMA) number and instructions on how to return this ori-
ginal Image-Prolicense.
Here is a sample RMA packing slip:

If you don’t have your original packing slip, please contact [email protected]

38
 Upgrading your License

Upgrading your License

If you are upgrading from a previous version of Image-Pro, you may need to upgrade your
license to authorize the new version you are trying to install. When you purchase your
upgrade, you will receive an unlocking code. You will need to have this unlocking code
handy during the upgrade process. Image-Pro includes a Licensing Wizard to step you
through the process of upgrading your Image-Pro license to the latest version. Note that
after the upgrade, the prior version license will still allow you to run previous versions.
In the Image-Proapplication, open the Help menu by clicking the down arrow next to the
Help icon, select Licensing Wizard.

39
Chapter 1

Configuring License Use

Image-Prouses a powerful licensing scheme allowing to provide the maximum flexibility
when deploying the product. Although the user usually doesn’t need to be involved with
configuring its license use, there are a few cases where it may be convenient to do so.
These include network configurations with shared licenses that may have to be assigned to
specific workstations as well as demonstration or evaluation licenses which may be con-
figured to select the adequate level of functionality. In order to help those users, a license
management tab is included in the About dialog.

l Each available product tier and module is displayed in a hierarchical view with its
contained tiers and features listed as child nodes.

40
 Configuring License Use

l Product tiers and modules can be excluded or left as available. Each selection gives
visual feedback based on the tier hierarchy and dependencies so that selecting a tier
will show exactly what features will be included or excluded. The three possible
licensing states are as follows:
l In Use: This tier or module is currently in use on this machine.
l Available: This tier or module is currently available on this machine
without being in use.
l Excluded: This tier or module will not be used on this machine, leaving
the license available for others to use.
l Once changes are made, the configuration can be saved using the Save Con-
figuration button and will take effect in the next session. The information is saved
in the identity so that different identities can use different licenses.

41
Chapter 1

Sending Feedback
Thermo Fisher welcomes your feedback about our products. If you have a suggestion, ques-
tion, or concern, please let us know. To send feedback, follow these steps:

1. Click the drop-down arrow in the upper right-hand corner of the application :
You will see this menu:

2. Choose Report a Problem or Send a Feature Request under the Image Analysis For-
ums.
You will see a form similar to the following according to your choice:

42
 Sending Feedback

3. Choose a general comment, feature suggestion, or issue from the dropdown list.
4. Enter your comments, and remember to include your email address.
5. Press Submit.
Your feedback is always appreciated. You should hear from us shortly.

43
Chapter 2: Getting Started
Getting Started
This chapter introduces you to the Image-Pro and Image-Pro with 3D Module systems,
including an overview of the interface components and features.

44
Chapter 2

The Image-Pro Application Window

When Image-Pro is loaded, its application window becomes active.

The in-product Help contains detailed information about all the features and functions of

Image-Pro. Click on the question mark at the right side of the application window
to open the help file. More information about using the Help appears at the end of this
chapter.
The center of the window contains a set of panels or panes. The far-left panel is the Over-
view panel, which contains the image strip and tools that provide an overview of the work-
space contents. The center panel contains the active content, which can be an image,
histogram, or other rendering. The far-right panel contains the image histogram as well as
additional dialogs that enable you to modify the contents of the workspace.
The following sections explain these features of the Image-Pro application window in
greater detail.

Floating Panels
One of the unique features of Image-Pro is the “floating” or “dockable” panels. The image
strip, details panel, mini-histogram panel, or other workspace areas can be moved inde-
pendently from the main image workspace. This may be particularly useful when working
with a dual-monitor system, as you may want to display the image on one monitor and the
details on the other. An example of floating or “undocked” panels appears below:

45
 The Image-Pro Application Window

Workspace Tabs
Image-Pro and Image-Pro with 3D Module display your images in tabbed workspaces, as
shown here:

The name of the active image in the workspace is highlighted. You can switch back and
forth among multiple images by clicking the image’s tab.
The small icons next to the name of the image indicate what type of image is open. Some
examples of common types are shown here:

Viewing Tooltips
Tooltips show the basic image information:

46
Chapter 2

l To view an image tooltip, move your mouse cursor over the image thumbnail
in the image strip.
The zoom and pan controls allow you to resize the image or a portion of the image.
When the active image is zoomed and doesn’t fit in the viewer, the viewing area is shown
on the image strip thumbnail as a semitransparent orange overlay.
l Click and drag the center of the viewing area on the thumbnail to control pan
and scroll of the image.

Tools and Layouts

The View tab contains specialized tools and graphs to display information about the act-
ive image or image set. Some of these tools allow you to see different aspects of the active
image also.

47
 The Image-Pro Application Window

Using the Multiple Document Interface (MDI)

The Switch to MDI button will toggle the workspace windows to MDI windows or back
to tabbed windows. This feature allows you to view more than one image at a time, and
move the images and panels to different locations in the application.
This feature is located in the Workspace group on the View tab’s ribbon:

Normally, when the workspace windows are set to Docking, the image appears like this:

When you switch to the MDI view, several images can be viewed at once, as shown here.
This example shows image windows tiled horizontally.

48
Chapter 2

The pull-down MDI menu allows you to select a window display:

This example shows image windows in the Cascade layout:

49
 The Image-Pro Application Window

Align Views lets you organize your images in rows and columns, as shown here:

The Tools in the Workspace group allows you to load and save custom tool sets and lay-
outs for your specific situation.

50
Chapter 2

Close All Views closes all the images and windows in the workspace.
Close All Tools closes all open dialogs and panels.
Tools redocks or resets any tools/ Click the drop-down arrow to see these features

Redock All Tools: Resets any open moveable panels that are undocked to their default
docked position.
Reset Tools Layout: Resets the application to its factory default tools layout.
Load Layout: This tool allows you to load a previously-saved tools layout. When you
click on this tool, the following dialog is displayed:

51
 The Image-Pro Application Window

This dialog shows all available tool layouts to be loaded. The Recent area shows a list of
the last loaded layouts. The Custom area shows a list of the custom layouts currently
stored in the Configuration Files folder. The Default area shows a list of the available pre-
defined layouts from installation directory \Resources folder.
SaveLayout: Click this tool to save the current layout of tools to a file that can be loaded
at a later time to restore the layouts to its current configuration. When you click this
option, you are prompted to give the layouts configuration file a name. Type a name for
the layout configuration file, then click Save. You can use the Load Layout tool (described
above) to restore the tools layout to its current configuration at a later time.

52
Chapter 2

The Image Context Menu

Clicking the right mouse button on an image displays the following context menu:

l The Zoom Scale Bar allows you to adjust the size of the image by moving the
slider to the right or left.

l The Undo and Redo buttons let you undo recent actions and reverse the effects of
the Undo.

l The Copy button will copy the part of the image under the ROI to the Win-
dows Clipboard.

l The Histogram button opens the image histogram.

l The Image Information button displays information about the image in a

table below the image.

l Local Zoom opens or closes the Local Zoom feature.

l The Red, Green,Blue, and White tint buttons apply the appro-
priate color to the image.

l The Remove Tint and Remove Tint All buttons remove tints from the active
image or all open images.

l The Toggle Nudge polygon/polyline editing mode button is enabled when the
selection tool is running. Nudge editing mode becomes active only when a polygon
or polyline is the selected object being edited. Pressing the <N> key has the same
effect.

53
 The Image Context Menu

l The Toggle Extended Vertex polygon/polyline editing mode button is enabled

when the selection tool is running. Extended Vertex editing mode becomes active only
when a polygon or polyline is the selected object being edited. Pressing the <E> key
has the same effect.

l Clicking the Convert button converts the selected graphic object(s) into poly form
if possible.

l The Toggle limiting graphic object button limits creation and editing to image
bounds on the active overlay. By default, graphic object creation and editing is lim-
ited by the bounds of the underlying image on all overlays except for the Annotation
overlay, where limiting is disabled by default.

l The Selection tool enables selection and editing of displayed graphic objects.

l The Draggingtool allows you to move the Pan/Scroll position by clicking and
dragging on the image.

l The Zoom tool allows you to zoom in or out on the image with mouse clicks or
by drawing a box around the area to be zoomed. Only one tool may be active at
any time, so starting one of these tools will stop any currently active one.
If your active workspace is an image set, you will see a different context menu.

More information about working with image sets appears in Chapter 5.

54
Chapter 2

Viewing Image Document Information

Click the symbol to see information about your active image. You will see the
DocumentProperties window displayed at the bottom of the Image-Pro workspace, as
shown here:

The image and document property information has been greatly expanded in from pre-
vious versions of Image-Pro. This expanded view allows you to find important inform-
ation more easily. You can also edit the metadata.
The Document Properties feature allows you to view your data in four different lay-
outs:

To compare information for more than one image, click the Lock icon.

55
 The Welcome Screen

The Welcome Screen

The first thing you see when you start Image-Pro is the Welcome screen. T The updated
and improved Welcome screen introduces a tabbed interface with 4 different tabs.
The Get Started tab displays a link to the Apps Library on the top part of the page. The
bottom part of the page shows the last three posts from the Image Analysis Forum. Click
any link to access the image analysis discussions and knowledge base.

The information on the left side of the screen is always available. From here, you can open
an image, sequence, or set. The list of recentimages displays a list of files that were
recently opened.
Check for Updates: Click this button to see if any upgrades are available from Thermo
Fisher.
Show page at startup: Check this box to see the Welcome screen each time that you start
Image-Pro.
The Training Resources tab displays a copy of the contents of the video tutorials page
on the website.

56
Chapter 2

The tutorials are in MP4 format. A media player is required to view these files.
The Latest News tab displays the latest news from Media Cybernetics.

A red notification displays the number of new articles and disappears when the tab is
clicked.
The Forums tab displays the most recent discussions from the Media Cybernetics Image
Analysis Forum.

57
The Welcome Screen

58
Chapter 2

The Image-Pro Ribbon

The Image-Pro Ribbon contains commands that affect or change the content in the main
workspace. The overwhelming majority of the commands in the Ribbon are buttons which
either perform some command directly or open a dialog panel for setting options and per-
forming related actions. The ribbon displays commands organized into tabs and groups.
The ribbon displaying the Capture tab is shown below:

The different ribbons will be examined in more detail later in this chapter.
Note that if you have not licensed capture, or are running Image-Pro with 3D Module,
some of the tabs and their associated ribbons will be different from this illustration.

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 Different Types of Content

Different Types of Content

Image-Pro is able to open and save a wide variety of files and formats, as shown in the
snapshot here:

In addition to different image file formats, Image-Pro can open different types of image
sets and Thermo Fisher experiment files.
Images, or single frames, come in many different formats and types. Image-Pro can open
and read many different formats. A list of available image formats appears in Appendix A.
Image sets are collections of images containing more than one variable dimension. A time
lapse movie is a simple example of the type of image sets that
Image-Pro is designed to handle. A more complex set may contain time lapse movies at
several different positions on the slide, with a Z-series at each time and spatial position. It
is possible to create image sets that have multiple channels, stage positions (user defined
or arrayed), Z slices, images per stage position (tiles), and time points. In an image set, we
still have multiple frames, but we know why – there are multiple channels, or the stage
moved, or the focus changed, etc.
Experiments are image files that contain different types of content stored in one, often very
large, file.

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Chapter 2

The File Menu

The File button is located in the upper-left corner of the application window. The File but-
ton menu contains commands that operate on the document as a whole, such as Print and
Save, as well as other basic functions.

Clicking the File button displays functions that are always available:
l New
l Open
l Save
l Save As
l Reload
l Print
l Close
Some of the items in this list have small menu arrows next to them. Hover your mouse
over the arrow to make the menu appear, as shown here:

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 The File Menu

The Recent Documents panel lists the files that you opened recently:

Click the name of the file to open it. You can use the push-pin icon to “pin” recently-
opened files to keep them in the list. The drop-down arrow

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Chapter 2

Opening an Image
To open a saved image:
1. Click the File button.
The pull-down application menu appears.

2. Choose Open. Click to go directly to the Open Images dialog, or hover your mouse
over the menu arrow to see the menu of choices.

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 Opening an Image

3. Select the location and type of image file that you want to open.
4. Select the image file that you want to open using the standard file open dialog as illus-
trated below.

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To open more than one image, hold down the <Ctrl> key and use your cursor to
select the images you want to open.
5. Click the Open button.
The selected image is opened in the Image-Pro workspace.

Opening an Image Sequence

Image sequences are like small movies. Image-Pro allows you to open, edit, and play image
sequence files (*.seq).
To open an image sequence:
1. Click the File button.
The pull-down application menu appears.

2. Choose Open. Click to go directly to the Open Images dialog, or hover your mouse
over the menu arrow to see the menu of choices.

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 Opening an Image Sequence

3. Select Open Images as a Sequence. Sequences have the extension *.seq.

4. Select the image sequence that you want to open using the standard file open dialog
as shown here.

6. Click the Open button.

The image sequence is opened in the Image-Pro workspace.

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Chapter 2

This example shows one frame of the heart.seq.

Opening an Image Set

An image set is a collection of images taken together to form a time lapse sequence, area
scan, or a Z-stack, or any combination of these. Image sets are often complex groupings
that include more than one dimension, such as both time and Z/focus dimensions, as well
as channel specificity and more. This would mean that a single image member file of a
given image set could, for example, represent a single channel at a single distance/focus
and a single moment in time. The collection of images would therefore need to be organ-
ized and reconstituted to be viewable and analyzable in any meaningful way. is designed
to work with the metadata and naming conventions found in many sets created by com-
monly used image acquisition tools and to organize the files according to the their sig-
nificant dimensions (or let you organize them) so that they can be saved into a single
image set file (with the extension *.mcs).
Note that there are five possible dimensions that can be used in the organization of files in
a set:
l Channel: Index number for the color channel.
l Slice: Index number for the relative position in the Z stack or series of focal adjust-
ments.

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 Opening an Image Set

l Site: Index number for the site. Used for organizing image data per area of interest
in a sample, for example.
l Time: Index number for the relative position in a time lapse sequence.
l XY Scan: Index number for the relative position in an XY scan (a series of images
that are taken across the surface of a sample).
Image-Pro and Image-Pro with 3D Module feature the SetBuilder. You can use this fea-
ture to create an image set from a collection of individual image files. Follow the instruc-
tions below:
1. Click the File button.
2. Choose Open Set from Files.

You will see the following dialog:

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Chapter 2

3. Select the folder containing your image source files.

Use the Select Source Folder browser in the far-left panel to navigate to the folder
where your image files are located. Your set files should be in numerical order if pos-
sible.
4. Select the method for determining the set. There are three modes of operation avail-
able:
l Automatically detect file sequence. Select this option if the files in the sequence
have matching internal dimensions and naming conventions that make their loc-
ations along each dimension distinctly identifiable. In such a case, Image-Pro can
reconstitute the entire set instantly and automatically. If you select this option,
you should:
a) Select a single file that is a member of the set you are trying to buld. analyzes
its name to tries to discern dimension information in the name. If any dimension
information is detected, it prompts you to confirm the meaning the data in the
name structure, as follows:

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 Opening an Image Set

b) Review the assigned dimensions. From the list boxes provided, select the cor-
rect dimension information if guessed wrongly.
c) For sequences that include numeric and non-numeric dimension information
(such as when a channel name is included in the file name), use “Check to add the
active selection as a partial set” to “lock in” the detected sequence to part of a lar-
ger set.
l Split sequence into dimensions. Select this option if the files in the sequence have
naming conventions that mix location information for multiple dimensions into a
single sequence. For example: a 50-slice, 2-channel set that includes files named
“Image001.tif” through “Image100.tif”In such a case, can automatically collect the
relevant files into a table, and perhaps organize them according to one dimension,
but it depends on you to specify the specify the allocation of the specified
sequence into the relevant dimensions. If you select this option, you should:
a) Select a single file that is a member of the set you are trying to buld. analyzes
its name to tries to locate sequence information in the name. If any dimension
information is detected, it prompts you to confirm the meaning of the data in the
name structure, as follows:

b) Adjust the dimension allocation as appropriate using the list boxes provided.
Note that additional dimension drop-downs appear as needed.
l Manually add files to a dimension. When you select this option, you are promp-
ted to select a dimension and position within that dimensions to which you want
to add files, and then select the files to be added: Follow the steps below to manu-
ally organize your files:
a) In pull-down list box below the Add selected files to... button, select the
dimension you want to define.
b) In the spin box to its right of the selected dimension, specify the position in
the selected dimension to which you want to add files.

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c) In the file list, select all files belonging to the specified position of the selec-
ted dimension.
d) Click the Add selected files to... button.
e) Repeat steps a) through d) until all relevant dimensions and positions are
defined.
Choosing a new operation mode will change the controls available in the control
panel at the bottom of the center region of the Set Builder dialog.
5. Select a folder to add images to the File Table in the center panel. The file table in the
center will list the supported image files that are present in that folder, whether or not
they belong to a set.
The Location columns of the file table display the location index fields that have been
assigned to that file. If the file has not been assigned to any location within the set,
this column will be blank. Once the file has assigned to a location in the set, the Loca-
tion column will be replaced with columns for each dimension that is currently active
in the set.

Each dimension is represented by an icon: Color Composite Channel, Location in the

Z –stack, and Time position. Each column header has a tool tip to indicate the dimen-
sion that the icon represents.
The dimensions of each set appear in a table on the right hand side of the dialog.
When you've selected a folder, the file table in the center will list the supported image
files that are present in that folder, whether or not they belong to a set are in the panel
on the right. This table indicates the extents of the set as currently constructed. In the
cases of incomplete (jagged) sets, the extents in this table will represent the longest
added set thus far:

6. Add channel information for the set.

The channels table displays one row per channel. You can set the channel name, tint,
or set them both by assigning a dye to the channel, as shown in the illustration below:

71
 Opening an Image Set

The channel name can be edited in-place. Use the Color drop-down arrow to see the
standard color picker. Click the “…” button in the Dye column to select a dye from
the favorites list, or from all available dyes:

l When Show all dyes is checked, all the dyes in the Dye Library will be included in
the list. If this option is not checked, you will see only the dyes in the Favorites
list. If the current channel name matches a dye in the list, that dye will be selected
in the drop-down list.
l Click Apply to set both the Channel Name and the Color for the selected dye. In
addition, the dye will be associated with the channel and will later be associated
with the image set channel when the set is imported.
7. Set the Z-spacing.
The Z- spacing panel on the dialog allows you to set the spacing between Z planes if
the selected set has Z stacks:

The selected spacing will be applied to both the set’s metadata and to the frames of
the images
8. Open the set.
The Open Set button will be disabled until you have selected at least one file to
define the set.
Here is an example of an image set open in the workspace:

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Chapter 2

The slider controls and other features of an image set are discussed in Chapter 5, Work-
ing with Images.

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 Saving an Image

Saving an Image
When you hover over the Save command in the application button drop-down menu,
Image-Pro displays two options:
l Save
l Save All.

Save
Use the Save command to immediately store the contents of the current window to its file
(the file listed on the window's title bar) while leaving the image active in its window. If
the image is in an untitled window, Image-Pro will display the Save File As dialog box.
The Save command can be used to save your most recent changes to disk. It is often per-
formed as a precautionary measure during lengthy or involved processes to reduce the
amount of reprocessing that might be required in the event of a system failure or oper-
ational error. When you close an image and choose not to save its changes, Image-Pro dis-
cards all changes made since the last Save operation.

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Chapter 2

Save All
Use the Save All command to immediately store the contents of all images currently open
in the Image-Pro workspace.
To save an image, follow the steps below:
1. Click the File button. The pull-down application menu appears.

2. Choose Save. You have a choice of saving only the active document (image) or saving
all modified documents or images. Your image is saved automatically.

Using Quick Save

In addition to using the Save to File feature of the image settings,
Image-Pro offers you a quick and easy method of saving your images. Note that Quick
Save does not work with image sets.
1. Go to the Quick Save group on the Capture tab.

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 Using Quick Save

2. Choose Quick Save for Analysis or Quick Save for Publication.

If you are saving a sequence of images, choose Save as Movie.
When you select Analysis, you will see the following drop-down menu:

l Quick Save for Analysis stores your images in *.TIF format so that they can be
retrieved for analysis at another time.
l Open Saved Images goes to the Open dialog and lets you select a previously stored
image.
l Options takes you to the File Options page where you can change your pref-
erences.
When you select Publication, you will see the following drop-down menu:

l Publication stores your image in a *.JPG format that can be easily incorporated
into a report or spreadsheet. It also burns-in any annotations, ROIs, or measurements
and resizes your image.
l Auto Open will automatically open your saved image in the workspace.
l Open Saved Images goes to the Open dialog and lets you select a previously stored
image.

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l Options takes you to the File Options page where you can change your pref-
erences.

Save As
Use the Save As command to store the contents of the current window, the current
ROI, or the active part of a sequence, to a file that you specify, in the format that you
specify. The Save As command serves several important uses beyond simply storing an
image to a new file name. It is also used to:
l Convert a single image from one format to another. For example, if you needed a
TIFF file converted to PNG format, you would simply open the TIFF image, and
then use Save As with the PNG format option to save it to a new file.
l Save an image with different compression or quality options from those it was ori-
ginally created with. For example, if your original TIFF file was uncompressed, you
can use Save As with the LZW compression option to store it in compressed form.
l Save an image to another disk or folder.

Note: Files containing multiple frames, or sequences, are supported only in the TIF,
SEQ, IPW, and AVI formats. All other formats will save all frames separately.
The Save As dialog is shown here:

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 Save As

Save in: Find the folder into which you want to save the file. You may create a new
folder using the New Folder button.
File name: Enter the name of the file you want the image saved to. To specify the
file's location, you can either enter its entire path (disk and folder), or specify its loc-
ation using the Save in list box.
Save as type: In this list box, select the format in which you want the image saved.
Compression: In this list box, select the form of compression you would like the file
stored in. The available compression methods will vary from one file format to
another. For example, LZW (Lempel-Zif encoding) compression is an option with TIFF
files, but it is not available in JPG. Moreover, some formats have no compression
options (None is the only choice).

Note: If you plan to use the image with another program, be sure it is compatible
with the selected compression method. Not all programs support compressed and
uncompressed format variants.
Frame By Frame This option allows to save the sequence into file frame by frame. The
frame’s file name based on next convention: <FileName> <FrameIndex> <Numer-
icSuffix>.<FileFormat> (<NumericSuffix> is an optional part and used to prevent files
from overwriting). If the file format doesn’t support sequences, then Frame By Frame
Only (always checked) will be displayed.

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Reload
Use the Reload command to fill the active window with a new copy of the file it is linked
to (the file listed on the window's title bar).
You might want to use this command if, after considerable editing, you wanted to restart
with an untouched copy of the image. Reload is also very useful in automated procedures
where a routine needs an image free from any effects of a preceding routine.
Note: Image-Pro loads the active window from the current copy of the file associated
with it. If you have made changes to the stored file (whether from this window or another)
after the image was opened, the changes will be reflected in the reloaded image.
Performing a Reload on an untitled image window has no effect.
Performing a Reload has no effect on the Lookup Table. If you have modified the Bright-
ness, Contrast, Gamma settings, they will remain as you set them. If you want to return the
LUT to the original settings, use the Reset button on the BCG tool bar.

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 Printing an Image

Printing an Image
Use the Print command to print one or more copies of the current image, or the current
ROI, to the selected output device. When you hover over the Print command in the File
menu, you are shown the following print options:

Quick Print: Click on this option to print the report to the default printer using the current
printer settings.
Print: Click this option to display the Print dialog box prior to sending the report to the
printer:

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Through the Print dialog box, you can select the printer you want to print to, define any
other print settings, such as number of copies. Click the OK button to send the page to the
printer.
Print Preview: Click this option to display the report in a preview screen prior to printing.
When you select this option, the preview is shown in a new image window with the fol-
lowing ribbon bar options:

Print: This is the same as clicking Print from the File menu (as described above).
Options: Click this tool to open the Page Setup dialog box:

Use the controls on this dialog box to specify the desired page layout options. Through
this dialog box, you can specify the paper size, page orientation, and margins for the page
containing the image you want to print. Click OK to apply your selections and return to
the print preview.
Close Print Preview: Click this tool to exit the print preview window without printing.

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 Quick Access Toolbar

Quick Access Toolbar

Quick Access Tool-
bardrop-down
arrows
This toolbar, located just above the ribbon bar tabs, contains controls which are usable
regardless of which tab is active. You may add a copy of any almost ribbon bar button or
menu item to the quick access toolbar. Added buttons will be retained from application
session to application session.

Clicking the drop-down arrows displays a drop-down menu that allows you to customize
the selections that appear. A copy of the drop-down menu appears here:

Use More Commands to add items to the Quick Access Toolbar. You can find information
about adding commands in Chapter 9.
In addition, right-clicking on any of the buttons in a ribbon displays a menu that allows
you to add that button to the quick access toolbar, as shown here:

l Note that there may be some functions that cannot be mapped to the Quick Access
Toolbar.

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Options Buttons
Image-Pro includes Options buttons throughout the workspace. Click on these gear but-
tons to adjust colors, font sizes, and other feature settings.

Close or Hide Dialogs for Extra Space

Open up your workspace by hiding open dialog boxes. If you are not using a panel to
close the dialog, or may need it again later, collapse it by clicking thumbtack button on
the top of each dialog. Hover over the dialog to expand and view it temporarily. When the
dialog is expanded, you can click the thumbtack again to “pin” it open for future use.

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 The Image-Pro Status Bar

The Image-Pro Status Bar

The Image-Pro status bar provides context-sensitive information about the status of your
activities.

l Status area. This is where the progress bar and other indicators will appear.
l Frame number of the image in the workspace.
l Date and time that the image was captured.
l Pixel location of the cursor.
l Pixel value.
l Spatial calibration if known.
The image view icons allow you to view your image or sequence in different present-
ations:

l Resource monitor. Clicking this icon opens the resource monitor to display import-
ant metrics about the management of images, and control over the application's
use of image memory.
l Single image viewer. This icon changes depending upon if the image in the work-
space is displayed as a single image or in a 3D view.
l Surface plot viewer. Click this icon to display the image as a surface plot.
l Gallery viewer. Click this icon to display an image sequence in gallery view.

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Chapter 2

l Slice viewer. Click this icon to display your image in slicer view.
l 3D viewer. Click this icon to display a 3D view of the image in the workspace.
This will be different if you are using Image-Pro with 3D Module.
l Image bitmap. Click this icon to display an image bitmap of the image in the
workspace.
l Zoom factor. Click this icon to adjust the size and scale of the image in the work-
space. You will see the following dialog.

l Scale to best fit automatically adjusts the image display to show the best fit
l Scale to width automatically adjusts the image to the width of the workspace
l Scale to height automatically adjusts the image to the height of the Image-Pro
allows you to enable or disable the status bar fields. Right-clicking on the status
bar displays a menu that allows you to select what type of data you want to dis-
play in the status bar.
A zoom slider and a fixed-zoom control list allow you to control the size of the image in
the workspace. The zoom slider is shown here:

When the magnification is known and the option active, the current zoom will be dis-
played as the effective magnification, as shown here:

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 The Image-Pro Status Bar

Image-Pro allows you to enable or disable the status bar fields. Right-clicking on the
status bar displays a menu that allows you to select what type of data you want to display
in the status bar.

Click the cursor next to the name of the field you wish to enable or disable. Enabled fields
are checked. You can also enable or disable fields in the status bar using the options
described earlier in this chapter.

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Chapter 2

Tabs and Ribbons

Tabs are used to display different groups of functions. The tabs are arranged in a pro-
gressive left to right order following a common workflow where a person using the applic-
ation acquires an image from a source, adjusts it in some fashion, selects objects in the
image for study, measures properties of those objects, visualizes those objects in multiple
potential contexts, then shares this information with others. If you have licensed a copy
ofImage-Pro with capture, the default tabs are named Capture, Adjust, Process, Select,
Count/Size, Measure, Review, View, Automate, and Apps. The Image tab is only
available when an image is open in the workspace. The Image tab is only available when
an image is open in the workspace. The Extras and Custom tabs are available only when
certain features are enabled. Each tab opens a specific ribbon displaying groups of related
tools.

If capture is not available with your application, the Capture tab is replaced by the Home
tab. If you are using Image-Pro with 3D Module (with or without capture) there are addi-
tional tabs: 3D View, 3D Measure, and 3D Animation. More information about the 3D
features appears in Chapter 12.
Some of the Image-Pro tabs are context-sensitive, which means that they are available only
when you are using certain features, like Line Profile, or opening certain types of images,
image sets, or Very Large Images (VLI).

Capture tab
The Capture tab contains groups for selecting dyes and lenses, calibration, and saving
your images.

Note that this tab does not appear if you are using the non-acquisition versions of
Image-Pro or Image-Pro with 3D Module. In this case, you will see the Home tab instead.

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 Tabs and Ribbons

This tab contains groups for selecting dyes and lenses, calibration, and saving your images.

Adjust Tab
The Adjust tab contains all the functions that allows you to manipulate the image for best
viewing and interpretation. Some functions may require making new or composite images
from other images or changing the pixel values within an image.

Process Tab
The Process tab contains the image processing tools that allow you to change an image
or extract information from it.

Select Tab
The Select tab contains the Clipboard, Annotation, ROI, and Mask features that enable
you to examine and annotate an image or part of an image.A

Count/Size Tab
The Count/Size tab contains tools that allow you to view, count, and measure objects in
a two-dimensional image. It also contains the tools to display the resulting data in a vari-
ety of formats. If you are using Image-Pro with 3D Module, this tab appears as 2D Coun-
t/Size.

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Measure Tab
The Measure tab contains features that enable you to measure and analyze features in all
or part of your two-dimensional image. If you are using Image-Pro with 3D Module, this
tab appears as 2D Measure.

Review Tab
The Review tab contains features that allow you to share your images and data with oth-
ers. You can also create an audit trail of all the recordable events during your image pro-
cessing session. The features on the Review tab allow you to create reports and export
your files.

View Tab
The View tab contains specialized tools and graphs to display information about the act-
ive image or image set. Some of these tools allow you to see different aspects of the active
image also.

Image Tab
The Image tab is only visible when there is an image or image set open in the workspace.
You can adjust the image display, show it in a gallery, or zoom/pan on a particular portion
of the image.

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 Optional Tabs

Optional Tabs
These tabs appear when you enable certain features using the Options described at the end
of this chapter.

Automate Tab
The Automate tab contains the macro scripting features. This tab is visible by default, but
you can turn it off by un-checking Show Automate Tab on the Display options page.

Apps Tab
The Apps tab contains any apps that you have loaded into your copy of Image-Pro. It
contains a link to the App Center, where you can find and download the newest apps. It is
empty until you have created or downloaded some apps.

Custom Tab
The Custom tab creates a ribbon for you to store the features that you use most fre-
quently. The first time you open it, the Custom tab’s ribbon will be empty. You can add
any group or tool to it.

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This tab does not appear until you check Show Custom Tab on the Display options page.

Extras Tab
The Extras tab contains advanced features, such as Find Shapes. This tab does not appear
until you check Show Extras Tab on the Display options page

Context-Sensitive Tabs
These tabs are available when you have opened certain features.

3D View Tab
3D View lets you load, save, and examine your 3D image in a variety of ways:

This tab is available when you have a 3D image open in the Image-Pro with 3D Module
workspace.

3D Measure Tab
3D Measure contains the tools and features you will need to make volume measurements
in a 3D image:

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 Context-Sensitive Tabs

This tab is available when you have a 3D image open in the Image-Pro with 3D Module
workspace.

3D Animation Tab
3D Animation lets you create animated images and movies from 3D images.

This tab is available when you have a 3D image open in the Image-Pro with 3D Module
workspace.

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Chapter 4 : Viewing Intensity Measurements

Viewing Intensity Measurements

This chapter explains how to:
n View an Image Histogram
n View a Line Profile
n View a Data Table
n Use the Saturation Warning

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Chapter 3

Image Histogram, Line Profile, and Saturation Warning

If you have a license for the Capture module, the Image Histogram, Line Profile, and Sat-
uration features appear in the Intensity group on the Capture ribbon. If you do not have a
Capture license, this group appears on the Home tab’s ribbon.

Image histograms measure, and illustrate in graph form, the distribution of pixel intens-
ities in an image. Histogram data can be created and viewed for data gathering and ana-
lytical purposes or can be manipulated for image enhancement purposes.
Line profile analysis allows you to collect the actual pixel intensities of the pixel values
under the profile line. A profile plot shows the pixel positions of the line along the X-axis,
and, along the Y-axis, indicates the pixel intensity for each position along the line. Either
or both axes may use calibrated units if the image has a spatial calibration (for the X-axis)
or intensity calibration (for the Y-axis). You can display a line profile on any type of
image, whether it’s the live image, a captured image, or an image opened from a file.
Saturation is used to show overexposed and underexposed areas with a given color. It can
be used with the live preview to set proper exposure time and avoid over or under- sat-
urated areas, so that intensity measurements on the image will be correct.

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 Viewing an Image Histogram

Viewing an Image Histogram

Image histograms measure, and illustrate in graph form, the distribution of pixel intens-
ities in an image. Histogram data can be created and viewed for data gathering and ana-
lytical purposes or can be manipulated for image enhancement purposes. To create an
Image Histogram:
1. Open the image that you want to measure if it is not already opened.
2. Select Image Histogram from the Intensity group.

A histogram window appears in the Image-Pro workspace:

The histogram area is displayed with a gradient tint that ranges from from black to
white for mono images, and from black to the color of the channel for color images.
Histogram & Profile options control the display of an image histogram or line profile.
More information about these options appears in Chapter 2.
The Reset button automatically resets the histogram display.

Best Fit applies the best fit display to the image .

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If you click the drop-down arrow, you will see the available best-fit options:

Auto-Range automatically adjusts the black and white levels on the image. This fea-
ture is available only if the current camera supports Auto-Range.

The Data Collector feature lets you accumulate data to display in the data
collector. Click the drop-down arrow to see these options:

Check the appropriate box to add or clear information for the data collector.
Right-click in the Histogram window to display the drop-down menu:

This menu lets you select a variety of options.

You can copy your graph, or export it to Microsoft Word, Excel, or Powerpoint. Other
options let you show or hide the different features of the image histogram.

Note that you can zoom in on a part of the histogram by using your mouse to draw
a region on the histogram graph.
The View sub-menu allows you to display the graph legend and statistics.

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 Viewing an Image Histogram

You can adjust the black and white levels by using the right and left sliders.As you
move the sliders, you will see the changes in the black and white levels reflected in
the image.
The Options sub-menu contains additional controls for displaying the graph.

n Ignore ROI: indicates that the histogram should reflect the entire image
n Active Frame: use only active frame for histogram data
n Mono interpretation: interpret color images as monochrome images
n Spatial Calibration: use spatial calibration to calculate histogram and statistics val-
ues
n Range Statistics: calculate statistics based on selected range only (selected range
displayed on the chart as yellow bar – you can move or adjust left and right edges
of the selected range with a mouse),
n Exclude Bins: exclude the number of bins to the left or right.
The Statistics option lets you hide or show the following information about the image
histogram:
n Mean: the calibrated Mean of all the bin counts (or selected Range)
n St. Dev.: the calibrated standard deviation of all the bin counts (or selected Range)
n Min: the calibrated minimum of all bin x values (or selected Range) (calibrated
intensity)

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n Max: the calibrated maximum of all the bin x values (or selected Range) (cal-
ibrated intensity)
n Sum: the calibrated Sum of all the bin counts (or selected Range) where calibrated
bin counts (Values) are multiplied by their x value (calibrated intensity), and accu-
mulated
n Area (…): the calibrated area of the pixels with intensity between Black and White
Levels (or selected Range),
n Area (%): the percentage of the pixels with intensity between Black and White
Levels (or selected Range) to the total image area.

Advanced Display
Additional histogram options are available when you select the Advanced button from the
Display group on the Adjust tab:

The Advanced context ribbon appears, with additional controls over image display and
brightness/contrast adjustment.

When the Histogram button is activated, the Advanced Display panel opens to show a lar-
ger version of the image histogram appears in the bottom panel of the application work-
space:

The drop-down menu of the Histogram group lets you control the appearance of the his-
togram display:

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The example above shows an area histogram. The Dynamic button adds a display of the
resulting or effective histogram after all of the current display adjustments (such as Bright-
ness, Contrast, or changes in the Black/White levels) are applied.
The Look-Up Table (LUT) is also displayed on the graph. The LUT determines how pixel
intensities are displayed on your monitor. Changes to Brightness, Contrast, or the freeform
LUT, will change the display of the image, but will not affect the underlying pixel values.
You can turn it on or off using the LUT button in the Look Up Tables group.

The LUT is indicated by a diagonal line on the histogram and by additional information in
the right-hand panel:

Clicking the Freeform button displays additional information on the histogram. Choose a
display option from the drop-down list:

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Here is a sample histogram with freeform and line information:

The Options group has the following features:

Ignore ROI creates a histogram using the entire image, even if an ROI is present.
Active Frame creates a histogram using only the active frame in a sequence.
The Log X, Y group uses logarithms to create the graph:

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Log X button is used to generate the graph’s horizontal axis using a logarithmic scale.

Log Y button is used to generate the graph’s vertical axis using a logarithmic scale.

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Viewing a Line Profile

Line profile analysis allows you to collect the actual pixel intensities of the pixel values
under the profile line. A profile plot shows the pixel positions of the line along the X-axis,
and, along the Y-axis, indicates the pixel intensity for each position along the line. Either
or both axes may use calibrated units if the image has a spatial calibration (for the X-axis)
or intensity calibration (for the Y-axis). You can display a line profile on any type of
image, whether it’s the live image, a captured image, or an image opened from a file.
To view a line profile:
1. Open the image that you want to measure, if it isn’t open already.
2. Select Line Profile from the Capture tab’s ribbon.

You will see the Line Profile ribbon:

There are many different drawing tools in the Line Profile ribbon. They can be used
to draw the sampling lines for the Line Profile..

3. When you select the Line Profile, Image-Pro will automatically draw an initial line
profile on your image.

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4. This profile is labeled “Line 1”. This is to help you get started with the Line Profile
tools. You can adjust the Initial Profile in the Histogram and Profile panel in the
Application Options. The Line Profile graph appears in a pane below the image:

As you draw more lines on the image, they will also appear in the profile pane. The
plot scale along the X-axis will be adjusted to display the longest line profile that
you’ve drawn. The currently selected Line Profile will appear on the plot and in the
legend as a thicker trace. Under the line profile plot is an image display of the intens-
ities of the pixels under the line profile at each position along the selected Line Pro-
file. The image display will be as long as the selected profile, which may be shorter
than the longest one that you’ve drawn.

Line Profile Tools

When the active image has one or more line profiles applied to it, the Line Profile context
ribbon is displayed. It contains many useful tools for working with your line profiles.

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There are many different drawing tools in the Line Profile ribbon that can be used
to draw the sampling lines for the Line Profile. The table below describes each
tool:

Select

The plot scale along the X axis will be adjusted to display the longest
line profile that you’ve drawn. The currently selected Line Profile will
appear on the plot and in the legend as a thicker trace. Under the line
profile plot is an image display of the intensities of the pixels under the
line profile at each position along the selected Line Profile. The image
display will be as long as the selected profile, which may be shorter than
the longest one that you’ve drawnUse this tool to select a line profile fea-
ture or group of features.

Line

The Line button will display the currently selected line. This tool
defaults to a simple line, but when you click the drop-down arrow on
the line tool, you will see additional selections. Use the default Line
tool to draw a sampling line on your image. Click on the first point and
drag to the last point of the desired line.

Polyline
This tool allows you draw poly-vertices lines and polygons, or freehand
lines and shapes.
To draw freeform lines and shapes
Use this technique to create lines and shapes that have smooth edges.
Hold the left mouse down while you draw with the cursor to create the
desired freeform shape. Double-click to complete the shape.

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To draw polyvertices lines and polygons

Use this technique to create lines and shapes that are made up of straight
line segments. Click the left mouse button at each vertex (including the
beginning point) of the polygon. Double-click to complete the polygon.

The auto-trace option for ploylines also appears when you click the drop-
down arrow.

Rectangle

Click the left mouse button and hold it down to drag the rectangle to
your desired size. Clicking the drop-down arrow will also display the
Polygon tool.

Polygon
This tool allows you draw polygons, or freehand shapes. Click the left
mouse button at each vertex (including the beginning point) of the
polygon. Double-click to complete the polygon.
Auto-Trace Polygon. Use this tool to trace polyshapes automatically.

Circle or Ellipse
Position the crosshair cursor anywhere in the image. Click and drag the
mouse from the insertion point to the desired destination. Hold down the
<Shift> key while dragging the cursor to create a circle.
Delete Selected
Use this button to remove the selected profile feature from your image.

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Delete All
Use this button to remove all the line profile features from your image.
Note that if you delete all line profiles, the Line Profile pane will be
empty until you draw another one.

Auto-Trace
Use this tool to trace the outline of an irregular object in the image auto-
matically. Place your cursor on the object and click two points. Image-Pro
automatically traces the object.

Width
Use the spin buttons to indicate the with of the profile line in pixel

Features Manager
Use this button to manage feature collections.You will find more inform-
ation about features and feature collections in Chapter 8 of this manual
and in the in-product Help.

Line Profile Options

Use this button to display the options that control the appearance of the
line profile labels in the line profile pane. You can control the size, opa-
city, direction, and color of the lines.

Line profile lines have a start and end point. Check the Flip Profile Dir-
ection box to reverse the direction of the profile lines.
Use the right and left arrow buttons to move the profile points in either
direction.
As noted above, each line profile has a beginning and an end. For example, lines
start at the first point at the left, and end at the last point, on the right. The last

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point gets an arrowhead by default. Closed figures are drawn in the clockwise dir-
ection, and an outline arrow indicates the direction, as shown here:

Checking the Flip Profile Direction box reverses the direction:

Background Tools

The Background combo box is used to select a background profile (values of back-
ground profile will be subtracted from all other profiles values). Background
smoothing is used to select the type of smoothing for background profile values.
The Points box indicates the number of points visible on the graph.

To Add a Line Profile to the Current Collection

Line Profile collections are created by adding defined Line Profiles (that is, Line Pro-
filess defined in the active image) to the list of ‘remembered’ items through the
Features Manager dialog box. You can add a single Line Profile definition, or you
can add a group of :Line Profile definitions under a single name.

1. Open the Features Manager dialog.

2. Draw the Line Profilen the active image.

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3. Click on the Add button.

The collection definition is added to the list box. Listed names represent the mem-
bers of the current collection.
When a single Line Profile is defined, a default name appears in the Name text
box of the Features Manager dialog box.

If you define more than one Line Profile on the image, the features manager gives
the entire group the default name, “Multiple.” Note that if multiple features are
defined in the image, they will be added to the list as a group regardless of which
feature among them is currently selected.
More information about the Features Manager appears in Chapter 8.

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Edge Detection Tools

The Edge Detection tools find peaks, valleys, and patterns in the line profiles. The
Edge detection tools are described below:

Peaks

Use this tool to find peaks on the profile.

Valleys

Use this tool to find valleys on the profile.

Rising Edges

Use this tool to determine rising edges in the profile

Falling Edges
Use this tool to detect falling edges in the profile.

Manual
Click this button to activate the manual edit edge tool (used to add/re-
move edges on the image)

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Edge Setting
Use the pull-down arrow next to the tools to open the Settings dialog.

Label lets you select the label style and color.

The Color Selector lets you change the color of the ticks and label
text.
Checking the Show Ticks and/or Show Labels boxes will display the
tick marks and labels on the image.
Smoothing: Use the spin buttons to apply the Gaussian smoothing fil-
ter to the luminance profile. The numbers indicate the degree of
smoothing that the filter uses. Smoothing of 1 means no smoothing.
If smoothing value is more than 1, a Gaussian smoothing kernel will
be applied to the luminance profile.
Offset: Use the spin buttons to indicate how many pixels to offset the
markers from the detected features.

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Threshold defines threshold level for marking an “edge” on a

weighted profile.
It uses the minimum difference between an extreme point and neigh-
boring opposite extremes. If the difference between the extremes is
smaller than the sensitivity threshold, the marker is ignored. The
Threshold is defined in percentage from 0 to 100 from the dynamic
range. The default value is 0, so that all peaks and valleys are detec-
ted, even when deviations of intensities are very small.
Min Edge indicates the minimum number of expected “edges” on
each profile.
Max Edges indicates the maximum number of expected “edges” on
each profile.

The pattern matching feature lets you select a pattern template or create your own pattern:
Pattern A
Use this button to select pattern template A

Pattern B
Use this button to select pattern template B.

Pattern Settings
Use the pull-down arrow next to the tools to open the Label Settings dia-
log.

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Match length indicates the number of profile samples over which

averaging or pattern matching is performed, i.e. this is the length
of the segment where edge finding method is performed.
Hotspots (%) indicates the point in a weighted peak to mark as
an edge, given as a percentage of the match length. This is an off-
set for the exact edge position as a percentage of the match
length. 0% - beginning if Match length segment, 100% - end of
the segment.
Threshold defines threshold level for marking an “edge” on a
weighted profile.
It uses the minimum difference between an extreme point and
neighboring opposite extremes. If the difference between the
extremes is smaller than the sensitivity threshold, the marker is
ignored. The Threshold is defined in percentage from 0 to 100
from the dynamic range. The default value is 0, so that all peaks
and valleys are detected, even when deviations of intensities are
very small.
Min Edge indicates the minimum number of expected “edges”
on each profile.
Max Edges indicates the maximum number of expected “edges”
on each profile.
Match Offset is an additional setting to creata a user- defined pat-
tern. This edge finding method is based on the same mean lumin-

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ance of a user defined pattern. You might use this option if you
were looking for a “W” shaped pattern on top of bright sections
of the profile, but you wanted to exclude such profile patterns
where the image was dark.
Size Weighting (%) is an additional setting for creating a user-
defined pattern. It defines the strength of the Match Size option.
Match Size is an additional setting for creating a user-defined pat-
tern. This edge finding method is based on matching amplitudes
in the user-defined pattern. The strength of the effect depends on
the Size Weighting property (stronger with greater Size Weight-
ing). You might use this option if you were looking for medium-
sized luminance peaks, but you wanted to exclude very bright
spots.

Lock Settings . If this setting is on, then edge detection set-

tings will be applied to the all profiles. This option could be
used for profiles imported from Feature Manager and edge detec-
tion should be done on all imported profiles.

Manual. This setting activates the manual edit edge tool

(used to add/remove edges on the image)
n Peaks – activates peaks manual tool,
n Valley – activates valleys manual tool,
n Rising – activates rising manual tool,
n Falling – activates falling manual tool,

n Pattern A/B – activates pattern A/B manual tool,

n Reference – activates reference manual tool

To use Edge Detection, follow these steps:

1. Open the image that you want to measure, if it isn’t open already.

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2. Select the Edges group from the Line Profile ribbon:

3. Draw a line or two on the image, as shown here. Line 1 is red and Line 2 is green.

4. Select the Peaks tool from the Edges group.

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Make sure that the tick marks option is turned on. The edges will be marked on the
image, as shown in the previous image.
A line profile displaying the variations in the edges will appear in the line profile win-
dow at the bottom of the Image-Pro workspace.

The Line Profile window contains additional graphic display tools:

The Graph button displays the line profile as a graph, shown above.

The Table button displays the line profile information in a table, as shown
here:

The Measurements button displays the line profile information with a picture
strip below:

5. Click the Plus button to add measurements to the table.

The Line Profile ribbon has other options available, which are described here:
The Classes group lets you apply labels to different features within the image.

You can select the color for each class label, and have the labels appear on the image,
graph, or data table.
The Measurements group in the Line Profile ribbon contains features that allow
you to add measurements to the Line Profile shown above.

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The Options group contains controls for displaying the image.

The Log group displays the X- and Y-axes in a logarithmic scale.

The Font group lets you select the font style, size and color for the class labels.

The File group allows you to save or load the current state of the line profile tools.

The Collect group allows you to accumulate data to display in the data collector.

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Click the drop-down arrow to see these options:

Check the appropriate box to add or clear information for the data collector.
To exit the Line Profile ribbon, click the Close button.

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Using the Saturation Warning

Use Saturation Warning to show overexposed and underexposed areas with a given color.
It can be used with the live preview to set proper exposure time and avoid saturated areas,
so that intensity measurements on the image will be correct.

Click the Saturation button to see the drop-down menu:

The colors are set using High level and Low level color pickers. Intensities that are greater
than or equal to the High Level threshold will be colored with the designated high color.
Intensities that are less than or equal to Low Level threshold will be colored with the des-
ignated low color.
The channels used for saturation test can be selected using Red/Green/Blue checkboxes or
by clicking the Channels button.
This is a sample of a partial image with the high saturation areas visible:

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The Reset button sets the controls back to their default values.

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Chapter 5 : Working with Images

Working with Images

This chapter introduces you to image processing in Image-Pro. It provides instructions for
opening, saving, and enhancing images through Image-Pro.

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Viewing Optical Characteristics

The Characteristics group on the Capture tab’s ribbon lets you choose a dye, lens, and/or
calibration to use while acquiring images.

Clicking the drop-down arrow next to the dye displays a list of available dyes:

Clicking the drop-down arrow next to the lens displays a list of available lenses:

The Lens is particularly important, as you can associate a spatial calibration with each
lens; in addition to the lens characteristics (such as Magnification) , a spatial calibration
will be applied to each captured image, assuring accurate measurements. Please refer to
the Using Spatial Calibrations section for more information.
This group can also display the magnification zoom level. You can select which char-
acteristics to display in the Display area on the Applications page of the Image-Pro
options dialog.

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 Viewing Optical Characteristics

The Mag. * Zoom display option controls display of the zoom magnification level (which
will always be at the top of the group, if displayed). As illustrated above, the group can
only hold 3 sets of controls, so when three options are checked, the fourth will be dis-
abled. To hide the Characteristics group, do not check any of the options.
Your selections will appear in the Characteristics group on the Capture tab’s ribbon:

1. To select optical characteristics, click on the drop-down arrow and select an item from
the drop-down list.

2. To edit one of the characteristics, click the edit button at the far right.
You will see an editing panel at the right side of the workspace.
The Lenses panel is shown here as an example:

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3. From this panel, you can edit or change the lens used to capture your images.
The Lens Library displays a list of available lenses with their calibrations.

You can search for a specific lens by manufacturer or magnification.

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Using Spatial Calibrations

Using a spatial calibration, Image-Pro's pixel-level measurements can be converted to any
unit of measurement. Spatial calibrations can also correct for irregularities in the image’s
vertical and horizontal spacing.
If you are working with an uncalibrated image, you will see this warning:

There are two primary components to a spatial calibration:

n Pixels per Unit
n Aspect Ratio

Pixels per Unit

A spatial calibration tells Image-Pro the size to which a given image is scaled, just as the
key of a roadmap tells us how many millimeters of image length represent one mile. Like-
wise, the spatial calibration tells Image-Pro how many pixels of image length represent
some more meaningful unit of length in the image, for example, one micron, millimeter, or
inch. To create a new spatial calibration, you must either know the number of pixels per
unit to specify for the image, or there must be a feature pictured in the image that has a
known length value in terms of the units of interest. If a feature of known length is pic-
tured, Image-Pro can calculate the number of pixels per unit by determining the number of
pixels it takes to represent the known length. (Companies often ‘plant’ an object of
known length into images for this purpose.)

Aspect Ratio
Aspect ratio refers to the ratio of vertical to horizontal lengths. For example, when a tele-
vision screen’s image appears flattened or squeezed, the ratio of horizontal length to ver-
tical length is out of proportion. Similarly, cameras often inadvertantly skew images in the
process of capturing them and translating them to digital format.

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Such skewing results in inaccurate lengths being ascribed to features that are measured in
the image. When a skewed aspect ratio has a flattening effect, vertical lengths will be
recorded as shorter than they actually are; when it has a squeezing effect, horizontal
lengths will be recorded as shorter than they actually are.
If an image has a known aspect ratio problem, Image-Pro can compensate for the flattening
or squeezing of vertical and horizontal lengths, and calculate the actual number of units
that measurements of its features represent. To do this, you must be able to supply it with
the value l/h (length divided by height) for a perfect square as represented in the image, or
there must be a feature pictured in the image that represents a perfect square. If a square
feature is pictured, Image-Pro can calculate the aspect ratio from that feature. (Companies
often ‘plant’ a perfectly-square object into images for this purpose.) The selected image
must have a reference feature of known size, which you will measure in step 5.

System and Reference Calibration

Image-Pro maintains a list of Reference Calibrations that will persist from session to ses-
sion and can be applied to multiple images.Image-Pro also maintains a list of all of the cal-
ibrations that have been used in your current session, which will include all the reference
calibrations, as well as any calibrations that have been loaded from any calibrated images
that you have opened. One of the Reference Calibrations can be selected the System Cal-
ibration. The System Calibration is automatically attached to images that you Capture or
Record using your camera, and may also be used as the default spatial calibration when
loading a 3D volume from uncalibrated images.

If you have configured your system using the Hardware Configuration Wizard (described
in Chapter 3), then you will not need to be concerned with Reference or System cal-
ibrations. A list of Reference Calibrations will be created and associated with your Lenses,
and when you select an active lens, the calibration associated with that lens will be made
the System Calibration.

Creating a Spatial Calibration

To create a new spatial calibration, you must either know the number of pixels per unit to
specify for the image, or there must be a feature pictured in the image that has a known
length value in terms of the units of interest (see “Pixels per Unit” under “About Spatial
Calibrations” above)..

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Quick Calibration Method

1. Open the image you want to calibrate or that you want to use to create the calibration
definition.
2. On the Capture tab’s ribbon, select the Calibration group.

3. Click the Create drop-down arrow.

4. Click the Quick Calibration button

5. Draw a line over a feature in the image.
You will see the Calibrate by Feature dialog:

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6. Select a calibration name in the drop-down list.

7. Select the units in the drop-down list, and indicate the number of units in the selected
feature.
8. Indicate if this is a system or reference calibration.
9. Click OK.

Auto-Calibration
To use Auto-Calibration, follow these steps:
1. Open the image of the Stage Micrometer (included with the Image-Pro Demo Images).

2. On the Capture tab’s ribbon go to the Calibration group.

3. Click the Create drop-down arrow.

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4. Select Auto-Calibration. You will see the Auto-Calibration dialog:

5. Click OK.
If the box is checked, you will see the Spatial Calibration dialog:

From here, you can rename your calibration and make further adjustments.

Calibrating by Aspect Ratio

If your calibration must also correct for aspect ratio, you must either know the value l/h
(length divided by height) for a perfect square as represented in the image, or there must be
a feature pictured in the image that is known to be a perfect square (see “Aspect Ratio”
under “About Spatial Calibrations” above).
To create a spatial calibration:

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1. On the Capture tab’s ribbon, select the Calibration group.

2. Click the Calibration options icon to open the Spatial Calibration dialog box
in the panel on the right.

The dialog box controls are enabled. This dialog has two view, Basic and Advanced.
In the list-box in the control window (Calibration Name) select the set of calibration
values you want to apply to the image. If you want to use the default values, select
(none). Any calibration sets that were loaded or created since the application window
was last opened, will be listed in this list box.
You will not be able to enter calibration values until a name other than (none) has
been selected in the Name list box. If (none) is the only set listed, you can create a set

by clicking the New button and specifying its calibration values.

By default, the name "Spatial Cal 0" will be assigned to a new calibration set; how-
ever, you may change this to a more descriptive name if you'd like.

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3. Type over the default name “Spatial Cal 0” with a name that is more meaningful.
4. In the Unit area, pull down the Name list box and select the units to which you want
the pixel-level measurements converted, as shown below:
For example, if the size of objects in the image is best expressed in terms of micro-
meters, select “Micrometer” from the list.
5. In the Abbreviation text box, enter the text you want to use as the abbreviation for
the selected units.
For example, a typical abbreviation for ‘Millimeter’ is “mm” as shown below. The spe-
cified abbreviation will appear on measurement reports.

6. In the Pixel/Unit group, do one of the following:

If you know the number of pixels per unit to specify:

1. In the X spin box, type the number of horizontal pixels it takes to rep-
resent one unit of length. For example, if you are converting to mil-
limeters and 4 pixels equals one millimeter, type “4.”
2. Ignore the Y spin box.

If there is a feature pictured in the image that has a known length value:

1. Click the From Image button in the Pixel/Unit area.

A dialog box appears in the Image-Pro panel, and a horizontal annota-
tion line appears in the image.
2. Click and drag the line over the reference object, and then adjust the
line’s control points so that the beginning and ending of the line cor-
respond to the beginning and ending of the reference object.

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TIP: For greatest accuracy, it is often helpful to zoom in on the reference

object. To zoom in on the reference object, click on the Zoom tool

from the View tab’s ribbon. With the Zoom tool selected, position the
cursor over the reference object, and click. Repeat these steps until the
object is large enough to determine its starting and ending pixels.
3. In the Reference length (units) spin box, type the known length of the
pictured object.
4. Click OK.
The Spatial Calibration dialog box is redisplayed and the calculated
pixels/unit value appears in the X and Y spin boxes.

If you have an image of a micrometer, you can calibrate the image auto-
matically:

1. Click the Auto button in the Pixel/Unit area.

2. A dialog box appears over the image of the micrometer.
3. Draw an ROI around the micrometer image.
4. Click OK., and the calibration will be calculated automatically.

7. Select the Advanced Options on the Calibration dialog.

8. In the Apect Ratio area, do one of the following:

If you know the aspect ratio value to specify, or if there is no aspect ratio
problem to correct for:

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 Creating a Spatial Calibration

1. In the Aspect Ratio spin box, type the aspect ratio value to be used.
For example, if there is no aspect ratio problem to correct for, enter “1.”
Image-Pro automatically adjusts the value for the X spin box to match
the entered Aspect Ratio value.

If there is a feature pictured in the image that represents a perfect square:

1. Click the From Image button in the Aspect Ratio area.

A dialog box appears in the Image-Pro workspace, and a diagonal
annotation line appears in the image.
2. Click and drag the line over the reference object, and then adjust the
line’s control points so that the beginning of the line corresponds with
the bottom-left corner of the reference object and the end of the line cor-
responds with the top-right corner of the reference object.

TIP: Again, for greatest accuracy, it is often helpful to zoom in on the ref-
erence object.
3. Click OK.
The Spatial Calibration dialog box is redisplayed and the calculated
aspect ratio value appears in the Aspect Ratio spin box. Additionally,
the X and Y spin box values are automatically adjusted to account for
the aspect ratio.

9. Click Apply to Active Image in the spatial calibration toolbar.

The spatial calibration is applied and saved for the session. To make it available across ses-
sions, you must save it to a file (see “Saving Calibrations” below).

Calibrating by Feature
Calibrate by feature option allow you to calibrate an image by a feature with known size.
Any line measurement can be used for image calibration.
Calibration by feature can be done using the following steps:
1. Create a measurement of an object of known size. For example, the length of a
bracket can be measured.

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2. Click the Calibrate by feature length button (the same function can be called from
the context menu in the Data Table).
3. Fill the feature size in calibration units and other calibration parameters, such as Units
and Calibration name in the shown Calibrate by feature dialog:

4. (Optional) Use the Show calibration dialog to adjust the calibration measurements.
5. Click Ok to create a new calibration and apply it to the active image.

Predictive Calibration
Predictive calibration lets you create estimated spatial calibrations based on the optical
characteristics of the camera and objectives. To use the predictive calibration feature, fol-
low these steps:
1. From the Calibration group drop-down menu, select Show Predictive Calibration.

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You will see the Create Predictive Calibration dialog:

Each camera is defined by the Manufacturer and Camera Model fields

2. Select your camera manufacturer and model from the drop-down lists. The pixel and
sensor size is determined by the camera make and model. If you want to set the pixel
and sensor size manually for the new calibration, choose Other in the list of cameras.
The Calibration prefix is also defined automatically the camera selection. You may
type in a new prefix in the space provided.
3. Select the lenses from the calibration from the Lenses panel.
All active lenses are selected by default, so if some lenses should not be cal-
ibrated,uncheck them using the button to select/deselect a lens.
4. Add additional Zoom settings in the Zoom panel. This list is empty by default.
5. Enter the calibration units in Units field under the Calibration parameters .
6. (Optional) Check the Show Calibration Dialog box if you want to edit the cal-
ibration after you’ve created it.
7. Click OK to create the predictive calibration.
Clicking the OK button will generated multiple calibrations for the list of selected
lenses and zooms for the given camera. If multiple zooms are present, the calibration
with zoom closest to 1 will be used as default lens calibration.

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The list of camera manufacturers and camera models is loaded from "Camera
list.cfg" file in the Resources folder of the application.

Saving Calibrations
Calibration definitions are saved per-session whenever they are applied to an image. If
you want calibration definitions to be saved between sessions, however, you must mark
them as Reference Calibrations or save them to a file. Once a calibration definition is
saved to a file, you can load it whenever it is needed in the future.
To save calibration definitions:
1. Click the drop-down arrow next to the Open Calibration button on the calibration set-
tings dialog.

The drop-down list box appears. If an active image is displayed in the Image-Pro
workspace that has a calibration definition assigned to it, that calibration definition
appears in the Spatial Calibration dialog box.
2. From the list box at the top of the Spatial Calibration dialog box, select the cal-
ibration definition you want to save.
3. Click Save from the Spatial Calibration group.

TIP: If you want to save all calibrations listed in the Spatial Calibrations list box,
skip Step 2 and click on the Save All from the Spatial Calibration group.
The Save Calibration dialog box appears.

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4. In the File Name text box, type the name you want to give to the current calibration
definition (or set of calibration definitions) being saved.
5. Click on the Save button.

Locking Calibrations
To ensure that your calibration measurements are not deleted or changed accidentally, you
can lock them. To lock a calibration, follow these steps:
1. Create a calibration using one of the methods described in this chapter.
2. Save your calibration.
3. Click the lock icon on the calibration ribbon:

You will see the Lock calibration dialog:

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4. Fill in your name and create a password.

5. Click OK. This calibration is now locked and protected against accidental changes.
The lock icon changes to an open lock.

6. To unlock the calibration, click the open lock icon. You will see the Unlock cal-
ibration dialog:

7. Enter the owner name and password.

8. Click OK. The calibration is unlocked and may be changed.

Importing and Exporting Calibrations

Import Calibration and Export Calibration have been added to the Calibration Options
drop-down menu, as shown here:

You can use these features to import/export the calibration and calibration/lens linkage as
a package that can be imported into other copies of Image-Pro.

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Applying a Calibration Marker to the Active Image

The calibration marker is like a distance key on a map – it shows a line and expresses
what that line represents in terms of meaningful units. In Image-Pro, it is composed of
two or three overlays, as shown below:

When you add a calibration marker, Image-Pro adds it to the currently active image based
on the number of pixels in the image.
To set the calibration marker to appear in the active image:
1. Choose the Auto-Marker button from the Calibration Marker drop-down menu:

A marker will be placed on all open and active images automatically.

2. Otherwise, click the Calibration Marker button [ ] in the Image-Pro toolbar.

A calibration marker will appear in the image.

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The Marker Options dialog box appears.

3. Use the Position buttons to place the marker on the image. You can resize the marker
and the length and the label will be adjusted automatically based on the current size.
The length is rounded to the nearest value from the list of 10 logarithmically
equidistant values (1, 1.2, 1.5, 2, 2.5, 3, 4,…).
4. Set the Control options the way you want them. The Length (units) spin box controls
how long the marker will be. Image-Pro renders a marker of the length you specify
here. Adjust the Length (units) value so that the resulting marker line is long enough
to give an indication of scale, but not so long that it takes up the entire image width.
Also, use round numbers. Keys generally use ‘reference’ lengths, such as multiples of
25. The size of the calibration marker can be adjusted manually by resizing the marker
on the image. Note that the minimum marker size is limited by the width of the cal-
ibration label text.
5. Set the Font and Size options the way you want them. Use the Size spin box to indic-
ate the size, in points, to be used for the text. To hide the text and only display the
marker, set the font size to zero. Use the Color button to change the text color.
6. Set the Background options the way you want them. Use the Color button to change
the color of the background. Uncheck the Show button if you want to suppress the
background.

TIP: You may need to experiment with the Text and Marker settings to get the
desired effect. Go with your best guess the first time you set the marker. Once the
marker is added to the image, you can easily delete it and define a new one (see
“Deleting an Annotation” in Chapter 7, “Annotating Images”). By refining the Text
and Marker settings on subsequent iterations, you can achieve the desired effect.

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The length of the calibration marker can be adjusted manually by resizing the marker
on the image. In this case the minimum marker size is limited by the width of the cal-
ibration label text.
7. Click OK.
The calibration marker is added to the image.
n To add a marker automatically to all active images, click the Auto Marker

button.

When calibration markers are initially added to images, they are added as annotation
overlays. This means that they can be moved and deleted the same as any annotation.
This also means that they will only be saved with the image if that image is saved in a
TIFF format.

Note that Image-Pro does not support calibration of the Z-axis. Where possible, the
location of each Z plane along the Z-axis will be determined from Z stack sequences
or image sets that you open.

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Using Intensity Calibration

The Intensity Calibration selections can be used to measure changes in intensity and
pixel values in an image or set of images. You would use this command if you wanted to
relate intensity to optical density, or relate intensity to temperature or relate intensity to
protein content, for example.
1. To start using the Intensity Calibration feature, click the Int. Cal button in the Cal-
ibration group on the Capture ribbon.

You will see the Intensity Calibration dialog:

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2. To create a new intensity calibration, click the New button on the Intensity
Calibration toolbar.
3. Choose a calibration name from the drop down list, as shown here:

The default is “Intensity Cal 0”. You can rename it if you choose.
A new intensity calibration curve will be displayed in the window.

The main toolbar contains the following buttons:

Apply: Applies the selected calibration to the active image.

New: Click this button to create a new set of calibration values. When this but-
ton is clicked, the Application will place the "Intensity Cal 0" name in the Name list
box (the 0 digit may be incremented to make the set name unique) and show the cal-
ibration curve. You can change this name to something more useful if you like.

Load calibration from file: Loads one or more saved calibration(s) from your
files.

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Save: Saves the current calibration to a file. Clicking thedrop-down arrow

will save all your calibrations.

Delete: Deletes the current calibration. Clicking the drop-down arrow will
delete all your calibrations.

Ref Only: Click this button to display only the reference calibrations.

Reset: Clicking this button resets the list of intensity calibrations.

Units: Type the name of your unit in this field (e.g., gray level,
density, degrees, disbursement). This name will appear when the application reports
intensity data.

Abbreviation: Enter the short name of the units (as OD, ng etc.).
Log scale X and Y turns the logarithmic scale along the corresponding axis on the
chart on or off.

Toggle Reference Calibration: Click this button to mark or unmark the current
calibration as a reference calibration.

Toggle System Calibration: Click this button to mark or unmark the current
calibration as a system calibration.
Intensity calibrations are handled in the same way as system calibrations. To learn
more about system and reference calibrations, please refer to the section on page 5-7 of
this manual.
To use the edit mode, select the Options/Edit calibration button to see the tools for
editing the intensity calibration:

Use these tools to add, delete, or edit points in the image for intensity calibration.

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Step Tablet Wizard

Intensity calibration can be created from a step-tablet image with known optical dens-
ity values using the Step Tablet Wizard .
1. Open the image called Steptab.tif

2. Click the Step Tablet Wizard button to display the following dialog:

3. Draw a line from center of the first to the center of the last step on the steptab image:

When the line is created, the bands on the step-tablet image are automatically detec-
ted and labeled:

4. Add the correct Output values for every Input value in the table.

Tip: you can create a file with Output values for certain types of step-tablets and use
it with multiple images using the Load and Save buttons.
5. Once you are satisfied with your values, save the calibration.

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The Image Tab

When you have an image active in the workspace, the Image tab appears in the applic-
ation, as shown here:

From the Views group, you can select one of seven different ways to view your image:
n Image is the default view, with the active frame of the image or sequence dis-
played in the image workspace.
n Surface represents the Surface Plot view, which is an advanced view discussed in
the next section of this chapter.
n Gallery displays all of the frames of the sequence in a grid, and allows you to
quickly and easily change the active frame.
n Slicer displays X/Y, X/Z and Y/Z projections of a sequence in the slicer view.
n 3D displays X/Y/Z sequences as a volume.
n Bitmap represents the Bitmap Analysis feature, which displays the pixel intens-
ities of the active frame of the image, and is discussed in a subsequent section of
this chapter.
n Clone allows you to open another view on the active image in any of the sup-
ported viewers.
The Zoom group lets you focus on specific parts of an image using the Zoom, Pan,
and Local Zoom features. The Lock button lets you synchronize two images or
frames together.

Viewing a Surface Plot

The Surface Plot (or 3-D Plot) tool creates a three-dimensional representation of the intens-
ity of an image. When using the Surface Plot tool, keep in mind that X = length; Y =
width; and Z = height. Learn more about Surface Plot by following these steps.
1. Open an image in the workspace.

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2. Go to the Views group on the Image tab.

3. Choose the Surface icon.

You will see the Surface Plot context ribbon:

A three-dimensional representation of the image appears in the workspace:

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The lighter and taller areas of the plot represent areas of greater intensity on the image.
Areas of lesser intensity are shorter and darker. You have many choices for adjusting
the appearance and features of the surface plot diagram, as described here:

Draw Mode
The options in the Draw Mode group control the appearance and display of the sur-
face plot diagram. One of the three modes can be selected: Texture, Color or Wire.

In Texture mode the surface uses an image for the surface plot texture.

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The Texture drop-down menu allows you to control the degree of transparency for the
surface plot. Transparency set to 0 indicates no transparency.

The Color group lets you select the colors to map to the gray values found in the sur-
face plot, as shown here.

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In Color mode the surface plot is pseudo-colored. The color is assigned according to
the height value. Click the drop-down arrow to see the context menu:

When Smooth palette is on the colors of palette entries are blended.

The Contour option adds black lines separating color bands.
The Zones control defines the maximum number of pseudo-color zones. Type a new
value and press Enter key to change it. Note that zones are created automatically
based on the total range of the height axis (see Auto Range option of Axes). The num-
ber of zones on the image corresponds to the number of major tick marks on the
height axis and can be smaller then the Zones value.
The Wire mode shows a wire frame version of the surface plot diagram.

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The number of wires along the longest image direction is defined by the resolution

option in the pull-down menu, shown here:

Resolution
The Resolution group enables you to adjust the display resolution of the surface plot:

Style
This group controls the visual presentation of the surface plot diagram.

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Clicking the Axes button toggles visibility of the axes and walls on or off. The drop-
down contains the options:

The Text Color and Transparency of the walls can be set using the pull-down con-
trols.

Tip:To hide the walls but leave the axes visible set transparency to 100.
When Auto Range is on (default) the range of height axis is calculated based on the
image data. When the option is off the height range is defined by the display range of
the topology image (ActiveImage.RangeMin and ActiveImage.RangeMax properties).

The Legend button turns the legend on or off.

The legend’s position can be selected from the drop-down menu

.
The Light button toggles lighting schemes in the order defined in the Scheme list.
When the Light Scheme is set to None, the illumination is switched off. Northern
Lights scheme uses three light sources, the restof the schemes use only one.

The ambient color can be selected using the Color picker.

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The Background control defines the style and color of the graph’s
background. Start Color and End Color define the colors of any background gradi-
ent.

Geometry
The Geometry group lets you view the surface plot from different angles.

Increase Height
Use this tool to increase the height of your surface plot image.

Decrease Height
Use this tool to decrease the height of your surface plot image.

Reset Height
Use this tool to reset the height of your surface plot image back to its
original size.

Perspective Projection
Use this tool to create a perspective projection of the surface plot
image.
Orthagonal Projection

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Use this tool to create an orthagonal projection of the surface plot

image.

XYZ Perspective
Use this pull-down menu to select the X, Y, or Z perspective

Output
The Output options determine where your surface plot settings and information will be dir-
ected.

Clipboard: Clicking this button sends your surface plot information to the Windows clip-
board, where it can be pasted into other applications that accept data from the clipboard.
Image: Clicking this button creates the surface plot in a new workspace. This function cre-
ates the surface plot in the same size as the original image. It is a good idea to create a
new image when you want to save or print the surface plot, because in this case the surface
plot has maximal resolution.
Animation: Clicking this button displays the Surface Plot animation dialog;

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Rotation direction: Indicate the direction of the animation movement, either horizontal or
vertical.
Number of steps: These spin buttons define the number of frames in the output animation.
Angle range: These controls define the rotation angle of the total animation around X, Y
and Z axes in degrees.
See-saw animation: Checking this box causes the animated image to move up and down,
just as the name implies.
OK: Clicking the Ok button creates an animation sequence in the workspace. The new
sequence then can be saved as a multi-frame *.TIF or *.AVI file.

Using Gallery View

The Image Gallery feature creates a workspace from an active image sequence. The indi-
vidual frames in the sequence are displayed in a gallery format. To see an image gallery,
follow the steps below:
1. Open an image sequence in the Image-Proworkspace.
2. Go to the Image tab.

3. Click the Gallery button.

Clicking this button creates a image gallery workspace from the active workspace.
This feature is disabled if there are no active images, or if the active image is a single-
frame workspace. Here is a sample image gallery:

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The red square in the gallery workspace indicates the active frame in the larger work-
space. Use your cursor to click on different frames in the gallery, and the red square
will move to the selected frame, and the active image in the workspace will change to
the selected frame as well. You can also use the following keyboard shortcuts:
l <Ctrl> +<Click> will select or deselect more than one frame.
l <Ctrl> + <Mouse Wheel> zooms the view in or out.
l Clicking the right mouse button opens the context menu and also displays
image information:

Gallery Ribbon
The gallery ribbon contains the following tools:

The Extract tools let you work with individual frames.

Extract. Clicking this button extracts selected frames into a new image or set.

Extract Frame By Frame. This button extracts selected frames into new indi-
vidual images (a new image is created for each frame).

Average. Clicking this button creates a new image by averaging the selected
frames,

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Difference. This selection creates a new image as the difference between the
selected frames.
l Wrap: Calculate the last frame as the difference between the last and first
frames.
l Diff Only: Returns only the difference frames (the result will be one frame
shorter than the original),
n Pad First - pad the result with first frame of no difference,
n Pad Last - pad the result with a last frame of no difference,
The Options let you specify how you will view the frames:
Show Range will show frames only from the range Start/End,
Show Every Frame will show each frame from the sequence,
Show Frame Number show or hide the frame number in the top-left corner
of the frame,
Show Frame Relative Time shows or hides the frame relative time
Show Frame Time shows or hides he frame date and time,
The Style tools let you customize the frame display:
Active Frame selects the active frame color
Selected Frame indicates the selected frames color
Active Range selects the active range color

Reset Style resets the colors to their default (Active frame – Red, Selected
frames – green, Active Range – yellow)

Reset Zoom resets the zoom of the frames to the default size

Clicking the Edit button opens an additional ribbon:

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Note: Edit Mode enables “Drag and Drop” operations with frames. This allows you
to (rearrange frames and add frames from the Image Strip.This feature is not usable
with image sets.
The Clipboard group contains the standard set of tools, applied to image frames:

Paste Click this button to paste frames from the clipboard into the image
next to the active frame.

Paste New exports frames from the clipboard into the new image.
Paste Frame by Frame exports frames from the clipboard into new
images (new image created for each frame).
Cut Click this button to cut selected frames from the image and place them
onto the clipboard.

Copy This selection copies the selected frames onto the clipboard.

Extract. Click this button to extract selected frames into a new image or set.

Extract Frame By Frame. Click this button to extract selected frames to new
images frame by frame (a new image is created for each frame).

The Frames group contains additional frame editing tools:

Add Frames:
Add Active frame only. When checked, only the active frame will be
added from the source image

Location
l First adds frames to the beginning of the sequence.
l Current adds frames at the current location (active frame).
l Next Current adds frames to the next from the Current location.
l End adds frames at the end of the sequence.

Add All adds all available frames to the sequence.

Add Blank Frame adds a blank frame to the sequence.
Remove is used to remove selected frames from the image.
Left moves the selected frames on one frame to the left.

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Right moves the selected frames on one frame to the right.

Close group:

Close Edit Mode closes the Edit ribbon.

Using the Slicer Viewer

Note: The section documents the simple Slicer Viewer that is included with Image-Pro.
Image-Pro with 3D Module includes a more advanced Slicer view, which is described in
The 3D Slicer Ribbon section in Chapter 12 and in the in-product help.
The Slicer Viewer allows you to view and examine a Z-stack volume using cross sections
of the XY,YZ , and XZ planes.The Slicer is located on the Image ribbon, as shown here.

Clicking the Slicer button will create a slicer view of the current image:

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The top left view is the XY plane. The top right view shows the ZY plane. The bottom
left displays the XZ plane. The bottom-right displays a 3D view of the image.

The Slicer Viewer Ribbon

The Slicer Viewer has the following ribbon bar:

The Composition group defines the parameters of the viewer.

Maximum intensity projection.

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Minimum intensity projection.

Note that the number of slides included in the projection is defined in the Thickness
group.
The Cross checkbox turns the crosshair marker on or off on all the views. The color of the
crosshair marker can be selected using the color-picker drop-down menu.
The Reset button resets the view and resizes the viewers to display the best-fit image.

The Duplicate button duplicates the active view in a new window.

The Voxel Size group defines the size of the voxel. Note that the X- and Y- size are defined
by the spatial calibration of the image. The Z-size is calculated from the distance between
frames in Z direction. Only the Z- size is editable.
Slicer Position defines the X, Y, and Z coordinates of the cross-section.
Slice thickness is defined in the Thickness group. Clicking the All button sets the thick-
ness to the specified volume dimensions. This button can be used to create maximum
intensity projections for the whole volume. Clicking the Thin button sets the thickness
to 1.

Using the 3D Viewer

The 3D Viewer window allows you to examine an image stack in a variety of different
ways: by volume, in thick or thin slices, and as an animated display. In the 3D Viewer, all
geometric objects are shown in 3D space. The 3D Viewer is located on the Image ribbon,
as shown here.

Clicking the 3D button creates a 3D view of the current image with the voxel size cor-
responding to the spatial calibration and the Z-distance between slices. Here is a sample
3D image:

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The image can be rotated by holding down the right mouse button and dragging the image
in the workspace. Pressing <Shift> + <Click> and dragging will pan the view. Pressing
<Ctrl> + <Click> and dragging the image up or down will change the zoom.
Note that if you are running Image-Pro with 3D Module, this feature is replaced by the
features described in Chaper 12 of this manual.

The 3D Viewer Ribbon

The 3D Viewer ribbon contains the following tools:

The Reload button allows you to reload the volume with different sub-sampling.
Clicking the drop-down arrow displays these choices:
l Reload
l Reload when Image changed

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l Reload when LUT changed

Reload when image changed or Reload when LUT changed indicates if the image must
be reloaded if the corresponding parameter changes.

You can also define the voxel size and sub-sampling manually. When the Auto box is
checked, the volume is subsampled automatically. Clicking the Reset button sets the sub-
sampling to 1x1x1.
The X, Y, and Z voxel size can be edited interactively in the Voxels Size group.

The Style group controls the display of the 3D image:

The Axes button turns the volume axes on or off. Activating the Smooth control enables
sub-pixel approximation of the texture.

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The Transparency slider of defines the volume transparency. The color of the background
can be changed using the Background color picker.
The Composition group defines the options to create the view:

Blended composition

Maximum intensity projection

Sum projection

Minimum intensity projection

The slider defines the number of slices in volume presentation. The larger number may pro-
duce better quality volume rendering, but may slow down the processing.
The Geometry group defines the camera parameters:

Activates the perspective view.

Activates the orthogonal view.

Clicking the Projection button activates following orthogonal views:

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Sets the XY projection .

Sets the ZY projection.

Sets the XZ projection.

The slider controls the Camera view angle in degrees.
The Output group lets you decide how to display the 3D image results:

Clicking the Clipboard button copies the current view to the WindowsClipboard.
Clicking the Image button creates a new image in the workspace.
Clicking the Animation button opens a dialog where you can define parameters for simple
animation.

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Rotation angle controls define the rotation angle of the total animation around X, Y and Z
axes in degrees. (X = horizontal, Y = vertical, and Z = perpendicular to the screen).
Number of steps defines the number of frames in the output animation.
Clicking the Ok button creates an animation as a new sequence in the application work-
space. The new sequence then can be saved as multi-frame a TIF or AVI file.
The Settings group allows you to load or save your 3D viewer selections:

Load 3D viewer options and Save 3D viewer options allow you to load or save your 3D
viewer selections to a file with V3O extension (the file is in XML text format). Loading
saved options allows you to restore a particular view point and composition parameters.

Bitmap Analysis
The Bitmap Analysis command in the View group on the Image tab is used to view the
pixel values of the active window (or a region of interest on that image) in numeric format.
These values can be saved to an ASCII file for later use with an external program, or
copied to the Clipboard and pasted into another application (a 3D plotting package, for
example).
Pixel values can be displayed as they actually exist in the image or in their calibrated form
. They are not interpreted through the display LUT however.
The Bitmap Analysis window imposes no limit on the number of pixels that can be
processed. There is, however, a maximum of 30,000 pixels that can be saved, copied to
the Clipboard or transferred to Excel ™. If you need to save more than this number of
pixels, you can define an AOI that is less than or equal to 30,000 pixels, and process the
image in chunks (i.e., move the AOI systematically across the image and save the data at
each interval). Or, you can use the sub-sampling option to read the pixel values at larger
intervals.
Note:Consider carefully how much data you actually need to save in ASCII format; a 100
x 100 segment of a Gray Scale image (10K in binary image form) will consume over 60K,
and a True Color segment will take three times that amount.
To use the Bitmap Analysis feature, follow these steps:

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1. Open an image in the Image-Proworkspace, as shown here:

2. Go to the Image tab.

3. Click the Bitmap button.

The button will be highlighted and you will see the hourglass icon and the Bitmap
context ribbon:

After a few moments, a data table with the intensity values of all the pixels in the
image or ROI will appear in the workspace.

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The Bitmap context ribbon appears in the ribbon bar above the workspace:

The Copy group allows you to send the table data to the Windows Clipboard, Excel,
Word, or save to disk in *.XLS or *.TXT (tab-delimited) formats for printing.
The Layout group defines the size of cells in the table (Cell Width and Cell Height)
and the color channels layout (if you are workign with a color image). If the image has
more than one channel, the channels can be reported in Rows or Columns:

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In column fornat, the channel index is displayed with a colon to indicate the pixel pos-
itions. For example, 392:2 indicates the Y coordinate is 392, in channel 2. Switching
to channels by Rows provides a pixel aspect ratio closer to the original image.
Sub-sampling is useful if you are working with a very large image. This feature lets
you analyze pixels at various intervals on the image.
The Options group let you display the pixel values as they actually exist in the image
or in their calibrated form.
Checking Image Background displays the image as a background for the bitmap data
table. This option can help visually identify the objects of interest.

Image Background on Image Background off

When Intensity Calibration is checked, the table shows intensity calibrated pixel
values. (The number of decimals is defined in the application options described in
Chapter 2).

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Intensity calibration bitmap table

Zooming Images
The Zoom group contains the controls that allow you to resize the image or a portion of
the image.

There are several ways to zoom in and out on an image, that is, to display it at different
levels of magnification. You can:
l Use the Zoom button in the Zoom group
l Use Local Zoom
l Use the wheel on your mouse (if available)
l Use the scale bar.

Zooming by Using the Zoom Button

This method of zooming allows you to zoom in on specific areas of the image.
1. Click the Image tab from the Image-Pro menu bar.
The Zoom group is displayed.

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2. Click on the Zoom button [ ].

3. Do one of the following:
n Position your mouse over the area of the image you want to magnify, and
click the mouse.
n Click and drag a rectangle around the area of interest on which you want to
zoom.
The image is magnified. If needed, you can click or drag the mouse again to magnify the
area of interest even further.

Zooming with Local Zoom

The Local Zoom feature provides a tool to show a zoomed area of an image in a separate
window with a given zoom factor. It can be used with manual measurements, the cal-
ibration marker, manual tagging, etc. You can find it on the Image tab, or by clicking the
button on the context menu.
When you select Local Zoom, a new window opens on the right side of the application
area.

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The cross hairs in the Local Zoom window show the current cursor location. This makes
placing accurate manual measurements very easy, even on large images:
Zoom: Choose the required zoom factor from the drop-down list.
Cross hairs: Checking this box turns the cross hairs on; un-checking the box turns the
cross hairs off. The color picker button selects the color of the cross hairs.

Zooming by Using the Mouse Wheel

If your mouse has a wheel on top between the left and right mouse buttons, you can use
this wheel to zoom in and out on the image. Simply roll the wheel while the image is act-
ive in the Image-Pro workspace. Roll the wheel toward you to zoom out on the image,
and roll the wheel away from you to zoom in on the image.

Zooming by Using the Scale Bar

This method for zooming allows you to take advantage of zoom presets, or to put the
image in ‘fit to window’ mode so that whenever you resize the image window, the image
is enlarged or reduced to the size of the window. For example, you can zoom the image to
a specific percentage of its actual size, such as 50% or 200%.
1. Position the cursor over the image and right-click the mouse.
You will see the scale bar.

2. Move the slider to the right to increase the size of the image, or to the left to decrease
its size. You may also click the arrow to see the drop-down list of zoom presets. The
scale bar also appears in the status bar at the bottom of the Image-Pro workspace.

Panning Images
The Pan tool is used to position an image that does not fit entirely within the image
window. You can:
l Use the Pan button in the Zoom group
l Pan in the Image Strip

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Panning Using the Pan Button

1. Click the Image tab from the Image-Pro menu bar.
The Zoom group is displayed.

2. Click on the Pan button [ ].

3. Use your mouse to move the image around in the frame.

Panning in the Image Strip Using Image Navigator

The Image Strip displays a thumbnail of your entire image. When the active image is
zoomed and doesn’t fit in the viewer, the viewing area is shown on the image strip thumb-
nail as a semitransparent overlay.

n Click and drag the center of the viewing area on the thumbnail to control pan and
scroll of the image.
The Image Navigator feature allows you to use your mouse to move the white rectangle
around in the image strip. The zoomed portion of the image in the workspace will move as
you move the thumbnail in the image strip.

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Enhancing an Image
There are several tools available through Image-Pro’s Adjust tab for visually enhancing
objects of interest in your images. The features on the Adjust tab include:

Resizing an Image
Image transformation allows you to rotate or resize the image in the Image-Pro workspace.
These options allow you to examine the image in more detail. To resize an image, follow
these instructions.
1. Open the castiron.tif image in the Image-Pro workspace, as described earlier in
this chapter.
2. Select the Geometry group from the Adjust ribbon:

3. From the Resize pull-down menu, select an image size:

4. Click the lock icon and select a measurement unit from the drop-down menu.

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 Rotating an Image

This will maintain the current aspect ratio of the image. To unlock the aspect ratio,
click the icon again. You can find more information about aspect ratios in Chapter 3,
Capturing Images.
5. From the second pull-down menu, select the type of smoothing you require in your res-
ized image:

The resized image will appear in the workspace, with a name derived from the original
image’s name and a suffix indicating that the image has been resized, such as
castiron_100resz*.

Rotating an Image
Rotating an image allows you many different views of the same image. For example, you
can rotate it 90 degrees counterclockwise, or flip it left to right. To try some of the image
rotation features, follow these steps:
1. Open the image in the Image-Pro workspace, if it is not open already:
2. Select Rotate from the Geometry group:

3. Use the pull-down menu to see the Rotate tools:

The tools are:

Rotate image 90° clockwise,

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Rotate image 90° couner-clockwise

Flip image left toright

Flip image top to bottom

Rotate image 180°

Swap corners
Free angle rotation:
l Angle indicates the angle of rotation counterclockwise.
l Preview displays a preview of the rotated ROI.
l Translucent displays the rotated preview in a translucent format.
l Ref. Grid displays a reference grid with the preview on the image at the spe-
cified grid angle.
l Trim will trim the rotated ROI to the same size as the original ROI.

l Rotate with trim into a new image

l Rotate without trim into a new image.

4. Choose the flip left to right tool.
The image will flip automatically, as shown here:

Original image Flipped image

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 Using the Canvas

You may want to experiment with the other Rotate tools. The Hint windows and tooltips
contain additional information about these tools. A sample hint window is shown here:

Using the Canvas

The drawing Canvas lets you add or remove space around an exisiting image.

1. To use the canvas, click on the Canvas button on the Adjust tab’s ribbon.
You will see the drop-down menu, as shown here:

2. Use the Canvas command from the drop-down arrow to position the image on the
canvas, as shown here:

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From here, you can add annotations and labels to the image on the canvas.You can
also adjust the background and color. You can crop the image by removing space
around it.

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Converting Images
Use the Convert group commands to transform an image to another color model, to extract
a specific color channel from a color image, or to merge an active, 8-bit gray scale image
into a True Color image. You might do this to perform editing or analysis upon the image
in another model or to save the image data in a different model for use with an external
program.
The Convert group is shown here:

To convert an image:
1. Select the Destination image type from the drop-down list.

2. Click Apply.

3. Select the type of conversion to perform.

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Your image is automatically converted to the new model. Once you have set the con-
version options the way that you want them, you can click the Apply button in the
Convert group to convert the active image using the current settings without making
furrther changes.

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Using Color Group Commands

Use the Color group commands to transform an image to another color model, to extract a
specific color channel from a color image, or to merge an active, 8-bit gray scale image
into a True Color image. The Color group is shown here:

Use the Extract selections to extract a specific color channel from a color image, and view
it as an 8-bit Gray Scale image. You might do this to reduce the saturation values in a
HSI image, or boost just the Blue values in an RGB image.
Use the Merge command to merge an active, Gray Scale image into a True Color image.
This is usually done to return a channel, extracted with the Extract Channel command, to
an image after the channel data have been manipulated. However, you might use it to
merge any Gray Scale image to a color channel in a True Color image.

Dye/Tint Images
The Dye/Tint feature lets you apply a color to your image(s) that corresponds to the emis-
sion color of your dye. If you are using color to explore the intensity distribution of your
image, please refer to the Pseudo Color feature later in this manual and in the in-product
Help. Click on the drop-down arrow to see the Dye/Tint dialog:

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Check the Tint Image On/Off box to show or hide the tint on your image.
Check Add to Color Composite to automatically add your tinted image to the color com-
posite image.
The Dye drop-down list displays a list of your current set of favorite dyes. You may select
a different dye from the drop-down list, and all the remaining controls will be updated to
show the characteristics of the new dye. If the dyes you want are not in the current set,

click the settings button to display the Dye Editor dialog, where you can view a com-
plete list of the available dyes. Here you can add select a new favorite dye, add a dye to
the list, or delete old ones.
The Emission Wavelength drop-down displays the emission wavelength for the dye you
have chosen. You can adjust the emission wavelength by clicking on the spectrum or enter-
ing a new value. The default Tint Color will change in response to changes in the emis-
sion wavelength.
The Tint Color box lets you choose a color to indicate a particular dye. Here you can
change the color associated with the selected dye, or define a custom color. The default
color is linked to the emission wavelength. When you change the emission wavelength,
the tint color and hue change. The default Tint Color can be adjusted to a different hue by
using the Hue slider or by entering a new Hue value. A completely custom Tint Color
can be selected by clicking the down-arrow next to the Tint Color box, and selecting a
standard or custom color.
The Emission Wavelength and Hue override a custom Tint Color, so to set a custom
color, you should first set the wavelength, and then set the color using either the Hue con-
trols or the Tint Color.
Click Apply to add your dye or tint color to the active image.

Using Color Composite

Color composite is a common operation for merging individual fluorescent images. For a
variety of reasons it is not always possible to acquire the images simultaneously, so image
processing tools are necessary to present a complete and accurate qualitative picture. The
Color Composite View is used primarily to merge and register fluorescent images acquired
as monochrome single wavelengths into a color composite image.
Use Color Composite to create and configure color composites using black and white
source images. You can access the Color Composite dialog box through the Color group
on the Adjust tab.

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 Using Color Composite

You can combine grayscale images into a color composite. Any group of grayscale images
that are of the same size can be mixed in a color composite. Images of 8-, 12-, 16-bit
integer or floating point format are combined into a 24-bit color composite. Each input
channel will have individual LUT adjustments, as well as a registration offset to line it up
with the rest of the images. You can also combine individual channels from a single
image. Each channel will be listed separately.
To create a color composite, follow the steps below.
1. Open the images that you want to combine. In this example, we are using the Blue_
DAPI_10.tif, Green_FITC_10.tif, and Red_TRITC_ 10.tif.
2. Click the drop-down arrow in the Composite area in the Color Group on the Adjust
tab.

3. Select Add AllImages from the drop-down menu.

The three demo images are combined into a single color image in the
Image-Pro workspace:

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The three original images and the combined image appear as thumbnails in the image
strip on the left side of the workspace.
The color composite dialog on the right side of the workspace contains information
about each image. Each of the original images represents a single channel in the com-
posite image:

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 Using Color Composite

Detailed information about each color channels can be displayed by clicking on the +
sign to expand the image information. You can make changes to the image inform-
ation, change the color display, or remove one image/channel entirely.
1. Click the eye button to show, blink, or hide the composite images, as shown here:

You can save your composite image as a single image or as an image set.
2. To save your composite, click the Apply button:

3. To save the composite as an image set, click the drop-down arrow and select image
set:

You will see the Save Image as Set dialog.

4. Click Save to save the composite as a *.mcs set.

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Pseudo-Coloring an Image
Use the Pseudo-Color command to “colorize” an active monochrome (Gray Scale 8, Gray
Scale 12, Gray Scale 16, or Floating Point) image. You might do this to highlight cer-
tain features in a gray scale image. For example, you might want to display all densities
above a certain point in red, or, if your imaging device recorded thermal information, all
temperatures below a certain point in blue.
You might also use it to visually amplify specific intensities that are very difficult to dis-
tinguish from their surroundings. For example, features produced by pixels with values of
122 in a field of pixels with similar values would be impossible to see in a gray tone
image, but would jump out if that value were to be rendered in color.
When you pseudo-color a monochrome image, you build a special palette through which
your monochrome image is displayed. Pseudo-coloring an image does not modify the
pixel values in your image bitmap in any way (it does not convert your image to True
Color or Palette, for example). It simply associates a pseudo-color palette with the image,
that interprets the gray-level values in the image as color.
Pseudo-colored images are very similar in structure to Palette class images, but they differ
in a couple of important ways. First, the pixel values in a pseudo-colored image actually
represent continuous-tone intensity information, whereas a Palette image's pixels carries no
intensity significance. Secondly, a Palette image includes a palette table that is actually
part of the image file.
The pseudo-color palette associated with a pseudo-colored image is not a permanent part
of the image. It is a palette that you assign to the image while you work with it in Image-
Pro. Pseudo-color palettes can be saved to a disk file, and later loaded for subsequent use
with any monochrome (Gray Scale 8, Gray Scale 12, Gray Scale 16, or Floating Point)
image.
The basic steps involved in pseudo-coloring the active, monochrome image are to:
1. Select the Pseudo button from the Color group on the Adjust ribbon:

2. Click the drop-down arrow to see the Pseudo-color dialog:

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3. Check the Pseudocolor On/Off to show (or hide) the pseudo-coloring on the image.
4. Change the color assignments and refine the intensity division widths. This is done
using the Spectrum and Type drop-down menus or the color bar.
The Spectrum drop-down list lets you choose which colors will be used for coloring
the images. Choices include Blue to Red, Red to Blue, Cyan, Magenta, and Yellow.
You can also define a custom color spectrum.
Type lets you choose RGB, HSI CW, HSI CWW, or a custom type.
5. Specify the range of intensities you want to pseudo-color. The intensity range is spe-
cified using the Start and End spin buttons. You can load saved pseudo-color set-
tings using the Load button.
6. Specify the number of distinct colors you want to associate with the selected intens-
ity range. The number of colors is selected using the Divisions spin buttons.
7. Indicate the number of spins. If you want the spectrum to repeat over the intensity
range of the image, you can set the Spins to the number of times to repeat the spec-
trum.
8. Save the settings for future use (with the current image, or with another). This step is
optional. You do not need to save the pseudo-color assignments in order for them to
be applied to the active image. If, however, you want to save it for continuing use or
to exchange with a colleague, you need to save it. When you click the Save button,
Image-Pro saves the current pseudo-color palette settings.

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Adjusting Color Channels

To adjust the color channels, or load a saved Look-Up Table (LUT) use the Display
group on the Adjust ribbon.

This feature contains the following tools:

1. Click the drop-down menu arrow.

2. Select the channel you want to adjust. The default is All channels.

3. Click the Advanced button.

You will see the Advanced tools ribbon:

This tab contains all the features you may need to make and view changes to the
Lookup Table.
As you make changes to the LUT, you will see them reflected in the LUT profile
graph at the bottom of the workspace:

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Note: You will not get a dynamic LUT profile graph for *.AVI and *.SEQ files unless you
select an active frame.
If you are working with a black and white image, you can adjust the black and white
levels by using the BL: and WL: spin buttons, or typing new values in those fields.

Changing the Display Range

The Display Range is the range of image intensities between the low and high values
(inclusive) of the black and white levels. This feature allows you to improve the visu-
alization of objects of interest in your images by:
l Excluding irrelevant image data
l Boosting the contrast between relevant intensities.
For example, the intensity values of an 8-bit gray scale image can range from zero to 255,
and often the useful data covers only a small portion of that range.
With the Display Range feature, you can set high and low thresholds for intensity values
such that all pixel intensities below the low level display as black and all pixel intensities
above the high level display as white. Image-Pro scales all intensities within the active
image to take advantage of the range of black and white values available on your monitor.

Display range enhancements are implemented through a Lookup Table rather than by
modifying the image bitmap. The original appearance of your image can be restored at
any time. If you want to make the enhancements permanent, you must write them to the
image with the Apply Display to Image button..

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To change the display range for an image:

1. Select the Display group in the Adjust ribbon.

2. Click the Best Fit button to find the best display range automatically. You can use the
BL and WL spin buttons to make further adjustments to the display range.

Adjusting Brightness, Contrast, and Gamma

The brightness, contrast, and gamma controls provide three ways to adjust pixel intensities
in order to enhance objects of interest in the image. They allow you to adjust the display
of image intensities between the black and white levels.
To adjust brightness, contrast, or gamma:
1. Click on the Display group in the Adjust tab’s ribbon:

As you move the sliders, the image in the workspace displays the changes in intensity.
2. Use your cursor to select the color channel that you want to adjust.

Brightness: Increases/decreases all intensity

values uniformly. In the example below, brightness has been increased from the ori-
ginal.

OriginalAfter Brightness Increased

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 Changing the Display Range

Contrast: Increases/decreases high and low

intensity values reciprocally (that is, as the high values are increased, the cor-
responding low values are decreased, and vice versa). In the example below, contrast
has been increased from the original.

OriginalAfter Contrast Increased

Gamma: Adjusts the contrast for the high and

low intensity ranges simultaneously. In the example below, gamma has been
decreased from the original.

OriginalAfter Gamma Decreased

Resetting the Image

Because the enhancement features (Equalize, Contrast Enhancement, Display Range, and
Invert Contrast) are implemented through a Lookup Table, the changes made to the appear-
ance of your images are not permanent. You can restore the image to its original appear-
ance at any time. To do this:

n Click the ResetDisplay button from the Channels group.

The Lookup Table is reset and the image appears as it did before any enhancement options
were defined.

Making Enhancements Permanent

Because the enhancement features (Equalize, Contrast Enhancement, Display Range, and
Invert Contrast) are implemented through a Lookup Table, the changes made to the appear-

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ance of your images are not permanent. This means they will not be saved with the image.
To make the enhancements permanent:

n Click the Apply Display to Image button.

All Lookup Table values are permanently applied to the image and the Lookup Table is
reset to its default state.

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Using the Process Tab Features

The Process tab’s ribbon contains many useful features for image processing.

Merge Files
Merge Files allows you to combine two sequence files into one file, or to add a new
image to an open sequence file. The Merge command always places the new sequence file
or image at the end of the open one (append). To use this command, follow these steps:
1. Choose Merge Files from the Combine group on the Process tab’s ribbon:

Selecting this option displays the Open File dialog box:

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If you have an image or sequence open, the files that you select will be merged with
the active image; otherwise a new workspace will be opened for the sequence. Select
the name of the file(s) you want to merge. If any of the files are sequences also, you
can select the starting frame of the sequence being opened, and number of frames that
you want to combine.

Sub-sampling
The Subsample feature in the Combine group on the Process tab allows you to create a
new sequence from a portion of the original sequence.

Sub-sampling builds a new sequence from copies of the selected frames.

Interval: This control allows you to specify the interval that will be used for the sub-
sampling. In the example above, the subsampled sequence will be composed of every 2nd
frame of the original sequence.
Difference: These controls calculate the frame-to-frame difference of selected portions of
your sequence. New images are created using the subsampled portion of your sequence.
Wrap Around: Calculates the last frame as the difference between the last and first frames
Diff only: Return only the difference frames (result will be one frame shorter than the ori-
ginal)

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Pad First: Pad the result with the first frame of no difference
Pad Last: Pad the result with the last frame of no difference
Average: Calculates the average of all frames of the selected images.
Sum: Calculates the sum of all frames of the selected images. Note that the output image
will be a floating point type with the number of channels corresponding to the source
image.
Motion Detection: This feature will detect only moving objects and extract them to a new
image for use with object tracking. There are 3 methods:
l Average Global
l Average Running
l Median Running
The operation subtracts a filtered image from the original, so all static objects become invis-
ible and only moving objects are detected. The Size parameter defines the number of neigh-
boring frames that are used for filtering. When using Median, the size has to be bigger
than the movement of the object through the given number of frames. For small and fast
objects this value can be small, for big and slow objects the value should be bigger.

Aligning Images
The AlignImages feature allows you to align individual images, or images in a sequence,
for translation, rotation, or scaling. This feature is useful if you are working with images
taken over time, images with some camera movement, or if you are aligning channels in
multiple images from a beam splitter, or correcting for offset angles through a stereo micro-
scope Z-stack.
To use the Alignment feature, follow the steps below:
1. Open an image sequence.
2. Chose Align from the Combine group on the Process tab.

3. Click the drop-down menu symbol and select Align.

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Choosing Align aligns the active image sequence image using the default method
(FFT). The function can be used to do a quick alignment without opening the Align
dialog.
Align Selected adds the image selected in the Image Strip to the alignment list and
opens the Align dialog.
Set Selected to Align adds an image selected in the Image Strip to the Alignment list.
Align by Feature aligns the active sequence by the location of a reference feature
defined by the ROI. You should draw an ROI around the reference feature.Then define
the Search Radius, which corresponds to the maximum shift between the positions of
reference features on neighboring frames.Click the Align by Feature button to create a
new aligned image. This function can be used when alignment by whole image can be
problematic due to significant changes between frames.
You will see the Align dialog on the right side of the workspace:

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 Aligning Images

4. Select images to align by clicking the Add button.

5. Choose the Anchor frame. By default the reference image (which will not be moved)
will be the first item on the list or the first image in a sequence. You can change the
default by highlighting an image with the cursor and clicking the Anchor button. All
other images will be moved to align relative to that image
6. Choose your alignment method:
l FFT
l User
l Manual
The FFT method allows you to set the alignment parameters, as shown here:

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n Rotation/Scaling/Translation: Click the correct box to choose which alignments

to perform.
n Rotation/Scaling angles and scales accuracy: These options let you choose how
many angles and scales to test. Rotation can be +/- 90 degrees, while scaling is
possible over a range of about 0.7 to 1.4. These ranges are divided by the number
of angles or scales to test. Higher numbers will require more computation and
higher accuracy. Note that these must be power of 2 factors for the FFT
algorithms.
n The max size control defines the maximum size of cross-correlation area used for
calculation of the alignment (in pixels). The alignment area is in the center of the
image. Limiting this size can speed up the alignment.
n Subpixel Accuracy: Check this box to calculate alignment between images with
subpixel accuracy (default). Use this option when images have small shifts com-
parable with pixel size. The option can be used for drift correction. Note, that
when this option is on, the output images are created with sub-pixel image shifts
using affine transform with bi-linear approximation.
n Full/Phase correlation: Select one of these radio buttons to perform the FFT trans-
lation correlation as a full correlation or as a phase-only correlation. Full cor-
relation works better for most images. Phase-only correlation ignores image
intensity, and may be preferable if the overall image intensities vary while the pat-
terns remain similar.

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 Aligning Images

n You can use the Calculate button to get transform values without creating an
image. The transforms then can be adjusted or saved in the Manual method page.
The User method allows you to set the degree of alignment in the X and Y directions:

n Shift Per Plane Xor Y: Indicate the number of pixels to shift the images in each
plane.
n Trim Borders: If the alignment is purely translation, clicking this button

allows you to trim the output to include only those pixels that are
valid in each image. Otherwise the output image is the same size as the input
images, and empty pixels are filled with black.
n Calculate - creates transforms based on the defined values. The transforms then
can be adjusted or saved in the Manual method page.
The Manual method provides tools to adjust or define alignment transforms manually.
The transforms can be saved to/loaded from a file.

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n The table contains the list of current transforms, which are created by the FFT or
User method.
n Load/Save Alignment allows you to loadi and save profiles. Using this method
transforms created on one set of images (e.g. Bright-field) can be applied to
another set of images (e.g. Fluorescence).
n The Reset button resets the list of transforms. The number of entries is set to the
total number of frames in the Selected images.
n Clicking the Adjust button opens the Manual Alignment dialog, where image
transforms can be adjusted manually:

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A pair of images is shown on the right side of the dialog. The preview window
can be zoomed in/out using the mouse wheel, or dragged using the left mouse
button. The images are combined using the selected Composition method.
The following compositions are included:
n Average . The average of two images is shown
n Red Green Pseudo-color. The first image is converted to Red and the second
to Green.
n Toggle Frames Fast. The images are toggled 10 times per second.
n Toggle Frames Slow. The images are toggled one time per second.
The Translation group allows the planes to shift in the X or Y directions.

The button resets the transform.

The button resets the zoom and translation of the preview window.
The Rotation defines the rotation angle in degrees.

Scale defines the magnification of the transform.

The Frame control allows you to change image pairs.

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The Composition control lets you select your translation method:

Click the drop-down arrow to see your choices:

Clicking Ok applies the new transform to the align table. Cancel closes the dialog
without changing the transforms.
7. Whatever method you select, click the Calculate button. The Manual Method dialog
appears.

8. Click the Adjust button. You will see the Manual Alignment dialog.

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 Using Extended Depth of Field

9. When the parameters are set to your satisfaction, click the OK button.
10. To align the images, click the Apply button.
The aligned images will be saved in a file, such as Paper_Aligned .

Using Extended Depth of Field

Extended Depth of Field (EDF) is used create an in-focus single-plane image from the
best-focus portions from different planes of a Z stack sequence. It also supports stereo
microscopes, where changing the focus position shifts the image. To use the EDF feature,
follow the steps below:
1. Select EDF in the Combine group on the Process tab:

2. Click the drop-down arrow. You will see the EDF dialog:

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The EDF Options dialog opens.

3. Click the Add button to add the images to the Selected Images list.

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The Selected Images list shows the images added for EDF. You can add images to the
list using the Add button or by dragging images from Image Strip and dropping them
to the list You can change the position of an image in the list by dragging and drop-
ping.
Clicking the Add button on the selected image in the Image Strip to addit ot he selec-
ted images list. The All button (in the Add button’s drop-down ) will add all open
images to the list. Images can be removed from the selected list by clicking the
Remove button. The selected list is reset by the Clear button.
Images of different sizes and classes are not accepted in the list. The output images
will have the same size and class as the first image in the list.
4. Click the Preview button.
Activating the Preview button will show EDF Test Strip which shows the preview res-
ults of all focusing methods in a separate window. A sample EDF test strip appears
here:

n Click Preview again to turn the Test Strips off.

5. Choose your output parameters.

(1) Indicate your output options:

l Generate composite best-focus image: Clicking this button creates a single
image composed of the best pixels from the set of input images, as determined
by the Focus Analysis Options.
l Return best-focus frame: Clicking this button creates a new workspace con-
taining a copy of the selected frame with the most in-focus material.

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l Activate best-focus frame: Clicking this button activates the frame that has
maximum contrast within the active ROI.

(2) Select the focus analysis options:

l Normalize Illumination: Use this option when the illumination or intensity

of emitted light will change in the time that it takes to acquire all of the
planes. Basically, this option corrects for uneven illumination as the Z plane
changes.
l Bottom Up: If you click this button, the order of the images is reversed. The
image at the top of the list will be the last image in the stack, and the image
at the bottom of the list will be the first image in the stack. Note that the per-
ceived “position” of the images will affect the Topographic Map, and may
affect which pixel is selected for some of the focus analysis Calculation.

(3) Choose your focusing method:

l Local Contrast: This method considers areas with high/maximum contrast to
be in focus. Pixels are analyzed in the area around the current pixel location.
The pixel from the plane with the largest variance or local contrast is selected.
l Stack contrast: The mean value of the pixels is calculated. The pixel from the
plane with the largest variance from the mean is selected.
l Max. Intensity: This method is used for samples that are more intense than the
background such as light emitting fluorescent dyes. The pixel from the plane
having the highest intensity at the current pixel location is selected.
l Min. Intensity: This method is used for regions that are darker than the sur-
rounding area to be in focus. The pixel from the plane having the lowest
intensity at the current pixel location is selected.
l Small Edges: Edges are extracted from the average of the stack assuming a
moderate blurring.

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 Using Extended Depth of Field

l Medium Edges: Edges are extracted from the average of the stack assuming a
mid-scale blurring.
l Large Edges: Edges are extracted from the average of the stack assuming a
large-scale blurring.

(4) Select an edge filter from the drop-down list:

Variance: Select this filter if you want to detect and emphasize edges and
textures. The Variance filter substitutes a pixel with the standard deviation for its
neighborhood. There are different sizes of this filter available.
l Variance 3x3 =Variance 3x3 filter
l Variance 5x5= Variance 5x5 filter
l Variance 7x7 =Variance 3x3 filter followed by Dilation 5x5
Dilated Variance filters come in two sizes:
Dilated Variance 3x3= Variance 3x3 filter followed by Dilation 5x5
Dilated Variance 5x5= Variance 5x5 filter followed by Dilation 5x5

(5) Indicate the significance threshold.

The Significance threshold defines threshold for significant edge pixels.
The pixels (after a edge filter was applied) above the threshold are considered in-
focus pixels. If, for a given pixel position, none of the frames contain edge intens-
ities above the threshold, the height of the pixel (in 3D) is assigned to the frame
with closest in-focus pixel. The threshold percent is relative to the total accu-
mulated value of edge image. The range of the value is from 0 to 100. The value
= 50 (default) will set threshold on level of Median intensity, cutting 50% of
pixels. The value 80 will take only 20% of pixels with highest contrast. Use
higher threshold on noisy images.
6. Indicate if you want to generate a topographic map:

l Generate Topographic Map: Clicking this button creates a topographic map from
the focused image.

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l Create Intensity Calibration: Check this box if you want to generate an intensity
calibration for the combined EDF image.
l Create Surface Plot: Check this box if you want to generate a surface plot image
of the combined EDF image.
l Smoothing: Choose a smoothing factor for the topographic map image.
7. When all the parameters have been set, click the Apply button.

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Tiling and Stitching

The Tiling feature allows you to align and stitch images together. This might be useful
when constructing panoramic views, or for linking together multiple images taken with a
motorized stage. To use tiling, follow the steps below.
1. Open the image or sequence. You will see your image in the Image Strip.

2. Select Tiling from the Process tab. The workspace will be blank.
3. Drag and drop a group of images or a sequence from the Image Strip into the work-
space. The tiled view will look something like this:

The Tiling View has its own ribbon, as shown here:

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The Add group lets to add or delete images from the tiling view. Using the drop-down
menu, you can add images individually or select Add All.

Add all adds all the images into the view frame by frame.

Delete deletes a selected frame from the view.

Delete All deletes all the frames from the view

Align contains options for displaying the tiled view.

You can display them as a cascading group, aligned by increasing or decreasing height, by
X, Y, and/or Z position, in columns or in rows. You can also flip the images from left to
right or from top to bottom to gain a different perspective. The settings let you set the dis-
tance between the tiles and the border.

Cascade displays all the frames in the cascading view.

Height decreasing aligns all the frames in the view by height decreasing.

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Height increasing aligns all the frames in the view by height increasing.

By XYZ aligns all the frames in the view according XYZ coordinates (available if all
frames in the view have valid XYZ coordinates).

By Columns aligns all the frames in the view by columns (quantity images in the
column set in “Images in Column”.

By Rows aligns all the frames in the view by rows (quantity images in the row set in
“Images in the Row”).
Flip Left-Right flips all the frames in the view from left to right.
Flip Top-Bottom flips all the frames in the view from top to bottom.
Z-Order uses the Z-stack order to align the images.
Images in Column numbers the images in the column and then aligns them by columns (0
– auto count).
Images in Row numbers the images in the row and then aligns them by rows (0 – auto
count).
Border indicates the border size in pixels.

Gap X indicates the horizontal gap between the frames in pixels.

Gap Y indicates the vertical gap between the frames in pixel.
Stitching lets you “sew” the individual images together.

None indicates no stitching.

Snap to Edge helps to position images in the workspace:

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l to create a mosaic image from unrelated images

l to create a tiled image from images which was taken without overlapping
l before using Auto stitch method,
Pattern Matching is a semi-automatic method to create a tiled image from overlapped
images. It helps to position images in the workspace using a pattern matching algorithm
(this is like Auto Stitching, but only for 2 images). You have the following choices. The
measurements are given in pixels.
l Correlation method selects the type of correlation method.
l Overlap X creates the horizontal overlap between the frames
l Overlap Y creates a vertical overlap between the frames.
l Search Radius (%) parameter, which defines maximum allowed shift from the expec-
ted position; the value is the percentage of Overlap. The default value is 50%. If the
value is set to 0 – limits are ignored.
l Matching Threshold parameter (range from 0 to 1), which defines the minimum cross-
correlation value between tiles that is accepted. If the cross-correlation value below
the threshold, the average shifts are used for these tiles. (when the parameter is 0 – any
cross correlation is accepted)
l When the Uniform X/Y Overlaps option is checked all tiles use the same average
shift, calculated from sampled set of well-correlated tiles. Use this option when many
tiles have insufficient texture, this option is also faster. Switch this option off to cal-
culate shifts for every tile individually, it provides more accurate tiling when shifts
between tiles varies.
l Multi-point Registration - when this option is on the tiles are aligned by multiple
points, when 2 or more anchors detected the tile is registered using elastic deformation
to match reference points. When the option is Off, the average shift from multiple
points is used, tile is not deformed. This option is recommended to use on samples
with variable surface levels, such as SEM images, where tiles may have variable over-
laps depending on the height of the surface level
l Calculate Overlaps - use this button to calculate overlaps based on the current pos-
itions of the tiles. Overlap X and Overlap Y will be computed as average overlap
between neighboring tiles in rows and columns. Note, that overlaps are also calculated
automatically when new images, that have XY position information, are added to the
tiling view.
Selecting Auto will create a stitched image from the tiled view automatically.

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Tiled image Stiched image

Shading group contains controls that correct illumination problems manifested itself as a
‘quilted’ effect where boundaries of every tile are visible due to uneven illumination or sig-
nal magnification on every tile.

Example of image with shading problem:

The Correct button drop-down has the following controls:

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Shading correction is performed by dividing tile images by background image. The back-
ground image can be either created by averaging of all tiles (default) or by using one of
the empty tiles (tiles without texture). When the Selected Tile option is active user can
select a tile on the view and click the Set Tile as Background button to set that tile for
correction. The tool-tip over the Set Tile button displays the name of background tile. The
Smoothing combo box defines the size of low-pass filter applied to the background image.
The size is defined in percent’s of the image size.
Clicking the Correct Background button applies shading correction to all tiles.

In some cases the tiling view will still have tile rows or columns visible due to differences
in average intensities between rows and columns caused by EM charge effects, lamp vari-
ation, etc. Clicking the Normalize button will compare intensities across tile borders and
rescale intensities to minimize this issue as much as possible.

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Display group contains controls for adjusting tile LUTs for better viewing while looking
at the results in the mosaic.

Best Fit stretches the display LUT to enhance contrast as much as possible, Brighten and
Darken scale the viewing gamma, and Reset will put the display settings back to defaults.
Note that contrast adjustment does not modify the data, just the display, also the LUT can
be adjusted on the source images and that will be synchronized with the tiling view.

The Result group lets you combine the tiles into a new image or image set.

Select your desired result from the drop-down menu.

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Add/Edit ROI activates the Add/Edit ROI mode. ROI defines the size of the result
image.

Delete ROI deletes an ROI from the view.

generates the resulting image or image set. Click the drop-down arrow to see the
options

Tiled Image: Tile frames within the ROI are stitched together into a single frame in a new
image. Stitching is done with the current tile placement and the desired Blending Method
(see below)
References Set: A new image set is created holding references to the tile frames that inter-
sect the ROI; these frames are placed into the XY (Scan) dimension of the set. The referred-
to frame X,Y positions are not modified from their original values and no copy of the
image data is made. If you save and then reload this image set into a tiling view, you will
get the original, unadjusted, tile placement.
Copies Set: A new image set is created holding copies of the tile frames that intersect the
ROI; these frames are placed into the XY (Scan) dimension of the set. The copied frame
X,Y positions are modified to reflect the current placement of the tile frames and any warp-
ing that needed to be done to support multi-point registration. If you save and then reload
this image set into a tiling view, you will get the current, adjusted, tile placement.
Tiled Set: A new image set is created holding equal-sized frames that cover the ROI com-
pletely with no overlap. The new tiles are created by stitching together frames in the tile
view with the current tile placement and the desired Blending Method (see below). The res-
ulting tile frames will have no overlap and are placed into the XY (Scan) dimension of the
new image set. Usually the tiling will be larger than the ROI, with excess portions needed
to fill out a full tile to the right and bottom set to black. If you save and then reload this
image set into a tiling view, you will get the a new, perfect (no overlap) tiling rounded out
to a full tile.

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Blending Method: This option is only significant if images are overlapping in the Tiling
View workspace. This option controls how the overlap is blended when the new stitched
image is created. The overlap can be blended by gradually fading it out. This is the "Gradi-
ent" method. The overlap can be blended by trimming the overlapping portions in half.
This is the "Trim" method. Alternatively you can use no blending method at all.

The Tiling Options let you specify more aspects of the tile view display and results:

Checking Allow Resize activates the Reset Size button. This will automatically reset your
images to their original size.
Keep Aspect Ratio maintains the aspect ratio of the original images in the tiled image.
Checking Transparency lets any background to show through.
Show Info displays the image information as an overlay on the tiled images.
Boundaries displays the outline of each tile in the image workspace.
Background opens the color picker, where you can change the color of the workspace
background.
The Font group lets you specify the appearance of any labels on your tiled image.

This option is available only when Show Info is checked.

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Comparing Images
The image comparison feature enables you to compare two images that may have changed
over time. You will find the Image Compare feature in the Compare group on the Cap-
ture ribbon.

To use Image Compare, follow these steps:

1. Open the two images that you want to compare.
2. Go to the Compare group on the Capture or Home tab’s ribbon.
3. Click the Image Compare tool. You will see the Comparison tools ribbon.

4. Click the Add button to add images to the view.

5. Use the tools in the Align group to adjust the position of your images.

6. Use the Parameters and Options groups to make changes to the transparency, back-
group, and other characteristics.

7. Click Apply A new image will be created from the comparison of the two selected
images.

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The darker areas indicate where the image has changed.

The results appear in a combined image.

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Image Math
The Image Math, or Calculator feature, lets you combine and contrast two images using
arithmetical and logical operations between images and numbers. This feature appears on
the Process tab.

Clicking the Calc button in the Math group opens the drop-down window with arith-
metical operations controls.

Image 1: Select one of the images in this list box to use as the first operand.
Image 2: Select one of the images in this list box to use as the second operand.
Operation buttons: Select the kind of operation you want to perform.

AND: Click this button to perform a logical “AND” to combine both images.
Only bit values that are “on” in both images will be “on” in the result.

OR: Click this button to perform a logical “OR” between the two images. Bit val-
ues that are “on” in either image will be “on” in the result.

XOR: Click this button to perform a logical “XOR” (exclusive OR) between the
two images Only when a bit value is “on” in one operand and “off” in the other will the
bit be “on” in the result. If bit values are “on” in both operands, or if they are “off” in
both operands, they will be “off” in the result.

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NAND: Click this button to perform a logical “NAND” between the two images.
“NAND” produces a result that is the complement of an “AND” operation. Bit values that
are “off” in either or both operands will be “on” in the result.

NOR: Click this button to perform a logical “NOR” between the two images.
‘NOR” produces a result that is the complement of an “OR” operation. Bit values that are
“off” on both images will be “on” in the result.

NOT: Click this button to perform a logical “NOT” on the bit values of your act-
ive image. Every bit value that is “on” in the active image will be “off” in the result.
Every bit value that is “off” in the active image will be “on” in the result. Use this com-
mand to invert the pixels in your image, producing a negative effect.

Add: Click this button to add the two images. If a pixel's result falls outside of
the image's intensity scale, it will be clipped to the highest /lowest permitted value. If you
have set the Add to result value, it will be added to the result before it is written.

Subtract: Click this button to subtract the second image from the first image. If a
pixel's result falls outside of the image's intensity scale, it will be clipped to the highest
/lowest permitted value. If you have set the Add to result value, it will be added to the
result before it is written.

Diff: Click this button to obtain the absolute value between the two images. If a
pixel's result falls outside of the image's intensity scale, it will be clipped to the highest
/lowest permitted value. If you have set the Add to result value, it will be added to the res-
ult before it is written.

Multiply: Click this button to multiply the first image by the second one. If you
have set the Scaling factor value, each result will be multiplied by the Scaling factor
before it is written.

Divide: Click this button to divide the first image by the second one. If you have
set the Scale value, each result will be multiplied by the Scale before it is written.

Average: Click this button to replace each pixel with the mean value of the two
images.

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Log: Click this button to replace each pixel with its natural logarithm. Pixel values
less than or equal to 0 are invalid.

Square Root: click this button to replace each pixel with its square root. Pixel val-
ues less than 0 are invalid.

Square: Click this button to replace each pixel with its second power.

Inverse: Click this button to replace each pixel with its inverse (1/pixel). Argument:
Number: new pixel = Number / original pixel. Pixel values equal to 0 are invalid.

X to the power of Y: Click this button to replace each pixel with its Yth power. Y
is limited to positive values. Pixel values less than 0 are invalid if Y is less than 1.

Set: Click this button to replace each pixel with the same given number or with the
corresponding pixel in the second image operand.

Exp: Click this button to replace each pixel with its exponent.

Maximum: Click this button to replace each pixel with the largest value of the
two operands.

Minimum: Click this button to replace each pixel with the smallest value of the
two operands.
Image: Set this option if you want to use another image for the specified operation. You
must also select Image 2 in the list box above. The two images need not be of the same
image class. The result has image class of the first image.
Number: Set this option if you want to use a constant value for the specified operation.
If a logical operation has been selected, numbers from 0 - 255 can be entered. If an arith-
metic operation has been selected, any value can be specified, including negative and frac-
tional values.
Image Equation summarizes the formula that will be used to perform the operation.
Apply: Click this button if you want to write the results back to the active image or
AOI. Results will be placed into the same pixel positions from which they originated.

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New: Click this button if you want to write the results into a new window. The new
window will be of the same size and class as the original image or AOI.
Float: Click this button if you want to save the results into a floating point image.

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Correcting the Background

Use the Background Correction command to make adjustments to the background of your
image. This tool is used to better distinguish image background from image objects, mak-
ing it easier to extract the objects during a counting or measurement operation. The Back-
ground Correction command can correct uneven background intensities, and compensate
for irregularities due to uneven lighting, nonuniform camera response or minor optic
imperfections. It might be used to remove evidence of dust on the lens, or to correct for
bright spots caused by the light beneath the microscope's stage.
Two types of background correction are provided by Image-Pro:
l Background Subtraction should be used for all images except those of transmitted
light experiments which will be used to measure optical density.
l Background Correctionshould be used for optical density applications, as it accounts
for the fact that optical density is not a linear function of the gray scale.
Image-Pro provides several tools that allow you to adjust the background of your image.
They appear in the Background group on the Process ribbon:

The Options allow you to create a new image from the background image.

Extract background image displays the background image during black and white Cal-
culation.
To use the Background Subtraction command, follow these steps:
1. Open the image that you want to correct.
2. Click the Subtract drop-down arrow .
You will see the following dialog:

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3. Choose a dark or light background to subtract.

The subtracted background will appear in the workspace.
You may also use the browse button to choose a saved image to work with; or create
a background image from existing points in the active image.
To use the Background Correction command, follow these steps:
1. Open the image that you want to correct.
2. Click the Correct drop-down arrow .
You will see the following dialog:

3. Choose a dark or light background for correction, and indicate the feature width or
black level. Your corrected image will appear in the workspace.
You may also use the browse button to choose a saved image to work with; or create
a background image from existing points in the active image.

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Applying Filters
Image-Pro filters are used to emphasize features of an image. The instructions below
demonstrate how to apply the Sharpen filter to accentuate the edges in the castiron.-
tif image.
1. Open the rice.tif image in the Image-Pro workspace, as described earlier in this
chapter.

2. Select the Filters group on the Process tab’s ribbon:

3. Choose the Sharpen filter from the drop-down list of available 2D filters.

4. Select Sharpen 50% from the pop-out menu:

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The filter is automatically applied to the image.

Advanced Filters
The Sharpen and Blur filters are only two of the 2D filters available in
Image-Pro. To see the complete set of available filters, click the arrow for the drop-down
menu:

1. Click the radio button next to one of the filters.

2. Click Preview to see how it will look on a sample portion of your image.
You may try this with different filters to see the various effects. The sample area
appears in the center of the image.

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You can also preview the filter in a strip. Cleck the box filter strip box in the drop-
down menu:

The filter strip shows how the image will appear after sucessive numbers of passes:

3. Click Apply to apply the filter to the whole image. To save your filtered image as a
new image, check the New Image box.

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Using the Grid Overlay

The Grid Overlay tool allows you to define and create precise grids for intercept counting
in either a manual (Manual Tag) or semi-automated (Image Math) fashion.
This tool allows you to create and save a grid of your own design, including such inform-
ation as length of the grid lines or number of points in the grid. You may also define the
following type of grids: point, lines (horizontal or vertical or both), random lines, line seg-
ments (horizontal or vertical or both), circles, and cycloids. In addition, you may set the X
and Y intervals for the line grids, spacing for point grids and combine multiple point grids
in one grid overlay.
The Grid Overlay tool will generate an overlay on the image for viewing purposes,
although you also have the option to create a new image mask of the grid. The grid is not
attached to the image, but stored in a separate file (*.grd).
Grid Overlay is located on the Process ribbon:

To use the Grid Overlay, follow these steps:

1. Open an image in the workspace.
2. Go to the Process tab.
3. Select the Grid Overlay group:

You will see the Grid Overlay dialog:

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4. From the Type panel select the style of grid symbol.

We are going to choose Points.
5. From the Layout panel, choose how you want your grid to appear. Select one of the
following:
l Orthogonal: The objects on the grid are displayed in a regular pattern of hori-
zontal rows and vertical columns.
l Concentric: The objects on the grid are displayed in a regular pattern of con-
centric circles.
l Random: The objects on the grid are displayed in a random arrangement.
Selecting Random allows you to choose a fixed seed parameter, so that the
same random pattern can be repeated.
6. Set the grid parameters using the Parameters panel.

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The example grid below displays the points marked as large squares in a pattern
spaced 20 pixels apart

7. Indicate the size and color of the grid using the options in the Margins panel:

8. When you are satisifed with your parameter selections, click Apply .
Your grid will appear on the image in the workspace. It will stay in place until you
choose Remove to erase it.

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Filtering with Fast Fourier Transforms

The FFT command transforms an image into a frequency spectrum. Image noise appears as
a repetitive pattern which is difficult to remove using standard spatial-filtering tools. The
FFT tool is especially designed to isolate and filter this noise, then transform and edit the
noise pattern out of the image in easy steps.
1. Load the Fftprint.tif file from the Fast Founier Transform folder.
The Fftprint.tif image window appears. Notice the strong diagonal noise pat-
terns.

1. Go to the Process tab.

2. Choose the FFT group.

3. From the drop-down menu, choose Remove Periodic Noise.

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A copy of the original image with the noise removed appears in the workspace.

Image with noise Image with noise removed

Viewing the Image Spectrum

1. To transform the image to its frequency spectrum, click the Forward button in the
FFT drop-down menu.

This process may take a few moments. When the transformation process is complete,
the fftprint-fft.tif containing the spectrum appears in the workspace.

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The spectrum appears as a centered cloud of points, with 4 bright points outside of the
central point. These 4 points represent the pattern noise you see in the image.
To eliminate the anomalous frequencies (the 4 outside bright points) you can defining
an AOI around them and cutt the frequencies from the spectrum.
1. From the FFT Filter Options dialog, select Spike Cut.

2. Click the Detect Spikes button.

The frequency spikes will be selected in the filtered image:

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3. Click the Apply button.

The frequency spikes will be removed from the image. The majority of the anomalous
frequencies have now been removed from the image.
4. To return the image to spatial form, without the noise, select New Image from the
Inverse drop-down menu:

A copy of the original image, without the noise, appears in the workspace.

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Image Sequences
Image-Pro allows you to open and navigate multi-frame images saved in the
.seq format. The sequence viewer is automatically used for images that have
more than one frame. The image sequence tools appear in the Combine group on
the Process tab’s menu:

To see the image sequence tools, click the drop-down arrow under Subsample:

Merge Frames merges the frames in all selected images into a sequence.
Subsample creates a subsampled sequence of selected images.
n The Interval control defines the sub-sampling frequency. The active frame range of the
sequence is used; the operation always starts from the first frame in the active frame
range. When Interval is less than 1, the input sequence will be oversampled.
n Offset defines the position of the first frame of subsampled sequence.

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Note that all sequence operations work with selected images. Multiple images can be selec-
ted in image strip using <Ctrl> or <Shift> clicks. If no multiple images are selected, the
active image sequence is used for processing.
Difference calculates frame-to-frame difference in selected images. The Options define how
the last or first frames will be processed:
Wrap Around calculates the last frame as the difference between the last and first
frames
Diff only return only difference frames (result will be one frame shorter than the ori-
ginal)
Pad First pad the result with first frame of no difference
Pad Last pad the result with a last frame of no difference
Normalize normalizes frame intensities on the whole sequence. The function uses the act-
ive frame as the reference and scales intensities on all other frames to match the mean
intensity of the reference frame. If the image contains ROIs, the sequence is normalized by
intensities within ROIs. The function can be used to normalize frame intensities on Z
stacks before segmentation (including 3D) and measurements. When the option By Gain
and Offset is active illumination is normalized by Gain and Offset, when the option is off,
only normalization by Gain is used. When the option is On the Gain is calculated based
on standard deviation of the intensity, when off - from the Mean value of histogram.
Average calculates the average of all frames in the selected images.
Sum calculates the sum of all frames in the selected images. Note that the output image
will be a floating point type with the number of channels corresponding to the source
image.
Maximum creates maximum intensity projection of all frames.
Minimum creates minimum intensity projection of all frames.
Motion Detect operation will detect only moving objects and extract them to new image,
then this image can be used with tracking. There are 3 methods: Average Global, Aver-
age Running and Median Running. The operation subtracts filtered image from original,
so all static objects get invisible and only moving objects get detected.
The Size parameter is used with Running algorithms and defines the number of neigh-
boring frames that are used in filtration. When using Median, the size has to be bigger
than the movement of the object through the given number of frames, basically the object
has to cross any point for the given number of frames. For small and fast objects this value
can be small, for big and slow the value should be bigger.

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Flip to XZ: Flips sequence to XZ plane, so the new sequence will have Y axis cor-
responded to Z axis of the original sequence.
Flip to ZY: Flips sequence to ZY plane, so the new sequence will have X axis cor-
responded to Z axis of the original sequence.
The last selected operation will be set as default for Sequence operations button:

The sequence viewer is automatically used for images that have more than one
frame. The sequence viewer has an on-screen toolbar, which appears only when
you move your mouse over the image. The example below shows the sequence
toolbar superimposed on the bottom of the image:

The toolbar becomes less transparent (more opaque) when the mouse is over the
toolbar:

The toolbar has the following buttons:

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Go to first frame

Go back one frame

Play sequence backward

Play sequence forward

Go forward one frame

Go to last frame

Use active range applies filters and other Calculation to the active range of
frames (on) or to the individual active frame only (off).

The playback options button opens the following options dialog:

Click Set Start or Set End to set the current frame as start or end of the active
playback range of the sequence. By default the range is set to all frames.
The Rate control defines the playback speed in frames per second.
Loop combo-box determines the action taken during playback when the end or
beginning of the sequence frame range is passed. Choose one of the following
options:

Use this button to click and drag the sequence toolbar

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Click this button to move the toolbar to the area above the status bar.

Clicking the button will move the toolbar back to the image.
This button also displays the Advanced Sequence Control toolbar.

This version of the toolbar contains all the functions of the smaller version and displays
the frame numbers also.
You can also use <Ctrl>+<mouse click> and <Shift>+<mouse click> to select active
frames for processing.

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Using the Active Data Overlay

The Active Data Overlay, also known as a heads-up display, is a method of displaying
changing numeric and graphic information over the active image so that you don’t need
to shift your eyes away from the primary view. The term, “heads-up display” was coined to
describe information displayed on windshields, camera view-finders, medical imaging sys-
tems, video games, and other presentations where it would be inadvisable if not dangerous
to distract the operator from the primary view.
To try out the active data overlay, follow these steps:
1. Open an image sequence in the Image-Proworkspace. Our example will use car-
crash.seq.

2. Go to the Data Overlay group on the View ribbon:

3. Click the drop-down arrow under the Show button:

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When you click Show, the data overlay will appear on the active image. In this
example, the overlay data appear on the upper and lower left sides of the active image.
A calibration marker appears at the lower right.

As you play the sequence, the overlay information updates the frame number and time
information.
4. (Optional) Clicking Marker will turn the calibration marker at the lower right of the
image on or off.

Marker Options let you adjust the position and appearance of the calibration marker:

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5. Click Convert to Annotation to convert the data overlay to an annotation that can be
saved with the image.

Customizing the Data Overlay

Click the Customize button to see the Data Overlay Options panel. Here you will
find tools to display and adjust their location and style of the overlays.

All of the properties can be expanded by clicking on the + sign at the left.

To show or hide an item in the overlay, click the button in the Show column. The
button will turn yellow when the item and its sub-items are visible in the overlay.
Hold down the <Ctrl> button when you click to prevent toggling of the sub-items.

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The item location can be adjusted by clicking the Location button.

The Location drop-down menu contains predefined locations (Top Left, Top Right,
Bottom Left, Bottom Right). You an create new locations using the Add button
and defining a name and an icon for the new location.

You can adjust the alignment of the location (Left or Right, Top or Bottom). Note that
alignment of predefined locations cannot be changed and they cannot be deleted. Off-
set defines the space between window border and location anchor. Width of both
columns (Title and Value) are defined in percents of window width.

Display style is adjusted using the Display style button

You can modify font, shadow and opacity in this dialog. Note that all changes are
applied to the current item and all its sub-items, as with the Show button..

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The layout and attributes of the overlays are saved between sessions. These settings

also can be saved and loaded using the Save and Load buttons.

Clicking the Reset button loads defaults.

The format of numbers and dates can be set on the Display page of the Application
Options, in the Formats group:

A note about measurement overlays: If you are making measurements on a Preview

image, and copying these measurements in an overlay to the captured image, you will see
the Live Preview Measurement Copy Warning if the captured image is a different size or
resolution than the previewed image.

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 Using Live Extended Depth of Field

Using Live Extended Depth of Field

Live Extended Depth of Field(EDF) allows you to create real-time EDF images using a
microscope with a manual focus control. It also supports stereo microscopes, where chan-
ging the focus position shifts the image.
1. To use Live EDF, go to the Capture tab. You will see the Live EDF group:

The Run button will be enabled only when a live preview or image sequence is act-
ive. Clicking the button will start the EDF acquisition on a live image. live EDF pro-
cess is active the button background turns orange and the text changes to Stop.
Clicking the button again will stop live EDF. If the active image is a sequence the
text changes to Run and clicking the button will execute an EDF acquisition on all
frames of the sequence.
2. Clicking the drop-down arrow displays the Live EDF options:

3. Click the Run button to start the Live EDF acquisition

4. Check the Auto Align (Stereo) option to enable auto align for every acquired frame.
Use this option on stereo microscopes where changing the focus position shifts the
image.

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When the Accumulate option is on every new live image is added to the com-
position. This option is preferable for Local contrast composition. In other modes you
can switch this option off and add images to a composite manually by clicking the
Add button.
5. Composition Mode: Make a selection from the pull-down combo box of composition
options:
n Local contrast: Choose this option for real time EDF using the Local Contrast
algorithm. This is most useful for high contrast brightfield images.
n Maximum: Choose this option for real-time EDF using the maximum composition
mode for sample imaging on a dark background. For example, imaging of fluor-
escent samples.
n Minimum: Choose the minimum composition mode for transparent mode illu-
mination of brightfield images
n Difference: Illustrates the difference between the base image and the current image
(image with a grey background)
n AbsDifference: Shows the absolute difference between the base image and the cur-
rent image. Background is black, but you can also activate Pseudo-color on gray
images, so you that can see changing objects in color on a black background.
6. Click the Options… button to see the Live EDF Options dialog:

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 Using Live Extended Depth of Field

Stereo Alignment:
n Alignment area width/height: Indicate width and height of the size of the area in
the middle of the image, which is used for stereo alignment of images in EDF. If
the images have alittle texture the area can be extended to achieve better cor-
relation
n Align by Previous: This option defines the alignment method, which is used with
Auto Align (Stereo) mode. When the option is on, the new image is aligned by
the last added to image, then the option is off, the start image is used to align all
following images.
Blending:
n Defocusing diameter defines the size of the filter that detects in-focus areas on the
image. The default value is 6.
n Plane blending sets the size of the blending area between in-focus zones from dif-
ferent planes. The default value is 4.
Output Window Position

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n Side by side: When this option is on, the workspace is split into 2 panes, where
the live preview window is on the left and the output window is on the right.
Task
n Run in background thread: When the option is on, the Live EDF is started in a
separate thread. This option should be used with multi-core processors, so Live
EDF will run in parallel to the live preview and not slow down preview frame
rate.
Reset
n Click this button to reset the Live EDF settings to the default selections.
7. Click the Reset button to restore the defaults for all options.
A sample dual-viewed, real-time EDF composite image is shown here:

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 Using Live Tiling

Using Live Tiling

The Live Tiling module for Image-Pro makes it easy to acquire a large tiled image without
an automated microscope stage.
1. To use Live Tiling, go to the Capture tab on the Image-Pro ribbon. You will see the
Live Tiling group:

2. The Run button will be enabled only when a live preview or image sequence is act-
ive. Click the button tostart tiling on a live image. When the Live Tiling process is
active, the button background turns orange and the text changes to Stop.
3. Click the button again to stop Live Tiling. If the active image is a sequence the text
changes to Run and clicking the button will will tile all the image frames in the
sequence.
4. Click the drop-down arrow to display the Live Tiling options:

5. Click the Run button to start or stop the Live Tiling.

To start the tiling procedure, move the microscope to the area of the specimen where
you want to start tiling and click theRunbutton. When you click Runthe current image
becomes the starting point for the tiled image. Yellow rectangles will show you the

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outline of the original frame when you move objects under the microscope. The tiling
uses cross-correlation to find the image shift. Use objects with texture for tiling. Note
that empty fields cannot be aligned correctly automatically. When you see that the yel-
low frame follows the image correctly, you can start tiling. Note that the maximum
image shift between slices cannot exceed 25-40%.
n Motion Detection:Motion blur is a common problem with capturing images of
moving objects. This problem can be eliminated by activating the Motion detec-
tion option. Activating this option is recommended for long exposure times.
When this option is active, a new tile in auto-tiling mode will be added only
when the camera is still.
n Use EDF Adding Tiles: If the image contains out-of-focus areas, use the Use EDF
Adding Tiles option. When this option is on, every new tile is added using the
EDF algorithm.The borders of the EDF image are blended with BlendingWidth
(see Tiling Options).When the option isoff, the tiles are added with full overlap
blending. The option can be activated temporary to merge several focus planes
adding tiles using the Add button.
n Auto EDF: When the Auto EDF option is on, the new images are added to the
output constantly and clicking the Add button is not necessary. You can simply
adjust the focus, and during the adjustment the EDF image will be created. EDF
images are not added when the stage is moving.
n Show Crop ROI: Shows the outline of any ROI on the image.
n Set New Position: Use this button if, in the middle of creating a tiled output, you
want to continue from another side of the tiled image. Clicking the button will
activate the position selection mode, where you must click on the output image
where you want to continue tiling. You will see the following message;

n Correct Background: If the microscope has uneven illumination, it can be cor-

rected using this option. Use the empty area on slide as the background, move the
slide under microscope, and click the Set backgroundImage button.

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n Background Color: This option is used for empty areas outside the tiles on an out-
put image. Clicking the Pick from Image button will activate the color picking
tool, which you can use to select a color for the image.

n The easiest way of tiling is with the Auto tiling option. In that case the
images will be added to the output automatically when the image shift exceeds
25% (whenan internal yellow rectangle touches the image border). Move the
stage slowly, and the images will be acquired and tiled automatically.
6. Move the object by 30-35% from the original position (when the inner rectangle will
touch image border) and stop the stage. The image will be automatically captured
when the object will stay still for 2 frames. It will eliminate motion blur and improve
the quality of tiled image.
7. Repeat step 2 (move-stop) until whole area is tiled.

The tiles can be also added manually using the Add button. If the tiled position is
not correct you can use the Undo button to remove the last tile and try other location.
Note that if you use Auto tiling the Add button will be switched off.

8. To use the Add feature, click Undo and move microscope to the location when
correlation is stable. Then activate Auto tiling option again.
9. When the tiling is done, click the Stop button. It will stop the output and any blank
areas will be filled with the background color.
10. Click the Options… button to open the Live Tiling Options dialog:

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n Tiling Undo depth: This option defines the number of undo buffers.
n MatchingThreshold: Use the matching threshold for new images. The matching
threshold is within 0...1 range, and the default value is 0.25. If the image cor-
relation is below the threshold, the tile is not automatically added, although it can
be added using the Add button.
n Color and Sound. Select the Normal color, Go color, and Error color: These
options define the colors use for the tracking box for normal, active, and error
states. You may also indicate if you want to play a sound when an error is detec-
ted.
n Blending: When the Use blending adding tiles option is on (default) new tiles are
added using gradient blending, so no edges between tiles is visible.
n Info Messages: Check these boxes to see helpful messages when clicking Undo or
Set New Position.
n Output Window Position:Side by side: When this option is on, the workspace is
split into 2 panes, where the live preview window is on the left and the output

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window is on the right.

n Task: WhenRun in background threadison, the Live Tilingis started in a separate
thread. This option should be used with multi-core processors, so Live Tiling will
run in parallel to Live Preview and not slow down the preview frame rate.
n Reset:Click the Reset button to restore the defaults to all options. Here is an
example of a tiled image:

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Creating an Audit Trail

The Audit Trail group on the Review tab allows you to create a record of all your image
processing events associated with a particular document. This enables you to reconstruct
your actions should your session terminate unexpectedly. Audit trail logging will be
turned off during macro execution, except that the act of running the macro will be recor-
ded. The Audit Trail group contains these features:

1. To create a new audit trail, click the Audit Trail button. You will see the following
dialog:

All of your previous Audit Trail sessions are displayed in the column to the left.
2. To see details, click the plus sign + next to the session name. The expanded Audit
Trail information apppears:

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 Creating an Audit Trail

3. To insert the Image Signature information into the Audit Trail, clicking the Image Sig-
nature button. A new line item appears in the Audit Trail:

The image signature contains only pixel data from the selected image.
4. Click the File Signature button to insert the File Signature information into the Audit
Trail.

The file signature contains all available data from the selected image, including head-
ers, metadata, and so forth.

Audit Trail Options

The Audit Trail is controlled by a set of options displayed on the Advanced tab of the
Options dialog.

Enable Audit Trail enables the application’s Audit Trail, which logs all the operations per-
formed by the software. The resulting file is saved automatically every five minutes as well
as at the end of the session. It includes all the sessions for the current day and has its own
digital signature used to guarantee its authenticity.
Enable Document Trail enables individual document trails, which collect operations done
by the software on each documents. When documents are saved, their history trail is saved
in a file with same name as the document’s but with the additional extension
“.audit.xml”, unless the following option is on:
Embed Document Trail enables embedding of document trails in document files when
supported; in this case there is no separate file.
Automatic File Signature inserts the file signature into the Audit Trail automatically.
Automatic Image Signature inserts the image signature into the Audit Trail automatically.

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Audit Trail Tools

The Audit Trail tools include controls to create new audit events or signatures, as well as a
way to export the audit trails to new files or MS Excel.

The Application Audit Trail opens the Audit Trails for this application.

The Document Audit Trails opens the audit trail associated with this image or doc-
ument.

Open Audit Trail loads a saved audit trail from a file.

Save Audit Trail saves your audit trail to a file.

Export to Excel sends your audit trail data to an Excel spreadsheet.

Image Signature inserts the image signature information into the audit trail.

File Signature inserts the image signature information into the audit trail.

Custom Audit allows you to insert a customized line item into the audit trail.

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 Very Large Images

Very Large Images

The Very Large Image (VLI) viewer is a new feature in Image-Pro designed to let you
view and analyze images that exceed 1 GB in size. The VLI viewer only works with the
64-bit version of the product. It is not supported in the 32-bit version. You should also
have a multi-core processor on your computer, as VLI processing can be very slow.
The VLI viewer supports these features:
l Status bar updates of the X/Y location and the intensity under the cursor
l A reduced context ribbon for VLI images
l Normal zoom, pan, and scroll.
l Local zoom
l Annotation and other simple overlays (like the active data overlay)
l Display group controls for BCG
l Image info and properties
l Some sub-set of measurements that do not use image data (simple manual meas-
urements, but no "caliper" or "snap to edge", no trace or magic wand), etc. Note that
some measurements that you might typically be able to get from the manual meas-
urement features will not be possible (like Density/Intensity).
l Annotations
l ROIs. VLIs support a single, non-rotatable, rectangular ROI. If ROIs are pasted into
the VLI, then they will be converted (if necessary) into single,non-rotatable rect-
angular ROIs, at the size of the bounding box of the source ROIs.
l Crop.
You should copy Very Large Image (VLI) sets to your local drive before trying to open
them.The processing and memory requirements prevent VLIs from opening over a network.
Other image processing features of Image-Pro are disabled in the VLI viewer.
Proper support for VLI images requires the following:
l The image is opened from a supported file format that supports the rest of the VLI
features, and

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l The image file contains multiple resolution images of the same subject matter,
from a whole-image thumbnail through other subsampled resolutions up to the nat-
ive resolution that was originally captured, and
l The image file supports tiled/random access to portions of the image.

Large Image Tools

When a VLI workspace is active, in place of the normal Image Tools context ribbon, a
Very Large Image Tools context ribbon will be available:

All of these controls are identical to the same controls in the Image ribbon, except for the
Crop command, which will behave in exactly the same way as the Extract button on the
on-image toolbar.
Other VLI tools include:

You are able to create a normal ROI on the image, and work with that part of the VLI.
The Extract button extracts a portion of the image to a new, normal image workspace.
The Resolution slider selects the sub-sampling resolution to use, or the size ratio.
The Emphasis switch allows you to select whether the toolbar should emphasis preserving
the size ratio (by auto-adjusting the size of the ROI), or the region of interest (by auto-
adjusting the resolution/size ratio).
The memory limit slider lets you set the percentage limit of the available image memory.
This could be set to 80-90% if only one copy of the extracted image is needed, or may
need to be 10-20% if multiple copies of the image are needed for processing, etc. This

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memory limit is used during the computations that the tool uses to auto-adjust either the
resolution or the ROI.
The memory usage bar will graphically display the percentage of the specified memory
limit that would actually be used when extracting an image with the current combination
of the image ROI and the selected resolution.
To work with VLIs, follow these steps:
1. Open the image from your hard drive. VLIs cannot be opened from a network drive to
due size and memory constraints. In this example, we are using the image called
breast cancer.tif:

Image-Prowill create a map from the VLI automatically:

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This map is a lower-resolution version of the VLI in a more manageable size. You can
use it to create tiled images for analysis using Image-Pro XYZ Navigator or another
application.
2. Use the Crop tool to extract a portion of the image for analysis.

From here, you can use the full range ofImage-Pro’s analysis features to analyze the
image.

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Image-Pro Experiments
When you open a VLI, Image-Pro also creates an Experiment document (*.mce). This file
can contains different types of content, in addition to images. A sample experiment file
description appears here:

The pyramid icon indicates the size and location of the original VLI.

The pentagon/ star icon indicates the size and location of normal images extracted
from a VLI.
These icons appear on the map and on the extracted image also.

Managing Mixed Content Experiments

Mixed content experiments are special Image-Pro documents that can be loaded from
.MCE files. These files link to other files that can contain images, image sets or even very
large images.
Mixed content experiment files are listed along with other file types in the standard “Open
File” dialog (see Different Types of Content earlier in this chapter).
Once loaded, a Mixed Content Experiment is represented by an experiment map that con-
tains one or several images combined together (the image maps). As shown above, the

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image contains icons that allow you to locate the various documents in the experiment. A
sample experiment image appears here:

Document sites in the experiment map are highlighted when the mouse hovers over them,
giving feedback to the user and displaying the document name(s).

A single click displays an overview of the selected document, and a double-click loads the
document directly.

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 Managing Mixed Content Experiments

The document overview contains a preview of the document, as well as information about
its content. Clicking on the preview or the load link will load the document in a new
workspace.

When multiple documents are located at the same position, the document overview allows
you to switch views from one to another.

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Working with Image Sets

Image-Pro and Image-Pro with 3D Module offer improved handling of image sets. In addi-
tion to opening and saving image sets, the improved support for multi-dimensional image
sets includes the ability to add regions of interest, annotations, crop, duplicate or extract
dimensions, and improved set navigation.
An image set is a collection of images taken together to form a time lapse sequence, area
scan, or a Z-stack, or any combination of these. Image sets are often complex groupings
that include more than one dimension, such as both time and Z/focus dimensions, as well
as channel specificity and more. This would mean that a single image member file of a
given image set could, for example, represent a single channel at a single distance/focus
and a single moment in time. The collection of images would therefore need to be organ-
ized and reconstituted to be viewable and analyzable in any meaningful way. is designed
to work with the metadata and naming conventions found in many sets created by com-
monly used image acquisition tools and to organize the files according to the their sig-
nificant dimensions (or let you organize them) so that they can be saved into a single
image set file (with the extension *.mcs).
Note that there are five possible dimensions that can be used in the organization of files in
a set:
l Channel: Index number for the color channel.
l Slice: Index number for the relative position in the Z stack or series of focal adjust-
ments.
l Site: Index number for the site. Used for organizing image data per area of interest in a
sample, for example.
l Time: Index number for the relative position in a time lapse sequence.
l XY Scan: Index number for the relative position in an XY scan (a series of images
that are taken across the surface of a sample).

Open Set from File

For the purposes of creating image sets from files, all elements of a given dimension are
identified initially by index numbers. For example, if there are three channels to the Chan-
nel dimension, they are identified by index numbers 1, 2, and 3.
To create an image set from individual image capture files,follow the instructions below:

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1. Click the File button.

2. Choose Open Set from File.

You will see the following dialog:

3. Select the folder containing your image source files.

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Use the Select Source Folder browser in the far-left panel navigate to the folder
where your image files are located. The set files should be in numerical order if pos-
sible.
4. Select the method for determining the set. There are three modes of operation avail-
able:
n Automatically detect file sequence. Select this option if the files in the sequence
have matching internal dimensions and naming conventions that make their loc-
ations along each dimension distinctly identifiable. In such a case, can recon-
stitute the entire set instantly and automatically. If you select this option, you
should:
a) Select a single file that is a member of the set you are trying to buld.
analyzes its name to tries to discern dimension information in the name. If
any dimension information is detected, it prompts you to confirm the
meaning the data in the name structure, as follows

b) Review the assigned dimensions. From the list boxes provided, select
the correct dimension information if guessed wrongly.
c) For sequences that include numeric and non-numeric dimension inform-
ation (such as when a channel name is included in the file name), use
“Check to add the active selection as a partial set” to “lock in” the detec-
ted sequence to part of a larger set.

n Split sequence into dimensions. Select this option if the files in the sequence have
naming conventions that mix location information for multiple dimensions into a
single sequence.For example: a 50-slice, 2-channel set that includes files named
“Image001.tif” through “Image100.tif”In such a case, can automatically collect
the relevant files into a table, and perhaps organize them according to one dimen-
sion, but it depends on you to specify the specify the allocation of the specified
sequence into the relevant dimensions. If you select this option, you should:
a) Select a single file that is a member of the set you are trying to buld.
analyzes its name to tries to locate sequence information in the name. If
any dimension information is detected, it prompts you to confirm the

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meaning of the data in the name structure, as follows:

b) Adjust the dimension allocation as appropriate using the list boxes

provided. Note that additional dimension drop-downs appear as needed.

n Manually add files to a dimension. When you select this option, you are promp-
ted to select a dimension and position within that dimensions to which you want
to add files, and then select the files to be added: Follow the steps below to manu-
ally organize your files:
a) In pull-down list box below the Add selected files to... button, select
the dimension you want to define.
b) In the spin box to its right of the selected dimension, specify the pos-
ition in the selected dimension to which you want to add files.
c) In the file list, select all files belonging to the specified position of the
selected dimension.
d) Click the Add selected files to... button.
e) Repeat steps a) through d) until all relevant dimensions and positions
are defined.
Choosing a new operation mode will change the controls available in the control
panel at the bottom of the center region of the Set Builder dialog.
5. Select a folder to add images to the File Table in the center panel. The file table in
the center will list the supported image files that are present in that folder, whether or
not they belong to a set.
The Location columns of the file table display the location index fields that have
been assigned to that file. If the file has not been assigned to any location within the
set, this column will be blank. Once the file has assigned to a location in the set, the

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Location column will be replaced with columns for each dimension that is currently
active in the set.

Each dimension is represented by an icon: Color Composite Channel, Location in the

Z –stack, and Time position. Each column header has a tool tip to indicate the dimen-
sion that the icon represents.
The dimensions of each set appear in a table on the right hand side of the dialog .
When you've selected a folder, the file table in the center will list the supported image
files that are present in that folder, whether or not they belong to a set. r in the panel
on the right.This table indicates the extents of the set as currently constructed. In the
cases of incomplete (jagged) sets, the extents in this table will represent the longest
added set thus far:

6. Add channel information for the set.

The channels table displays one row per channel. You can set the channel name, tint,
or set them both by assigning a dye to the channel, as shown in the illustration
below:

The channel name can be edited in-place. Use the Color drop-down arrow to see the
standard color picker. Click the “…” button in the Dye column to select select a dye
from the favorites list, or from all available dyes:

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 Open Set from File

n When Show all dyes is checked, all the dyes in the Dye Library will be included
in the list. If this option is not checked, you will see only the dyes in the Favor-
ites list. If the current channel name matches a dye in the list, that dye will be
selected in the drop-down list.
n Click Apply to set both the Channel Name and the Color for the selected dye. In
addition, the dye will be associated with the channel and will later be associated
with the image set channel when the set is imported.
7. Set the Z-spacing.
The Z- spacing panel on the dialog allows you to set the spacing between Z planes if
the selected set has Z stacks:

The selected spacing will be applied to both the set’s metadata and to the frames of
the images
8. Open the set.
The Open Set button will be disabled until you have selected at least one file to
define the set.
Here is an example of an image set open in the workspace:

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Image Set Controls

Once you have an image set open in the workspace, you will see an additional set of con-
trols at the bottom of the viewer.

C indicates which channel is being examined in the viewer.

T indicates the time that the image was acquired,
Z indicates the position of the image in the Z-stack.

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 Image Set Controls

A limited context menu is available for image sets in composite view:

The Zoom Scale Bar allows you to adjust the size of the image by moving the slider to
the right or left.

The Undo and Redo buttons let you undo recent actions and reverse the effects of the
Undo.

The Histogram button opens the image histogram.

The Image Information button displays information about the image in a table below
the image.

Local Zoom opens or closes the Local Zoom feature.

The Draggingtool allows you to move the Pan/Scroll position by clicking and drag-
ging on the image.

The Zoom tool allows you to zoom in or out on the image with mouse clicks or by
drawing a box around the area to be zoomed. Only one tool may be active at any time, so
starting one of these tools will stop any currently active one.

The Toggle Nudge polygon/polyline editing mode button is enabled when the selec-
tion tool is running. Nudge editing mode becomes active only when a polygon or polyline
is the selected object being edited. Pressing the <N> key has the same effect.

The Toggle Extended Vertex polygon/polyline editing mode button is enabled when
the selection tool is running. Extended Vertex editing mode becomes active only when a
polygon or polyline is the selected object being edited. Pressing the <E> key has the
same effect.

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Clicking the Convert button converts the selected graphic object(s) into poly form if
possible.

The Toggle limiting graphic object button limits creation and editing to image
bounds on the active overlay. By default, graphic object creation and editing is limited by
the bounds of the underlying image on all overlays except for the Annotation overlay,
where limiting is disabled by default.

Extract dimension will extract a new image from the set using images along the cur-
rent dimension and at the current location along all other set dimensions.
The Set Navigator controls are similar for the controls for an image sequence.

The toolbar has the following buttons:

Go to first frame

Go back one frame

Play sequence backward

Play sequence forward

Go forward one frame

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 Image Set Controls

Go to last frame
Select indicates the portion of the set to process. The options are:

Color composite displays the set in color composite view.

Playback options offer the following choices:

The Rate control defines the playback speed in frames per second.
Loop combo-box determines the action taken during playback when the end or
beginning of the sequence frame range is passed. Choose one of the following
options:

Play all Frames will play the entire set.

Extract dimension will extract a new image from the set using images along the cur-
rent dimension and at the current location along all other set dimensions.
Most of the other features of Image-Pro will work with image sets, such as ROIs, annota-
tions, and filters. Image sets may be moved, measured, and changed using the features
described in Chapter 12, Using the 3D Features.

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Chapter 6 : Using Regions of Interest

Using Regions of Interest

You can define one or more Regions of Interest(ROIs) in a variety of shapes, and you can
control the positioning, size, appearance and display status of ROIs. This chapter provides
step-by-step instructions for defining and working with ROIs to isolate areas of interest in
your images.

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The Select Tab

The Select tab’s ribbon contains those functions that allow you to mark up an image with
regions of interest or annotations, and share it with another person or work group.

You can find the ROI feature on the Select tab’s ribbon:

If you are working with Image-Pro with 3D Module, this tab is called 2D Selection.

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 What is an ROI?

What is an ROI?
An ROI is a Regionof Interest that is isolated from the rest of the image. Certain
Image-Pro commands can be constrained by an ROI. Once you have defined an ROI, the
command applies to only the pixels within the ROI.
Examples of commands that are constrained by an ROI are: Save As, Cut, Copy, Duplicate,
Rotate, and Resize.
For example, in Figure 6-1 below, a square-shaped ROI has been drawn around a particular
object of interest in the image. When the Duplicate command is applied to the image, the
ROI portion of the image is copied to a new image, as shown in Figure 6-2.

You can also apply a filter to an ROI. Figure 6-3 show an image with an ROI drawn on it,
and a Sharpen filter applied to the area defined by the ROI:

Figure 6-3
More information about applying filters appears in Chapter 5.

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Defining an ROI
Before defining an ROI, be sure the image you want to work with is opened in the Image-
Pro workspace. To define an ROI in the active image:
1. Go to the ROI group on the Select tab’s ribbon:

2. Click on the ROI tool you want to use. Clicking this button starts the drawing of this
type of ROI. The different ROI tools and types are described below.

Selection Tool: Use this arrow to select an ROI on the image.

Click the drop-down arrow and use the Select All tool to select
more than one ROI.

Rectangle ROI: This tool allows you to define a rectangular or

square ROI. Click and drag the mouse from one corner of the
Region of Interest to the opposite corner. To force a perfect
square, hold down the <Shift> key as you click and drag the
mouse. Click the drop-down arrow to see additional ROI shapes.

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Circle or Ellipse: This tool allows you to

define a circular or elliptical ROI in the
image. To force a perfect circle, hold down
the <Shift> key as you click and drag the
mouse.

Rectangle by Numbers: This tool allows you to define a rect-

angular ROI by entering the left, right, top, and bottom locations.

Polygon: This tool allows you define a

polygon-shaped or freeform ROI in the
image.
To define a freeform ROI
Use this technique to create smooth edges.
Hold the left mouse down while you draw
with the cursor to create the desired free-
form shape. Double-click to complete the
shape.
To define a polygon-shaped ROI
Use this technique to create polygons that
are made up of straight line segments.
Click the left mouse button at each vertex
(including the beginning point) of the
polygon. Double-click to complete the
polygon.

Auto-Trace. Use this tool to trace the outline of an irregular object in the
image automatically. Place your cursor on the object and click two

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points. Image-Pro automatically traces the object.

Display Options
Use this button to see the Display Options panel.

Delete Selected/Delete All:

Use the buttons to remove one or more of the ROIs from your image.

Multiple ROIs: Click this button to place mutiple ROIs on your image.
This option is always on by default.

Options
Use this button to see some additional ROI options:

Invert ROI: Click this button to invert the ROI; for example, a black
ROI on a white background.
XOR Pixels: Click this button to include or exclude pixels in over-
lapping ROIs. This option is always on by default.

.Order: Use these buttons to arrange your ROIs from back to front, or
front to back.

Features Manager
Image-Pro allows you to create collections of predefined items called Feature Collections.
Click the Add button to create a new Feature Collection from your active (live) annota-
tions, ROIs, Measurements, Line Profiles, or Grids.

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Each feature definition included in a collection can be applied repeatedly to a single

image or can be applied to different images. Feature collections can also be saved so that
they are available across sessions, and so that you can manage your collections in different
files. Using the Features Manager, you can:
n Delete feature definitions from the current collection
n Apply any feature to the current image
n Save the current collection to a file
n Load a previously saved collection.

Features Manager: Clicking this button opens the Features Manager

described here:

In addition to the instructions below, you can read more about the Features Manager later
in this manual. "Features Manager" on page 441

To Add an ROI to the Current Collection

ROI collections are created by adding defined ROIs (that is, ROIs defined in the active
image) to the list of ‘remembered’ ROI shapes through the Features Manager dialog
box. You can add a single ROI definition, or you can add a group of ROI definitions
under a single name.

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1. Define the ROI shape you want to add to the list in the active image. If you want
to add a group of ROI shapes under a single name, define all ROI shapes belong-
ing to the group.
In the following example, a single polygon-shaped ROI has been defined.

Figure 6-12

When a single ROI is defined, a default name for the ROI shape appears in the
ROI Name text box of the Features Manager dialog box.

If you define more than one ROI in the image, the features manager gives the
entire group the default name, “Multiple.” Note that if multiple ROIs are defined
in the image, they will be added to the list as a group regardless of which ROI
among them is currently selected.
2. Click on the Add button.
The collection definition is added to the list box. Listed names represent the mem-
bers of the current collection.

To Apply an ROI to the Active Image

1. In the ROI collection list box on the Features Manager dialog box, click on the
name of the shape you want to place in the currently active image.
The selected name is highlighted in the list. The shape named “Polygone” is
selected in the above example.

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2. Click on the Add Feature to Image button.

An ROI in the selected shape is added to the currently active image.

To Save an ROI Collection

1. Click on the Save button of the Features Manager dialog box.

The Save ROI List dialog box appears.

2. In the File Name text box, type over the existing text with the name you want to
give the ROI collection.
For example, name it “My Collection.”
3. Click on the Save button.
The current collection is saved with the specified name, and the Features Man-
ager dialog box is redisplayed.

To Load an ROI Collection

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1. Click on the Open button of the Manage page of the Features Manager dialog
box.

The Load ROI List dialog box appears.

2. Click on the ‘*.ROI’ file you want to open.

For example, click on “My Collection.ROI”.
3. Click on the Open button.
The collection list box of the Features Manager dialog box is populated with the
shape names contained in the selected file.

Placing Multiple ROIs on an Image

If you want to define more than one ROI in a single image, you can use the Allow Mul-
tiple ROIs tool.

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 Placing Multiple ROIs on an Image

l Click on the Allow Multiple ROIs [ ] button in the ROI group so that it is
highlighted. This is the default selection.
When this tool is selected, you can define multiple ROIs in a single image.

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Arranging ROIs
After you have defined an ROI, you can move it, resize it, and delete it.

Moving an ROI
To move an ROI:

1. Click the Selection tool [ ] from the ROI group.

Click on the ROI you want to move. The ROI is selected.
2. Position the cursor over the selected ROI.

If the cursor is positioned correctly, the Dragging tool [ ] appears.

3. Click and drag the ROI to the desired location.

Resizing an ROI
ROIs can be resized by moving their control points. To resize an ROI:

1. Click the Selection tool [ ] from the ROI group.

2. Click on the ROI you want to resize.
The selected ROI is selected.
3. Position the cursor over the control point that you want to move.

If the cursor is positioned correctly, the Resize tool [ or ] appears over the con-
trol point.
4. Click and drag the control point to the desired location.

Using XOR Pixels

Use this feature to examine pixels in overlapping ROIs on your image. To apply a filter
using XOR pixels, follow these steps:
1. Draw two or more ROIs on your image, as shown here:

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Figure 6-4
2. Go to the Adjust tab and select the Filters group. Click the drop-down arrow.

3. Choose a filter from the filters list:

4. Your filter will be applied to the area between the flags on the image.

Figure 6-5

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Rotating an ROI

You can rotate a selected ROI without

rotating the image. The Rotate tools are shown here:

1. Click the Selection tool [ ] from the ROI group.

2. Click on the ROI you want to resize.
The selected ROI is selected.
3. Position the cursor over the center point in the ROI that you want to rotate. You will
see the rotation cursor.
4. Click and drag to rotate the ROI.

Improvements to the ROI controls allow you to rotate the ROI with or without trimming
and place it a new image. When the Trim box is checked, the rotated size of the ROI is
the same size as the original ROI bounds, which usually requires trimming the rotated
ROI. The icons displayed are:

n Rotate with trim into a new image

n Rotate without trim into a new image.

An example appears here:
The below shows a translucent preview of a -20 degree rotation with the Trim option
unchecked. The green rectangle is the ROI to be rotated. The red rectangle shows the
rotated ROI. The yellow rectangle shows the bounds of the new, rotated result image.

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 Rotating an ROI

Figure 6-6
The rotated result is shown in the following image. Note that no part of the rotated ROI
has been trimmed off, but as a consequence the new image is larger than the source ROI.
Also note that areas outside the source ROI are shown as black in the new image.

Figure 6-7

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The next example shows a translucent preview of the same -20 degree rotation with the
Trim option checked. The green ROI rectangle is not visible here, because the yellow rect-
angle showing the bounds of the new image lies exactly on top of it (this would not be
case if the ROI were a non-rectangle, as in the next example). The red, cut-off rectangle
shows the rotated ROI with its trim.

Figure 6-8
The rotated result is in the following image. Note that part of the rotated ROI has been
trimmed off in order to keep the new image the same size as the bounds of the source ROI.

Figure 6-9

Deleting an ROI
To delete an ROI:

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 Deleting an ROI

1. Click the Selection tool [ ] from the ROI group.

2. Click on the ROI you want to delete.

3. Click the Delete [ ] button or Press the <Delete> key.

Note that you can also use the delete button in the image context menu (click the
right mouse button on the image when an ROI is selected) to delete the selected ROI.

TIP: You can use the Delete All tool in the ROI group to instantly remove all ROIs
defined in the active image.

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Setting the Appearance of ROIs

These instructions explain how to change the appearance of an existing ROI and how to
set the default appearance attributes for the different ROI types.

Changing the Appearance of an ROI

Sometimes you might want to change the border or fill color or size of your ROI to make
it easier to see on your image. To change the appearance of an ROI:

1. Click the Selection tool [ ] from the ROI group.

2. Click on the ROI that you want to change.
The ROI is highlighted.

3. Click the Display Options button in the ROI group.

You will see the Display Options dialog box in the panel along the right side of the
workspace.

4. Use the controls available through this dialog box to change the appearance of the
ROI.

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Drawing Properties

Some common controls available for changing the appearance of graphical annotations
are described below.
n Border: Allows you to change the visibility, color, width, and line style of a bor-
der around your annotation.
n Fill: Allows you to choose the fill color for your annotation, including opacity
and/or transparency.
n Options: These options allow you to change the size of the annotation when the
size of the images changes, adjust the opacity/transparency of the annotation, and
layer annotations on the image. If you are working with an image sequence, you
can specify the first and last frrames where the annotation will appear.
n Color: Click on the Color button to change the color of the bounding box border
or fill, or to change the color of the text.
n Zoom with Image: Check this box to make the annotation larger or smaller as the
image is resized.

Label Properties

Some common controls available for changing the appearance of label annotations are
described below.

n Text: Click on the Text tab to change the text of the annotation.

n Font : Allows you to change the font to be used for rendering text.

n Size : Allows you to change the font size to be used for rendering text.

n : Allows you to make the text shadowed, bold, italics, under-

lined, or shadowed.
5. When you have made the desired changes, click the ROI Display Options panel.
The changes are applied to the selected ROI.

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Setting the Default Appearance for ROIs

The default appearance attributes for ROIs are set by designating the appearance attributes
of an existing ROI as the default for future ROIs of that type. Note that defaults exist inde-
pendently for each type of ROI you can draw: rectangle, rounded rectangle, ellipse, poly-
gon, point, and text. To set the default appearance for an ROI type:

1. Click the Selection tool [ ] from the ROI group.

2. In the active image, click on an ROI of the type for which you want to set appearance
defaults.
For example, if you want to set the default appearance for ellipse-type ROIs, click on
an ellipse-type ROI in the active image. If one doesn’t exist, draw one.

3. Click the Display Options button in the ROI group.

You will see the Display Options panel along the right side of the workspace.

4. Click Save as Default

The default appearance values are set. Future ROIs of that type will appear as the
selected ROI.

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 Using the Magic Wand

Using the Magic Wand

The Magic Wand tool allows you to select regions in an image based on the color of
pixels in the image. You can use it to select irregular regions and convert them into ROIs,
Measurements, or Annotations. The Magic Wand tool appears on the Select tab’s ribbon:

The Magic Wand group includes the following features:

l Options

New Selection. All previous selections will be replaced with a new selection. Hold
down the <Alt> key to temporarily activate this option.

Add Selections. A new selection will be added or merged with any previous selec-
tions. Hold down the <Ctrl> key to temporarily activate this option.

Subtract Selection. A new selection will be subtracted from any previous selec-
tions. Hold down the <Shift> key to temporarily activate this option.
l Magic Wand button

Interpretation (Mono, RGB, HSL or HSI). Converts the image into a different inter-
pretation before thresholding (not available for mono images).
Mono Ranges. Indicates the number of gray levels used for thresholding (combination
of gray ranges depends from type of active image and selected interpretation).
Filtering: Indicates the intensity of any filter to be applied to the selected outline.

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Smoothing. Indicates the degree of post-filtering of the outline (0 – no filtering, 9 –

high degree smoothing).
Fill Holes: Check this box to automatically fill any holes in the outline and include
the hole area in your measurements.
Outline color. Allows you to change the color of the object’s outline using the color
selector.
l Validate button

as ROI. Converts and validates the selections as ROIs.

as Measurement: Converts and validates the selections as measurements.
as Annotations: Converts and validates the selections as annotations.
To use the magic wand, follow these steps:
1. Open an image with irregular areas in the workspace:

2. Select the Magic Wand tool.

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 Using the Magic Wand

3. Place the wand cursor inside the feature containing the area(s) that you want to trace,
and click the left mouse button once.
The magic wand will automatically trace the outline of the object(s) based on the
color similarities or difference of intensity ranges between the pixel under your cursor,
plus or minus a specified tolerance interval.

Figure 6-10
To outline a different region, place the cursor on that region and click with the left
mouse button. To outline more than one region, hold the <Ctrl> key down while
clicking on the region you want to outline.
4. To save the outline areas as irregular ROIs, click Validate as ROI.
The outline color changes, as shown here:

Figue 6-11
The Regions of Interest group becomes active, and you can now use all the ROI fea-
tures to modify or save this ROI.

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Polygon Nudge Tool

The Polygon Nudge tool allows you to adjust the size and shape of irregular ROIs and
measurements.
The nudging technique for reshaping polygons and polylines allows you to push control
points around, rather than clicking and dragging them. This technique makes it easy to con-
tour curves in curvy polyforms by 'nudging' the control points into place.
To use nudging mode:
1. Enable nudging mode by right-clicking the mouse in the image window, and clicking
on the [ ] tool in the popup context menu.

Initially the nudge tool will be a dashed circle and has no effect on the selected
object.
2. Use the mouse wheel to adjust the size of the nudge tool
The size of the nudge tool determines how many controls points it can nudge at one
time.
3. Press the mouse button and move the nudge tool into the control points you want to
nudge.
Notice that, when you press the mouse button, the circle becomes solid and shows an
arrow tracking the direction of mouse movement, and the nudge follows the direction
of the mouse movement. The nudge tool is designed to operate only at slow speeds.
So by moving quickly, you can skip the tool from one side of a polyline to the other
or from the exterior to interior of a polygon without needing to let up the mouse but-
ton and without disturbing the boundary as it is quickly crossed. This may take some
practice to master, but it is often necessary to nudge from both sides of a poly line sec-
tion to get a boundary placed correctly, so using this technique can speed things up.
Holding down the <Ctrl> key while editing a polygon or polyline temporarily leaves
nudge editing mode to allow you to insert (<Ctrl> double-click) or move (<Ctrl>
drag) a single control point.

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4. To turn nudge mode off, press the space bar.

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Using Overlays
The Overlay feature allows you to apply Live Overlay features to the active image. Types
of live overlays include grids, measurements, and ROIs. The Overlay feature appears in the
Compare group on the Capture tab’s ribbon:

When click the drop-down arrow, you will see the Overlay dialog box:

The Crosshair feature is described in Chapter 3 of this manual.

Live Overlay: Click this button to apply the overlay to the active image. This but-
ton remains disabled if there is no active image or Live Overlay features are not selected,

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Using Annotations
Annotations are used to add text or drawings to your images. The annotations are in a
graphics overlay that is displayed over the image without modifying the image data itself.
This chapter provides instructions for creating text and graphical annotations, arranging
them, and saving them with your images.

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The Select Tab

The Select tab’s ribbon contains those functions that allow you to annote an image and
share it with another person or work group. The functions in this chapter can be found in
the Select ribbon’s Annotate group.

If you are working with Image-Pro with 3D Module, this tab is called 2D Selection.

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Creating an Annotation
Using the tools on the Annotate group, you can draw an array of graphical and textual
annotations. This section provides instructions for drawing various shapes and for placing
text overlays in images. The Annotate group looks like this:

Creating a Graphical Annotation

There are a number of options for annotating your images with graphical overlays. You
can define points, lines, rectangles, rounded rectangles, ellipses, polygons, and polylines.

To define a graphical annotation:

1. Click on the Annotate group in the Select tab’s ribbon.
The Annotate group contains these tools:

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2. In the toolbar, click on the annotation tool you want to use, and draw the annotation
in the image as described for that tool below.

Select/ Select All

Use this tool to select an annotation object. Click the drop-down arrow to see
the Select All tool to select a group of objects.

Text Box

Use this tool to add a text box annotation. Click the drop-down arrow to add
a text call-out annotation:

You can add a callout line using the callout tool.

Rectangle or Rounded Rectangle

Click the left mouse button and hold it down to drag the rectangle to your
desired size. Use the drop-down arrow to see the rounded rectangle, circle, and
polygon tools.

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Circle or Ellipse
Position the crosshair cursor anywhere in the image. Use the drop-down arrow
to see the other tools.

Click and drag the mouse from the insertion point to the desired destination.

Line or Polyline: This tool allows you draw poly-

vertices lines and polygons, or freehand lines and
shapes.

Note: Whether this tool draws a polygon or a poly-

line depends on the current setting for the Connect
Start/End option. To change the status of this
option, simply right-click the mouse over the object
after it is drawn, and select Annotation|Object
Properties…. The Connect Start/End option
appears on the Polygon tab of the Graphic Object
Properties dialog box.
To draw freeform lines and shapes

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Use this technique to create lines and shapes that

have smooth edges. Hold the left mouse down
while you draw with the cursor to create the
desired freeform shape. Double-click to complete
the shape.
To draw polyvertices lines and polygons
Use this technique to create lines and shapes that
are made up of straight line segments. Click the
left mouse button at each vertex (including the
beginning point) of the polygon. Double-click to
complete the polygon.

Auto-Trace. Use this tool to trace the outline of an irregular object in the
image automatically. Place your cursor on the object and click two points.
Image-Pro automatically traces the object.
Point Position the crosshair cursor in the image where you want to place the
point marker. Click the mouse to place the point. The drop-down menu for the
point tool displays a wide variety of point and arrow styles.

Delete Selected/Delete All

Use the buttons to remove one or more of the
annotations from your image.
Group/Ungroup
Use these buttons to collect your annotations into a
group or to ungroup them.

Order
Use these buttons to order your annotations front to
back or back to front
Cut, Copy, Paste
Use these tools to cut, copy, or paste annotations.

Properties

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Use this button to see the Display Options information.

Burn In
Click this button to make your annotation a permanent part of the image.

Annotation Shapes
The drop-down menu for the point tool displays a wide variety of point and arrow styles:

You can use your mouse wheel to adjust the size of your annotations.

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Creating a Text Annotation

To create a text annotation:
1. Click on Annotate ….
The Annotate group is displayed.

2. In the toolbar, click on the Text tool [ ].

3. Click and drag the mouse in the image to draw a bounding box for the text.
When you release the mouse, the Text tab of the Display Options dialog box is dis-
played. An examples appears on the next page.

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4. Type the desired text in the Text tab’s textbox.

5. Click OK.
The specified text appears as an overlay in the image. For instructions on changing the
font, color, or bounding box attributes of the text annotation, please refer to “Setting the
Appearance of Annotations” later in this chapter.

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Inserting a Bitmap Annotation

To insert a bitmap annotation:
1. Open the desired bitmap image in the Image-Pro workspace, that is, open the bitmap
image you want to use as an annotation. (See “Opening an Image” in Chapter 4,
“Working with Images.”)
The image appears in the application workspace.
2. Choose Paste Bitmap from the drop-down menu in the Cut, Copy, and Paste group.

3. Right-click the mouse in the image to which you want the bitmap added as an annota-
tion.

4. Click the Paste button [ ].

The bitmap is added as an annotation to the image. For instructions on moving the bitmap
annotation, please refer to “Moving an Annotation” later in this chapter.

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 Arranging Annotations

Arranging Annotations
After you have created a set of annotations, you can move them, resize them, layer them,
and delete them.

Moving an Annotation
To move an annotation:

1. Click Select [ ] from the Annotate group.

2. Click on the annotation you want to move.
The annotation is selected.
3. Position the cursor over the selected annotation.

If the cursor is positioned correctly, the Dragging tool [ ] appears.

4. Click and drag the annotation to the desired location.

TIP: You can also move the annotation by pressing the shift key along with the
arrow keys (up/down, right/left). For example, press the <Shift> and <Up Arrow>
keys and notice the annotation moving upwards.

Rotating an Annotation
Annotations can be rotated by using the Select tool and the <Control> key.

1. Click Select [ ] from the Annotate group.

2. Click on the Annotation you want to rotate.
The Annotation is highlighted.
3. While the Annotation is selected, move your cursor to the center point of the Annota-
tion. The rotate cursor (a double-ended arrow) will appear, enabling you to rotate the
Annotation either clockwise or counter-clockwise. Holding down the <Shift> key
while rotating an Annotation will move the Annotation in 15-degree increments.
You can also rotate an Annotation while creating or editing it by holding down the
<Control> key as you move the Annotation.

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Resizing/Reshaping an Annotation
Annotations can be resized and reshaped by moving their control points. To resize or
reshape an annotation:

1. Click Select [ ] from the Annotate group.

2. Click on the Annotation you want to resize or reshape.
The Annotation is highlighted.
3. Position the cursor over the control point that you want to move.

If the cursor is positioned correctly, the Resize tool [ or ] appears over the con-
trol point.
4. Click and drag the control point to the desired location.

Grouping Annotations
The Annotation feature allows you to group annotations in the image. Annotations that
are grouped are fixed in positioning relative to each other and they are otherwise treated as
one object. To group annotations:

1. Click Select [ ] in the Annotate group.

2. While holding the <Ctrl> key down on the keyboard, click on the annotations you
want to include in the group.
For example, annotations #2 and #3 have been selected below.

3. Click on the Group button [ ] on the Annotate panel.

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The selected annotations are grouped.

Ordering Annotations
The Annotation feature allows you to control the order in which annotations appear. To
change the order in which an annotation appears:

1. Click Select [ ] in the Annotate group.

2. Click on the annotation you want to relocate in the order.
For example, annotation #3 has been selected below.

3. Click the Order back to front button in the Annotate group.

The annotations are arranged in the order specified.

Deleting an Annotation
To delete an annotation:

1. Click Select [ ] from the Annotate group.

2. Click on the annotation that you want to delete.
The annotation is highlighted.

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3. Click Delete [ ] from the Annotate group, or press the <Delete> key.

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 Setting the Appearance of Annotations

Setting the Appearance of Annotations

These instructions explain how to change the appearance of an existing annotation and
how to set the default appearance attributes for the different annotation types.

Changing the Appearance of an Annotation

To change the appearance of an annotation:

1. Click Select [ ] from the Annotate group.

2. Click on the annotation that you want to change.
The annotation is highlighted.

3. Click the Options button in the Annotate group.

You will see the Display Options dialog box in the panel along the right side of the
workspace.

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4. Use the controls available through the Display Options dialog box to change the
appearance of the annotation.

Drawing Properties

Some common controls available for changing the appearance of graphical annotations
are described below.

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NOTE: For complete descriptions of all available controls, refer to the “Graphic
Object Properties (Dialog Box)” topic of the Image-Pro Application Help system.
n Border: Allows you to change the visibility, color, width, and line style of a bor-
der around your annotation.
n Fill: Allows you to choose the fill color for your annotation, including opacity
and/or transparency.
n Label position: If you are including a label (different from the text box) in your
annotation, you can adjust the position of the label here.
n Callout line: Allows you to show or hide the line attached to a callout box and to
adjust the line thickness and end style.

Label Properties

Some common controls available for changing the appearance of label annotations are
described below.
n Text: Click on the Text tab to change the text of the annotation.

n : Allows you to change the font to be used for rendering text.

n : Allows you to change the font size to be used for rendering text.

n : Allows you to make the text shadowed, bold, italics, under-

lined or in a different color.
n Autosize: Allows you to have the box surrounding the text automatically resized to
fit the text.
n Fill: Controls whether the box surrounding the text appears as opaque or
transparent. This attribute does not apply to the text.
n Color: Click on the Color button to change the color of the bounding box border
or fill, or to change the color of the text.
n Zoom with Image: Check this box to make the annotate larger or smaller as the
image is resized.

Additional Options

Additional display options have been added to Image-Pro:

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n Zoom with image automatically adjusts the size of your annotation as the size of
the image changes.
n Opacity adjusts the transparency of a line or shape as you move the slider.
n Angle lets you specify angle of display for you annotation.
n Layer lets you create overlay layers on your annotation. This can be very useful
when displaying a 3D or Z-stack image.
n Start Frame/End Frame lets you specify on which frames of a sequence the
annotation will appear.
5. The changes are applied to the selected annotation.

Setting the Default Appearance for Annotations

The default appearance attributes for annotations are set by designating the appearance
attributes of an existing annotation as the default for future annotations of that type. Note
that defaults exist independently for each type of annotation you can draw: rectangle, roun-
ded rectangle, ellipse, polygon, point, and text. To set the default appearance for an
annotation type:

1. Click Select [ ] from the Annotate group.

2. In the active image, click on an annotation of the type for which you want to set
appearance defaults.
For example, if you want to set the default appearance for ellipse-type annotations,
click on an ellipse-type annotation in the active image. If one doesn’t exist, draw one.

3. Click the Options button in the Annotate group.

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 Saving Annotations

You will see the Display Options dialog box along the right side of the workspace.

4. Click Save as Default.

The default appearance values are set. Future annotations of that type will appear as
the selected annotation.

Saving Annotations
When annotations are originally placed on an image, they exist as graphical overlays and
are not part of the image. If you want them to become part of the image, however, you can
save them with the image.
1. Click Save from the File menu:

2. Answer Yes when the question, “Do you want to save the changes with [image
name]” appears.
The annotations will be saved with the image.

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Note: Annotations can be saved only with TIFF format images.

Note Regarding Zooming Annotations:. Zoom with image is a per-view overlay display
feature, but burn-in affects all views equally (since it changes the underlying image data).
Thus, burn-in is always done at 100% zoom. This will make those graphic objects with
"Zoom with image" off (such as labels) look larger (if the view is zoomed in) or smaller
(if the view iszoomed out) after they are burned in.

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Chapter 8 : Measuring, Counting, and Tracking

Making Measurements
This chapter describes how to measure, count, and track features in images.

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Overview of the Measurement Feature

Image-Pro provides tools for measuring features in images. For example, suppose you
want to know the distance between two points in an image. Using the Measurement
tools, you can simply draw a line between the two points and acquire an instant distance
measurement. All of these tools are found on the Measure ribbon.

Measurement operations in Image-Pro are performed in terms of image pixel positions. For
example, the length of a line feature is determined by the number of pixels along the line.
Note: If you are running Image-Pro with 3D Module, some of the tab names may appear
as 2D Count/Size or 2D Measure.

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Data Measurements
Image-Pro offers you a variety of measurement types to analyze your images. These appear
in the Measurements group on the Count/Size ribbon as well as on the Measure ribbon:

To select your measurements, follow the steps below.

1. Click the Types drop-down arrow. You will see the Measurements dialog:

2. Using the Add or Add All buttons, select the measurements you want to make.
The Available measurements list contains the measurements that can be grouped by
Measurement type (All Types, Regions, Lines, Points, Best-Fit, Angles) and Meas-
urement Category:
l All Categories
l Basic
l Housekeeping
l Luminance
l Density - Optical density related measurement
l Histogram - A histogram or pixel-counting-based measurement
l Size - Size related measurement

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l Position - Position related measurement

l Shape - Feature shape related measurement
l Reference - Measurement relating to a reference feature
l Nesting - Nesting relationship measurement
l Marker - Some marker or landmark measurement
l Holes - Measurement ignores holes
l Texture - A texture-related measurement
l Classified - A classification-related measurement
l Color - A color related measurement
l Manual Measure - Available as one of the Manual Measurements
l Polyline - The measurement is only relevant for lines with more than one segment
l User Defined - The measurement is a user-defined one
l Angle Result - The measurement results are angles (have units of Degrees).
l Uncalibrated Result - The measurement results do not use the Spatial Calibration
or Intensity Calibration (length units are Pixels, area units are Pixels squared and
intensity units are raw pixel luminance values).
Tooltips display additional information about each measurement, as in the example
shown here:

The Selected Measurements list contains the measurements that will be reported in
the data windows (Data Table, Histogram and ObjectWindow). If the checkbox in
the Filter column is active, the measurement will be used as filter and only the
objects with parameters within the defined limits will be counted. Additionally the fil-
ter limits can be set in the Histogram window, in the Measurement filter pane.
Note that the measurement filters are applied only when the Ranges button is
checked:

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Edit Ranges
Adjusting filter ranges interactively can be done using the Edit Range button:

Min and Max filter values can be adjusted by dragging the corresponding markers on
the histogram or editing their numerical values. The mask of objects in range is shown
on the active image. The number of objects in range/total objects and percentage are
shown below the histogram.

Measurement Options

The Options button shows the measurements appearance options panel.

The first section contains the Segmentation options:

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Connectivity, either 8-connected or 4-connected, defines the number of pixel neigh-

bors segmenting the objects.
CleanBorders specifies if the objects touching the selected borders will be eliminated
from the result.

Fill Holes option indicates if the holes inside objects should be included into the
object area.
Smoothing defines the degree of smoothing of used for the object outlines. The default
is zero when no smoothing is applied.
Segmentation bias automatically adjusts the calculated threshold values by the given
bias value. More about segmentation bias appears later in this chapter.
Grow defines the size increment (in pixels) of segmented objects that is applied after a
Count operation using dilation or erosion of the segmented regions. More about grow-
ing or shrinking appears later in this chapter.
Auto Threshold allows you to define the method that is used to segment objects in
Auto Bright/Dark segmentation modes. More about Auto-Threshold appears later in
this chapter.

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Load and Save buttons load and save threshold segmentation options. These
buttons can be used to save and then restore counting environment in a new session.
Record enables you to save your segmentation as a sequence or movie. Reset changes
your settings back to their default state.

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Manual Measurement Tools

This section describes how to use the measurement tools and view your measurement data.
These tools appear on the Measure ribbon:

Selection Tools
These tools allow you to select, hide, or show objects and measurements:

Clicking the Select Object button activates the selection tool. You can select measurement
objects on the image and edit them. Additionally, when one or more objects are selected, a
semi-transparent floating bar will appear on the image that allows you to perform some
operations more conveniently (delete, hide, show, create relative measurements, calibrate).

The Select All tool lets you select all the measurements at once:

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The Show button and its menu allow you to toggle hide or show the overlay and selected
features on the image display. It also allows you to unlock the counted objects.

All objects created by a Count operation are locked by default and cannot be moved or
adjusted. Clicking the Unlock objects button will unlock counted features and enable you
to move or edit some of the objects.

Direct Measurements
This panel contains a “toolbox” of measuring instruments. These tools should be sufficient
to take most measurements.

Manual Tag

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Clicking this button activates the manual tag tool

Line
Activates the line tool, where you can draw a line by clicking and
dragging. A activating any of the measurements from the drop-down
makes it the default for the main button. Clicking the drop-down
arrow displays additional line tools:

Point to point
Activates point to point tool. A line feature will be created after two
clicks.
Polyline
Activates the polygonal line tool. As you click, new points will be
added to the line. (You can stop adding points by double-click and
by pressing the Enter button; pressing the Esc button cancels the line
creation.) There is also an option to auto-trace the polyline.

Auto-trace Polyline activates the auto-trace polyline.

Pitch with line

Activates pitch with line tool. First two points define the reference
line and every next point adds a perpendicular line from the point to
the reference line. (You can stop adding points by double-click and
by hitting the <Enter> button). The drop-down menu contains the

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Show base line option, which turns the base line (first 2 points)
of the pitch group on or off.

Circleor Ellipse
Activates the Circle tool, where you can create a circle by clicking
and dragging a rectangular area with the inscribed circle. Clicking the
drop-down arrow displays the Ellipse and other circle tools.

Ellipse
Activates the Ellipse tool, where you can create an ellipse by clicking
and dragging a rectangular area with the inscribed ellipse. There are
the following options for creating circles:

Radial drawing– center and radius (only for circles) or

Box drawing - box diagonal points (for circles and ellipses).

Find circle – activates Snap to Circle mode (see Snap to Image).

Polygon
Activates the Polygon tool, where you can create an n-sided polygon
by clicking n times. Clicking the drop-down arrow displays the Rect-

angle and Auto-Trace Rectangle tools.

Angle (joined)
Activates angle tool, where you can create an angle measurements
adding 3 points to the image. The angle range is defined in the drop-
down menu :

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Edge Measurements
In most cases, manual measurements are done measuring the distances from one edge of
object to another. Locating object edges manually is tedious work and the resulting meas-
urements may have subjective factors. The Snap to Edge feature locates image edges
within a cursor neighborhood automatically, and either moves the cursor to the calculated
position or snaps the measurement features to the calculated position. It provides greater
measurement accuracy, reduces the chance of subjective errors, and generally makes taking
measurements easier.

When you select the Snap to Edge tool on the Measurements ribbon, you will see the fol-
lowing dialog:

There are a number of methods available to making the edge measurements:

Snap to straight edge
The closest straight edge is located within the cursor area defined by
a radius. If the mouse stays still for a short interval (default is 0.4 sec),
the cursor moves to the calculated position. This method is available
with all manual measurement tools.

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Snap to any edge

The closest edge is located within the cursor area defined by a radius.
The option can be used on small objects or on objects with curved
outlines. If the mouse stays still for a short interval, the cursor moves
to the calculated position. This method is available with all manual
measurement tools.

Adjust line length

The measurement feature is snapped to the sharpest rising and falling
edges underneath the line. This tool can be used when the object is
brighter or darker than background and there are no holes or other
sharp edges in the middle of the object. The Width control defines
the width of the caliper line and Length is the matched length of the
edge detector. This method is available with the Line and Point to
Point measurement tools

Adjust line ends

The created line is snapped to the object’s edges along the line. The
line ends are also adjusted by edges. The width of the snap area is
defined by a radius. The Width control defines the width of the cal-
iper line and Length is the matched length of the edge detector. On
noisy images Width can be set to higher values to smooth the profile
averaging pixels in perpendicular to the line direction (default is 3).
This method is available with the LineandPoint to Point meas-
urement tools.
If Adjust line length or Adjust line ends option is active a line is
drawn on image using Line or Point to Point tools will be auto-
matically snapped to the object’s edges as soon as it’s created using
the caliper functionality.

Adjust along edge

The created line is snapped to the object’s edges in the neighborhood

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of the line ends. The area length is defined by a radius. The Width
control defines the width of the caliper line and Length is the
matched length of the edge detector. The Length defines how sharp
the detected edge can be. Low values (3 to 7) are recommended for
high contrast images with uneven intensity of objects. If objects have
even illumination and they are relatively wide, Length can be
increased smoothing noise along the line. This method is available
with the Line and Point to Point measurement tools.

Find circle
Finds a circle within the radius from the click position. Click inside
the circular object and the position and radius will be calculated auto-
matically, analyzing the object edges in the neighborhood.
This method is available with the Circle measurement tool.
The template button activates the mode, which can be used to create
measurement templates. When Template mode is active the Snap
functionality is not applied immediately after creating the line fea-
tures, but only when you click the Auto Adjust button. Only
Adjust line ends, Adjust along edge and Find circle can be used in
Template mode.

Best-fit measurements

Best-fit measurements can be used to create features from a number of manually placed
points.
Best-fit Circle
The Best-fit Circle tool allows you to add points to the image and a
best-fit circle will be generated. Stop adding points by double-click-
ing or by pressing the <Enter> key.

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Best-fit Arc
This tool allows you to add points to the image and a best-fit arc will
be generated. You can stop adding points by double-clicking or press-
ing the <Enter> key. The angle of the created arc is defined by the
positions of the first and the last points.

Best-fit Line
The Best-fit Line tool allows you to add points to the image and a
best-fit line will be generated. You can stop adding points by double-
clicking or pressing the <Enter> key.
Display points for best-fit features
When the Display points for best-fit features isON the points, which
define best-fit features are shown on the image and included in the
data table. When the option is off the points are hidden.

The Points edit boxes in the drop-downs for Line, Circle, and Arc
features are used to set the maximum number of points for best fit
lines, circles, and arcs. Lines will allow from 2 to 1000 points, while
circles and arcs will allow from 3 to 1000 points. When creating a
best-fit feature, the feature will be automatically created when the cur-
rent maximum is hit. This makes it possible to set feature creation to
accept 2-point lines and 3-point circles and arcs (without requiring
double-click on the last point) while still supporting multiple-point
fits when desired.

Relative Measurements

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The Relative Measurements group provides tools to create measurements between existing
features. You have to create one or more objects first, and then create a derived meas-
urement.

Distance between objects

Use these tools to create distance measurement between two objects.
Point group
These tools are used to create point features at the start of the
l First point creates a point feature at the start of the selected
object.
l Center point creates a point feature at the center of the selected
object.
l Last point creates a point feature at the end of the selected
object.
Distance between centers
This tool creates distance measurements between the centers of two
selected objects. Note, that if two lines (polygonal lines) are selected,
the Average gap between traces will be measured.

Minimum distance
This tool measures the least distance between two selected objects.
Note that if two lines (polygonal lines) are selected, the Minimum
gap between traces will be measured.

Maximum distance
This tool measures the greatest distance between two2 selected
objects. Note that if two lines (polygonal lines) are selected, the Max-
imum gap between traces will be measured.

Angle between lines

This tool creates an angle feature between two selected lines. When
drawing lines, the order in which the lines are selected and the dir-
ection of the lines is taken into account. The shortest distance is cal-
culated when the lines are drawn in the same direction.

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Merge Objects
Clicking the Merge button will combine all selected objects into one
by connecting all the objects by the shortest line. Note that the
Merge function is available only for making region measurements of
objects (polygon, ellipse, circle, count/size objects). The Merge but-
ton is also displayed in the on-image context bar.

Incremental Distance
Clicking this button creates a set of vertices between two selected fea-
tures (lines or regions). This feature is useful when measuring coating
(between 2 poly-lines) or a doughnut (between external and internal
regions) thickness distribution. It is also possible to create incremental
distance measurements for a single object (closed polygon). If the
object contains a hole, incremental distance will be calculated
between the object’s outline and the hole (doughnut thickness meas-
urement). If more than one hole exists, the largest hole is used. If the
object doesn’t contain holes, the incremental distance will be cal-
culated between 2 opposite sides of the object along the given angle.
The step size offset on both sides of the object is used to avoid zero
length lines. When auto-angle is on, the angle is calculated auto-
matically.

Additional information about the Angle between Lines measurement follows:

The angle range is determined by the option in the Angle tool dropdown and can be
0…180, -180…180 (default) or 0…360.

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It is computed as the difference between the vector angles (measured counterclockwise

from the positive, rightward, horizontal axis) of the first selected line, tail-to-head, minus
the second selected line, head-to-tail, limited to +/-180 degrees. The measurement thus
depends both on the order in which the lines are selected and the directions in which they
are drawn (in an opposite sense).
The arc is drawn from the center of the second line in most cases, unless the center is close
to the intersection point. In that case the arc is drawn from the point with fixed offset from
the center:

The arc is always drawn from the mid-point of the second selected line to the point where
the arc intersects the first selected line or its projections. The arc is drawn in either a clock-
wise or counterclockwise direction to create an arc that covers an angle equal to the abso-
lute value of the measurement (computed as described above). The arc starts at the
midpoint of the second selected line and ends on the first selected line or its projection.
The direction that the arc is drawn is not determined by the sine of the measurement value.
Rather, it depends on the location of the midpoint of the second selected line relative to
the trajectory of the first selected line.

Incremental Distance
Incremental Distanceautomatically measures distances between two irregular edges to
quantify changes in surface size and shape. The drop-down contains the following options:

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Step Size defines the distance between vertices in pixels.

Type selects the incremental distance measurement type that defines how vertices are cre-
ated. It can be one of the following:
l Shortest measures the shortest distance between point and line. Points along the
object line are equally distributed with the interval defined by the step size. The
shortest distance to the reference line is created from every point on the object
line.
l Equidistant measures the distance between points along the object line. Step Size
defines the number of vertices. The interval between points on the reference line is
calculated based on the number of points on the object line. Vertices connect a
point on object line with the corresponded point on the reference line.
l Directional is used when all vertices are parallel. The distance between the ver-
tices is equal to the steps Size.
When using the Directional type, the orientation of vertices is defined by an
angle (in degrees from X axis counterclockwise). When auto-angle is on, the
angle is calculated automatically based on the object and reference lines. Created
vertices skip non-overlapping line ends.
l Shortest bi-directional measures the distances between points on the line and
another line calculated as two sets of lines: from the object line to the reference
line and from the reference line to the object line. This algorithm is used in
Image-Pro to calculate distance between traces. The distances for object and ref-
erence lines are calculated in the same fashion as the shortest method.
l Distance map calculates thickness of the layer using a distance map. The source
points are placed on the skeleton line between two traces created by the distance
map. New lines perpendicular to the skeleton line and with a length cor-
responding to the thickness based on the distance map are created. The method
can be used to measure thickness of bent profiles.
When Hide Base Lines is selected, the source base lines are hidden after the incremental
distance lines are created, so that they don’t affect statistics and histogram distribution of
visible objects.
To measure incremental distances, follow these steps:
1. Go to the Measure tab and draw two polyline measurements on an image.
2. Select both polyline measurements
3. Select the Incremental Distance button in the Relative Measurements group.

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Note that all relative features are adjusted when their base features are changed. Relative
features cannot be moved manually; they will be moved by changing positions of the base
features.
Note also that the order of selection also affects derived measurements.
Morphological Operations
The selected region-based features can be automatically modified using four morphological
operations: Grow, Shrink, Open, and Close
Grow
Grows selected objects using a Dilate operation.
When the "Use Restricted Grow" option is on, the
Grow operations (with Count or Grow selected) do
not merge objects (default is on).

Shrink
Shrinks selected objects using an Erode operation.
The size increment for Grow and Shrink operations
can be adjusted in the dropdown of the Shrink but-
ton. The increment size is defined in pixels.

Open
Applies morphological Open operations to selected
regions. Use this operation to separate neighboring
features if they are connected by a narrow path. The
size of the morphological Open or Close operation
is defined in the dropdown of the Open button. The
size is in pixels.

Close
Applies morphological close operations to selected
regions. Use this operation to merge neighboring fea-
tures.

Note that all relative features are adjusted when their base features are changed. Rel-
ative features cannot be moved manually; they are moved by changing positions of
the base features.

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To make line measurements in an image:

1. Open the image that has features you want to measure.
The image appears in the Image-Pro workspace.

2. Select the Direct group on the Measure tab’s ribbon.

3. Select the measure line tool.

This tool allows you to make distance measurements directly on the image
4. Apply or define the spatial calibration that you want to use. See “Managing Cal-
ibrations” earlier in this chapter for more information.
5. Draw a line on the image.
The measurement information will appear in a label on the image. The label will
appear near the center of the line without covering up the end symbol or much of the
line itself. The measurement values will change as you drag the line.
You can make changes to the appearance of the label using the Options button.

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6. Click the Options button. You will see the Measurements Options dialog:

The text area of this dialog allows you to change the font and size of the text that will
appear in the measurement label. The border and fill areas control the display of the
border and background of the label. The options area lets you resize the label when
you resize the image, and control the opacity/transparency of the label.

Note: Measurements and Manual Tagging can be applied to live preview images for
quick measurement previews. However, these measurements are not saved with the
captured image.

Setting the Default Appearance for Measurement Labels

The default appearance attributes for measurement labels are set by designating the appear-
ance attributes of an existing label as the default for future measurement labels. To set the
default appearance for an annotation type:
1. Click Select from the Measurement group.
2. In the active image, click on a label of the type for which you want to set appearance
defaults. Most labels appear at the top-left or bottom-right positions near the marker.

3. Click the Options button to display the Measurement Options panel.

4. Expand the Appearance Options section of the panel:

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5. Go to the Font panel.

6. Choose a font from the drop down list:

You will see the measurement labels change to the font you select. Use the color,
style, and size buttons to make additional changes to the measurement labels.
Shadow (S) lets you add a background shadow color to the label text.
Bold (B) displays the font in bold.

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Italic (I) displays the italic font.

Underline (U) underlines the label text.
Class Color uses the class color for the label.
A lets you select the text color.
The number in the spin box indicates the font size.
7. In the Labels panel, select the text, position, and units of measure for the measurement
labels:

The choices for Text include:

n None – no labels
n Name – measurement object name is displayed
n Measurement – the label measurement is displayed
n Name And Measurement – full name and measurement are displayed.
Units of measure and measurement abbreviations can be added to the measurement
label by checking the appropriate boxes. You can also indicate the number of decimal
points for each measurement. Position indicates the location of the measurement label.
If the Dynamic box is checked, the label information will change as the information
in the image changes. Measurement labels will also be resized automatically. When
you select a feature, its associated label will be highlighted. If the Auto Size box is
checked, the labels are scaled proportionally to object area.
Here is an example of measurements with labels:

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8. Use the Outlines panel to define the style of the each object class and shape:

9. The Cursor panel lets you customize the measurements cursor:

n Cross Bars are useful adjusting line ends on the features. They are only active in
the line editing mode.
n Crosshair helps you to center horizontal or vertical measurements.
n Tooltips show measurement data on the image when you are moving the mouse
over an object on the image.

Using the Auto-Trace feature

Activating the Auto-Trace tool for an ROI or Annotation will cause the Polyline tool
in Line Profile to behave the same as the Auto-Trace tool. When you click on the
Polyline feature and activate Auto-Trace, you will see the following dialog:

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The message at the top of the dialog indicates if auto-trace has started, or not.
Tracing speed: Use the slider to control the speed of the auto-trace.
Edge quality: Indicate the number of pixels to use for the edge trace.
Noise reduction: Use the spin buttons to indicate the degree of noise reduction in the
traced object.
Momentum: Use the spin buttons to indicate the degree of momentum in the traced
object.
Smoothing: Use the spin buttons to indicate the degree of smoothing in the traced
object.
Check the box next to the auto trace feature that you want to use.
AutoStart Tracing: If this box is checked, the second mouse click made when cre-
ating a polygon or polyline will start the auto tracing. If uncheck, start auto trace by
holding down the <Ctrl> key and clicking where you want the trace to start. The
lightning bolt cursor shows you the auto trace path.
AutoAccept Final Trace: When this box is checked, the auto trace automatically
returns to the starting point to finish tracing the object. If unchecked, you must press
the <Enter> key or double-click the mouse to place the final point and complete the
auto trace.
Trim Leading Points: Frequently, a trace will not return to its starting point but will
interest with itself some distance from the starting point. If this box is checked, the
points before the intersection point are removed if the intersection is close to the start-

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ing point. If this box is not checked, you must edit the trace manually to remove
extraneous points.
Remove Redundant Points: If this box is checked, all horizontal, vertical, and diag-
onal runs leading to the auto trace are replaced by their end vertices in the final result.
If this box is not checked, these points will be retained.
Monochrome Interpretation: When this box is checked, image color is ignored for
edge detection; only average luminance is used. If unchecked, each color channel’s
luminance gradient is considered when doing edge tracking.
Assume Clockwise: When this box is checked, the trace will begin moving clockwise
around the object. This information is used to determine which side of the trace is the
interior color. This option is useful when working with colored images. If this box is
not checked, no assumption is made about which side of the trace is the interior.
UseRangeMinimum/Range Maximum: When checked, the range for full-scale lumin-
ance swings is assumed to be the full minimum/maximum range of the entire image. If
this box is not checked, range for full-scale luminance swings is assumed to be the dis-
played range of image luminance. This option is usually left clear.
Bound Trace by ROI: When this box is checked, the trace object must stay within
the bounds of the ROI. If this box is not checked, the boundaries of the ROI are
ignored.
Disable Auto Trace: When this box is checked, auto-trace will be disable and you
cannot use it until you uncheck the box.

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Manual Tagging
Use the Manual Tag tool to select individual objects or groups of objects in an image with
your mouse. You can create different classes of objects for analysis, and “tag” each object
with a specific marker. To use Manual Tagging:
1. Open your image if it is not already open.
2. Select the Manual Tag tool from the Direct group.

3. Select a shape and color from the Class group.

4. Click on the points in your image to tag them with a color and label:

Information about your tagged points appears in the Data Table below your image:

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5. Save your data to a file or export it to Excel.

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Viewing Measurement Information

Viewing a Data Table

The Data Table collects all the information from the measurements taken on your image.
To view a data table:
1. Open the image if it is not already open.
2. Select the data type that you want to view.
3. Select the Data Table from the View Data group.

4. The data table will appear in a pane below your image:

The data table default names for measurements are:

Abbre- Meas- Abbre- Meas- Abbre- Meas-
viation urement viation urement viation urement
Name Name Name
AN Angle AB Angle BFA Best-fit
between Arc
Lines
BFC Best-fit BFL Best-Fit
Circle Line
C Circle CCD Center to CPT Center
Center dis- Point

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tance
E Ellipse FPT First Point L Line
LPT Last point MMD Minimum MXD Maximum
distance Distance
P Polygon PL Polygon PR Count/Size
Line Polygon
region
PPL Point to PWL Pitch with
Point dis- Line
tance
R Rectangle T Tag

You can manually rename any features in the data table.

The Data Table provides the following tools:

Load Data Table Measurements: This tool allows you to load meas-
urements saved in a previous session from a *.ipm file.
Save Data Table Measurements: This tool allows you to save your
measurements in a *.ipm file for use at another time.

Send Measurements to File: This tool allows you to save your meas-
urement data to a file or export it to an Excel spreadsheet. The pull-
down menu lets you choose one of the following:

Send to Report: This tool allows you to save your measurement data
to Image-Pro report. The pull-down menu lets you create a new report
or choose an existing one.

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Show all Statistics: This tool allows you to hide or show the statistics
panel for the data table measurements. A sample statistics panel is
shown here:

The drop-down option Statistics per Group defines what is displayed

in the statistical pane. When this option is off, the statistics of all vis-
ible features is shown. When the option is on, the statistical values are
shown per group. This option has affect only when Grouping is active
Setup Group Statistics display the statistics by group. It will activate
grouping by class, add Percent Area Parent to the list of measurements
and activate statistics per group
Grouping: Click this button to see options for grouping your data in
the table. You can choose one of the following:

Show All, Show Selected Measurements, Hide Selected

Measurements: These tools let you choose to display or hide some of the meas-
urement statistics.

3D Mask: Click this button to create a mask of volume objects.

Load Data Table Measurements: This tool allows you to load meas-
urements saved in a previous session from a *.ipm file.
Save Data Table Measurements: This tool allows you to save your
measurements in a *.ipm file for use at another time.

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Send Measurements to File: This tool allows you to save your meas-
urement data to a file or export it to an Excel spreadsheet. The pull-
down menu lets you choose one of the following:

Send to Report: This tool allows you to save your measurement data
to Image-Pro report. The pull-down menu lets you create a new report
or choose an existing one.
Show all Statistics: This tool allows you to hide or show the statistics
panel for the data table measurements. A sample statistics panel is
shown here:

The drop-down option Statistics per Group defines what is displayed

in the statistical pane. When this option is off, the statistics of all vis-
ible features is shown. When the option is on, the statistical values are
shown per group. This option has affect only when Grouping is active
Setup Group Statistics display the statistics by group. It will activate
grouping by class, add Percent Area Parent to the list of measurements
and activate statistics per group
Grouping: Click this button to see options for grouping your data in
the table. You can choose one of the following:

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Show All, Show Selected Measurements, Hide Selected Meas-

urements: These tools let you choose to display or hide some of the measurement
statistics.

Viewing a Data Histogram

Histograms measure, and illustrate in graph form, the brightness and contrast characteristics
of an image. Histogram data can be created and viewed for data gathering and analytical
purposes or can be manipulated for image enhancement purposes. To create a data his-
togram:
1. Open the image that you want to measure if it is not already opened.
2. Select the type of measurement data to view. For example, point position X
3. Select DataHistogram from the View group.

A histogram of the data for the x position of the selected points appears in the right-
side panel of the workspace:

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More information about histograms appears in Chapter 4 of this manual.

Viewing an Object Window

The Object Window allows you to view information about a specific object in an image.
To view the object window, follow these steps:
1. Open the image that you want to measure if it is not already opened.
2. Select the type of object data to view. For example, point location T2.
3. Select Object Window from the View Data group on the Measurements ribbon.

The Object Window containing data for point location P1R12 appears in the right-
side panel of the workspace:

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Colocalization
Colocalization describes the existence of two or more molecule types in precisely the same
space. The material under examination may be treated with different types of dyes to indic-
ate the presence or absence of areas of colocalization or overlap. The Colocalization com-
mand can be used to measure the amount of colocalization in a color image, in a pair of
gray images, in an image sequence, or in an ROI on a color image.
The Colocalization feature appears on the Measure tab’s ribbon:

Colocalization provides tools to measure the colocalization parameters of images and

objects. You can define colocalization ranges using thresholds or drawing outlines manu-
ally using the available ROI tools. The Colocalization button is enabled when the active
image has multiple channels or if there are more than one single channel images open. If
you are working with a monochrome image, you will be able to define a second channel
for colocalization analysis.
To use this feature, follow the steps below.
1. Open your image(s) in the Image-Pro workspace:

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2. Click the Colocalization icon on the Measure tab’s ribbon:

You will see this this drop-down menu:

3. Indicate the first and second images:

You can also specify a Parent Image that can be used for object segmentation. If there
is no parent image, then the segmentation will be done on the combined image of
colocalization channels. For multi-channel (color) images the segmentation on a parent
image is enabled when Use Parent Channel option is on. In that case, the third chan-
nel that is not used for colocalization will be used as the parent image.

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4. Choose the colocalization method.

The Color Pair combo-box defines the combination of color channels for analysis. If
two monochrome channels are used, the color pair defines the colors for the coloc-
alization plot. The list contains all combinations of Red, Green and Blue pairs.
The Result Type combo-box defines the type of colocalization plot:
n Color indicates the resulting image is a scatterplot with all color combinations
between 2 channels. The point is added if there are one or more pixels with a
given color combination.
n Frequency indicates that the resulting image is a 16-bit monochrome scatterplot
where the intensity of every pixel corresponds to the number of pixels on the
source image with a given color combination.
Threshold indicates what type of threshold parameters to use in the colocalization pro-
cess.

n Auto-Bright. The thresholds for both channels are calculated independently using
the Otsu algorithm. This is the same method that is used in Count/Size for Auto-
Bright segmentation.
n Zero Pearson. The method is based on finding threshold levels below which Pear-
son correlation is zero. This method produces very low threshold levels and
should not be used for object-based colocalization analysis, since it complicates
object segmentation.
5. Click Create.
Clicking the Create button creates a colocalization plot for the selected color pair. In
case of two monochrome images, a new color composite image will be created and
used as a source for colocalization. A sample image is shown here:

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If the active image is a sequence and the Apply to sequence option is selected, the
colocalization scatterplot and the Measurement Data table are calculated based on the
active frame range of the sequence (by default all frames are included, optional ROIs
can be added). In this mode, the colocalization is reported for the whole Z-stack.
If you want to have colocalization results for the current frame, the Apply to sequence
option must be off. In that case the colocalization scatterplot and the Measurement
Data table will show data based on the active frame.

Colocalization Panel
The colocalization panel is opened when a colocalization image is created, or when an
image with colocalization data is activated. The panel shows the colocalization plot
and provides controls to adjust and analyze colocalization parameters.

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You can make further measurements and adjustments to the colocalized image by fol-
lowing these additional steps.
6. Define the colocalization plot.

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The Mode control defines the selection mode for colocalization. Your choices are
Thresholds (default) or Free ROI.
In Thresholds selection mode the colocalization thresholds are defined by intensity
thresholds on the first and second channels. Initially the thresholds are calculated
using the selected Threshold method. The thresholds can be adjusted clicking and
moving the mouse cursor on the colocalization plot or using the threshold sliders and
edit controls. The pixels are colocalized when the intensity values for both channels
are equal or above the corresponded threshold value.
Use the red and green sliders to adjust the colocalization threshold values.
Switching to Free ROI mode allows you to use the ROI editing tools to adjust and
draw free ROIs.

Using the Image button displays the colocalization scatterplot in the main applic-
ation workspace. It also allows you to define areas using any ROI tool in the Select
tab’s ribbon.
7. Define the parent outlines.
This group contains the controls that are used to define the outlines of parent objects
for object-based colocalization analysis.
When a parent channel or image is defined, the group has the following buttons:

The Method group includes Segmentation (default) and Manual Regions methods.
In the Segmentation method the objects can be define by setting the threshold on the
Parent channel by clicking the Threshold button.
After adjusting the parent threshold, you can either close the threshold dialog and con-
tinue adjusting colocalization parameters or just click the Count button to segment par-
ent objects and measure their colocalization parameters.
When a parent channel is not defined, the group has the following buttons:

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In this mode the colocalization mask can be shown in Multichannel or coloc-

alization only modes. In Multichannel mode 3 types of pixels are shown: Channel1
(e.g. Red), Channel2 (e.g. Green) and Coloc. That multichannel mode can be used for
object-based colocalization analysis. Clicking the Count button will segment objects
that have connected pixels in Channel 1 or Channel 2.
When Multichannel is off (or in Free ROI mode, or when a parent image is defined)
the mask only shows colocalized pixels.
Parent outlines can be created manually by drawing regions on the image using the
measurement tools:

Clicking the Show Measurements Tab button will display the Measurements tab.
Measurement information is available throughout this chapter or by clicking the Help
link in the tooltip.

The mask of colocalized pixels is shown on the original image. The mask can be
toggled on and off using the Show button. The mask can be created as a separate
image clicking the Mask button. Note that if the source image is a sequence, a 3D
sequence mask will be created.
8. Review your results.

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Colocalization parameters are calculated automatically and are updated when changes
occur to the colocalization thresholds or Free ROI. The data for whole image is
shown in the table when Data is set to Measures.
These parameters are for the whole source image (respecting ROIs). Parameters for indi-
vidual measurement objects are reported in the Measurements table. All colocalization
measures are added to the measurement list by clicking the Add Measures button.

Object-Based Colocalization Analysis

The colocalization data for each measurement object is reported in the Measurement
Data table. You can draw objects manually on the image using the Measurements
tools, or segment objects automatically based on the Parent Image or Multi-channel
colocalization mask. Click the Count button to segment objects, measure coloc-
alization parameters, and optionally classify them by colocalization degree.

The Limit control defines the minimum value of the Colocalization intersection (range
0...1) to classify objects as colocalized. When the value is 0, objects that have at least
1 colocalized pixel are classified as colocalized.

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The classification is based on the colocalization limit defined in the Classify button
dropdown.
When Auto Classify option is on the objects are automatically classified into 3
classes:
Channel 1 (e.g. Red): objects that have Coloc Intersection parameter below Limit
and M2>M1. (Objects are predominantly red)
Channel 2 (e.g. Green): objects that have Coloc Intersection parameter below Limit
and M2< M1. (Objects are predominantly green)
Coloc: objects that have Coloc Intersection parameter above the Limit (colocalized
objects).
The classification statistics are shown in the table:

The table can be sent to Excel or copied to Clipboard .

Using the Report buttons will create a Colocalization report for the active image
based on the pre-defined template.

All colocalization measures and mean intensity measurements per channel can be
added to the measurement list by clicking the Add Measures button.
The measurements data table provides detailed data for every object and can be
grouped by statistical values per class:

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Object segmentation using Parent Image

When a Parent Image is defined (or Use Parent Channel is on) you can create a
colocalized image from a multi-channel image.

The segmentation threshold for the parent image can be set clicking the Set Parent

Image Thresholds button. It will open the Threshold dialog with predefined
thresholds corresponding to Auto-Bright segmentation for parent channel:

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Object segmentation using colocalization channels

When a parent image is not defined (or Use Parent Channel is off) you can create
colocalization from a multi-channel image

Activate multi-channel mode and adjust the colocalization thresholds. The coloc-
alization will be done on the mask that includes BOTH red and green objects. This
method can be used when no parent image/channel is provided. Note that the Count
operation respects all options defined in Count/Size or Measure tabs including meas-
urement filters and auto-separation options.

Manual object drawing

It is possible to draw objects manually using measurement tools. You can activate
Manual Regions mode and use the drawing tools on the Measure tab.

All colocalization parameters are reported in the data table.

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L*a*b* Color measurements and Color Correction

Color measurements and color correction tools are located on the Measure tab, in Color rib-
bon group:

The module is designed for absolute color measurements in CIE L*a*b* or XYZ color
coordinates. This module also provides easy to use tools for color correction.
The module can be used in cosmetic, textile, pharmaceutical or food industries, those that
require accurate measuring of colors or color differences.
The dropdown button contains 3 items that open dialogs with Color measurements and
Color correction tools:

Color Measures
Color measures dialog allows measurement of colors under cursor and also extracting color
channels as monochrome images.

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CIE L*a*b* color measurements can be executed using 2 methods:

1. sRGB color model
2. Color profile
sRGB (standard Red Green Blue) is an RGB color space created cooperatively by HP and
Microsoft in 1996 for use on monitors, printers and the Internet, and subsequently stand-
ardized by the IEC in 1999. It is often used as the "default" color space for images that do
not contain any color space information, especially if the images are stored as 8-bit
integers. sRGB color model assumes that RGB colors are captured with D65 illuminant.
More info can be found here.
sRGB color model can be used with images that do not have associated color profiles or
when only measurements of color differences are required after executing of color cor-
rection to sRGB space.
If image has own color profile, the second option Use color profile should be used. In that
case color profile associated with the image or the working color profile provide the cor-
rect XYZ and L*a*b* measurements.

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Image color profile can be loaded clicking the Load button and selecting ICC or CMM
color profile file from the disk.
The Reset button will remove color profile from the image and activate the working color
profile for measurements. The working color profile can be selected using the Select work-
ing color profile button. It can be reset to the default sRGB IEC61966-2.1 clicking the
Reset working color profile button.
If image has own color profile, it can be converted to the working color profile using the
Convert image to working profile button.
ICE L*a*b* measurements are also available for any Region objects using Count/Size or
manual Measure operations:

Note, that L*a*b* color measurements generated by Count/Size use the color measurement
method, color profile and Illuminant selected in the color measure dialog.
The bottom part of the dialog provides measurement values under cursor on the active
image:

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RGB group shows Red Green and Blue values under cursor and Color Space/Illuminant
defines the active color space/illuminant and corresponded measurement values.
There are following color spaces available:
l Lab – CIE Lab color space
l XYZ – intermediate color space between RGB and Lab spaces
l YIQ - the color space used by the NTSC color TV system, employed mainly in
North and Central America, and Japan. I stands for in-phase, while Q stands for
quadrature, Y component represents the luma. (https://en.wikipedia.org/wiki/YIQ )
l CMY - subtractive color model, used in color printing, cyan, magenta, yellow.
(https://en.wikipedia.org/wiki/CMYK_color_model ).

Note, that YIQ and CMY are not absolute color measurement spaces and listed in the dia-
log for completeness of color measurements on the image.
The Illuminant defines the type of light source used when the image was taken. The fol-
lowing standard illuminants are listed:
l D50 - CIE standard illuminant D50. Simulates warm daylight at sunrise or sunset
with correlated color temperature of 5003 K. Also known as horizon light. (all
color profiles use it as PCS colro space)
l D55 - CIE standard illuminant D55. Simulates mid-morning or mid-afternoon day-
light with correlated color temperature of 5500 K.
l D65 - CIE standard illuminant D65. Simulates noon daylight with correlated color
temperature of 6504 K. (used with sRGB color space)
l A - CIE standard illuminant A. Simulates typical, domestic, tungsten-filament light-
ing with correlated color temperature of 2856 K.
l C - CIE standard illuminant C. Simulates average or north sky daylight with cor-
related color temperature of 6774 K.
l E - Equal-energy radiator. Useful as a theoretical reference.

The Illuminant affects XYZ and Lab color measurements. It's preferable to use the native
Illuminant for specific color profiles, for example sRGB model used D65 illuminant, while
Color profiles use D50 as intermediate white point. Conversion to other illuminates are
done by using Bradford transform in form of chromatic adaptation.

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Every channel of a color image can be extracted to a monochrome image clicking the
Extract button.
Reference colors
Color measurements can be verified using some reference color images called color check-
ers, one of them is Gretag-Macbeth Color checker.
The patch has 24 colors covering visual gamut and has known colors published on the net.
The application installs the color checker image as ColorChecker_sRGBD65.tif to the Col-
ors folder of demo images:

The RGB values of every are generated with D65 illuminant. The list of patch values can
be found in wikipedia .

Color Correction
The Color Correction tool is closely related to the CIE L*a*b* measurements. The goal of
the color correction is to adjust colors of one image to colors of the reference image with
known Lab coordinates or just to some reference color patch, so the colors can be meas-
ured or compared.

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Typically, the reference image can be a color checker (e.g. Macbeth ColorChecker) or just
an image with color patches.
User should place manual Tags as reference points on corresponded color patched of the
reference and active images and execute color correction.
There are 2 types of transforms are available:
l Polynomial
l Thin Plate Spline

With Polynomial type the images may have from 3 to 20 points for color correction. A
polynomial transform is created matching colors on the active and reference images. The
type of the polynomial depends on the number of tags:
l 3-point calibration corrects changes in color gain
l 4-point calibration corrects color shift and gain
l 7-point calibration corrects the linear color mix

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l 10-point calibration corrects the color mix using polynomial approximation of

power 2
l 20-point calibration corrects the color mix using polynomial approximation of
power 3 .

Polynomial transform is recommended to use with small number of reference points (3-7) as
high degree polynomials may produce unexpected color distortions for colors in between
references.
Thin Plate Spline transform can be used with 3 or more reference points. This method pro-
duces smoother color space transforms comparing to Polynomial with large number of
points, though reference point colors may not match exactly when Regularization is more
than zero.
It is recommended to use Thin Plate Spline transform for large number of tags as in case of
full 24-point matching of Macbeth ColorChecker.
Point Radius - radius of the measurement circle at the tag position in pixels. The average
RGB values of the circle region are used for color matching. When the value is one -
single pixel value is extracted.
Regularization – is a smoothing parameter used only with Thin Plate Spline transform
method. Increasing the value will smooth color transform, though the reference values may
not match exactly.

Typical color correction work-flow

Use image of Color Checker or image with color patches as a reference, add manual tags
on the color patches using tag tools:

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Tags can be added activating the Manual Tag tool and clicking at the color patches on
the image. Tag positions can be modified activating the Select tool, selecting and moving
or deleting them using the Delete key.
You can also add the whole grid of color tags clicking the Add ColorChecker grid but-
ton and drawing polygon clicking the centers of the corner color patches in the following
order: top-left, top-right, bottom-right, bottom-left:

then close the polygon either by double click or hitting the Enter key.
The tag pattern will be created:

The tags on corresponded color patches on the active image should also be created and
then color transform can be saved to a file or applied to the image.

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The transform can be saved to a file using the Save button. When the transform is saved, it
can be used correcting the current or other color image taken with the same conditions.
Clicking the Correct Colors button will apply transform to the active image.

The correction can be undone using the Undo button.

After the correction the RGB values of the color patches at tag positions will be trans-
formed to RGB values of the reference image and L*a*b* or XYZ measurements can be
performed properly.

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Color Validation App

Transform verification can be done using the pre-installed Color Validation app. The
screenshot below shows the results of Visualize Color Transform operation:

The app contains documentation describing the available tools, refer to the app's help file
for more info.

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Counting Objects in Images

An Object is considered to be any contiguous group of pixels that share a specified range
of intensity values. You can use the Count/Size commands to identify, count, and meas-
ure these objects. These commands appear on the Count/Size tab’s ribbon:

The basic steps involved in a counting task are to:

l Set the range of intensities (monochrome images) or the colors (color images) that
identify the object you want to count using the tools in the Intensity Range Selec-
tion group box.
l Select the kinds of measurement you want to record in the Select Measurements
command.
l Count and measure the objects using the Count and/or Measure buttons.
Then, using the Count/Size menu commands, your measurements can be viewed, saved,
and edited. You can also classify, sort and graph your data using the menu commands.
In the following example, we will count the number of dark objects in the spots.tif
image.
1. Open the spots.tif image in the workspace:

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2. Go to the Count/Size tab:

3. From the Segment group, choose Dark (automatic dark objects).

4. From the Split drop-down menu, choose Split All.

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This option allows Image-Proto separate objects that are touching or overlapping auto-
matically.
Clicking Split All will split all existing region objects.
The method can be Watershed or Boundary Shape, which define the splitting
algorithm. When Ridge size is on, only objects wider than the given width get sep-
arated. This option can be used to avoid splitting of elongated objects.
When Split Objects after Count is active, the objects get split automatically after a
Count operation, so no additional Split step has to be performed manually.
To split objects manually, activate the Split tool, draw some separation lines and then
click the Apply split button.
5. From the Count drop-down menu, choose Count:

Use Count by ROI if you want to group segmented objects by ROI.

The counted and numbered objects will appear on the image:

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The number of objects counted will appear in the Results group:

n Total indicates the total number of objects segmented in the most recent counting
operation.
n In Range indicates the number of objects remaining after applying filter ranges to
the results.
The Select group also displays the counting results:

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Note that adding or removing objects manually will not change the count results,
since those numbers are related to the count operation. However, the current object
count will be shown in the label on the Delete All button.
Now that we’ve counted the spots, we’re going to sort them.
6. Select Sort from the Image group on the Count/Size tab.

7. Choose Create Sorted Objects Image from the pull-down menu.

In a few moments, you will see a new image with the spots sorted (in this case, from
largest to smallest).

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In the next part of this exercise, we will examine one individual spot.

Displaying Individual Objects

Image-Pro gives you the ability to select a single object after the counting operation, and
to see its outline in the Object Window.
In the example below, object #P1R16 has been selected:

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The enlarged display of that object appears in the Object Window on the right side of the
screen.

The statistics associated with that object are also displayed.

To display an object, follow these steps:
1. Open the spots.tif image in the workspace:

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2. Go to the Count/Size ribbon:

3. From the Segment group, choose Dark (automatic dark objects).

4. From the Split group, choose Split All.

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5. From the Count group, choose Count.

The counted objects will appear in the image:

Statistics relating to the objects appear in the measurement table:

6. Select an object by clicking the Feature Name. In this example, select P1R1.
7. Open the Object Window in the View group:

Object P1R1 is displayed in the Measurement Window:

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Note that there are limits associated with the display of large numbers of objects. You
can set these limits in the Applications Options dialog. When you set these limits, you
may see this message:

Count Options
The Count options appear in the Measurement options panel on the right side of the
workspace:

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When you perform a count/size operation, you can have Image-Pro find whole
objects, or portions of objects such as convex hulls, holes, and so forth. Click on the
Objects drop-down menu to see the choices:

The Preserve existing dataoption allows you to accumulate multiple Count/Size oper-
ations for the same image. When it is active, existing Count/Size results will be
retained when new Count/size operations are executed. By default, Reset existing
data is on, so old data is deleted when a new Count operation is executed.
Checking Close Threshold After Count will close the Threshold dialog auto-
matically after a count operation is complete.
You can collect measurements from the active frame or all frames to Data Collector
clicking the Collect button

When you click Collect , the application synchronizes active measurements from the given
data provider (Measurement types for Count/Size tab), executes Count on every frame
(when All Frames option is on) and collects measurements data for all frames in the
sequence in the Data Collector. The Data Collector table will be opened automatically
after the collect when the Show Table option is on (default).
When the Clear Collector option is active the data is removed from the Data Collector
before the Count. When theClear Measurements is active, the selected measurements list
of Data Collector is cleared before synchronizing of active measurements .
You can access all Data Collector tools and windows clicking the Go to Data Collector
button.

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Thresholding
Thresholding allows you to reduce your image to just two colors: black and white. This is
done by specifying a range of intensities to be emphasized (set to white), and converting
all others to black (0). Thresholding is often used to segment an image in order to extract
its important features, or to reduce an image to two intensity levels in preparation for a
watershed or thinning filtering operation.
Thresholding is performed using the Mask button on the Select tab.

When the Mask command is selected, you will be asked to specify the range of values
you want emphasized (set to white).
The Threshold command operates upon gray scale values, so if you are working with a
True Color or Palette image, it must be converted to Gray Scale before it can be threshol-
ded.
To use the Threshold tool, follow the steps below:
1. Open the image, if it is not already open.
2. Click the Mask button on the Select tab.

You will see the Threshold Tool panel.

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The dialog shows the histogram of the active image and the colored area shows the
range within thresholds. Using the mouse wheel will zoom in the histogram in and
out.
3. Use the following tools to set your threshold range:
The Class combo-box shows the active segmentation class. You can add new seg-

mentation classes by clicking the Add button or delete classes using the Delete

button. All classes can be reset using the Reset button. The class names that
you defined here are assigned to thresholded objects created by a Count operation in

Count/Size. Class color is defined by the color button.

The visibility of the threshold overlay can be toggled using the Show Overlay button

. The Show All Classes check box in the drop-

down defines the preview overlay context, when the control is on, all classes are
shown; when it is off, only the active class is visible. When Allow Overlap option is
off, multi-range segmentation ranges are not allowed to overlap. You can enable this
option to create overlapping ranges.

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The Mask button creates a new binary image of thresholded areas for all classes of the
active class depending on Show All Classes control.
The Log Y button switches the Y axis to a logarithmic scale.
The Reset zoom button resets the histogram.

The Set Global button sets threshold segmentation ranges to Global. These ranges
can be applied to another image using the Apply Global button.

The segmentation ranges can be saved to a file using the Save button and loaded
from a file using the Load button.
The bottom part of the dialog displays these thresholding tools:

Color Picker (eyedropper)

Use this tool to pick intensities from the image. The pixel area that is
used for threshold definition is shown in the cursor window, as in a mag-
nifying glass. The area in the magnifying glass is defined by the Size
parameter.
Start/End

The Size parameter determines the pixel used to sample the intensities.

Undo/Reset

Use the arrow to undo the most recent operation. Use the Reset button

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to reset the threshold.

Minimum/Maximum
Use these buttons to set the minimum and maximum threshold values.
Auto Threshold Min selects only bright objects, while Auto Threshold
Max selects dark objects.

Auto-Threshold
Auto-Threshold allows you to define the method that is used to segment objects in Auto
Bright/Dark segmentation modes. This feature appears when you click the Options button
in the Measurements group on the Count/Size tab’s ribbon.

You can find Auto-Threshold on the Segmentation Options ribbon, which appears on the
panel on the right side of the workspace:

The auto-threshold methods include multi-phase auto segmentation methods and a his-
togram method that should be used with Bias to segment objects above or below the back-
ground. The methods are:
l Min Variance - The ratio of between-phase to total variances minimized (method of
Otsu). This is the default method.

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l Legacy method. A version of the Minimum Variance method with a bias towards low
values. .
l Mean - Threshold is the Mean value of histogram. Use Bias to segment the objects
above or below background. .
l Mode - Threshold is the Mode value of histogram. .
l Median - Threshold is the Median value of histogram. .
l Minimum - Threshold is the Minimum value of histogram. Maximum - Threshold is the
Maximum value of histogram. .
l Best Fit Min - Threshold is the Minimum of best-fit range of histogram. .
l Best Fit Max -Threshold is the Maximum of best-fit range of histogram. .
The methods from Mean to Best Fit Max should be used with Bias to segment the objects
above or below background. These methods can be used with batch macros to count and
measure objects with intensities above or below threshold by fixed value.

Segmentation Bias
Segmentation Bias is defined by the Bias control:

You can use this option to adjust automatically calculated threshold values by the given
bias value. The bias is applied only to AutoBright/Dark segmentation methods. There are
3 bias methods:

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l Range Max - where bias is calculated as a percentage of the maximum range of the
given image type.
l Best Fit Range - bias is calculated as a percentage of the best-fit range. This is the
default method.
l Standard Deviation - bias is calculated as percentage of standard deviation of the
image intensity distribution.
Note that the Bias value, Bias method and Auto-Threshold methods are automatically
reflected on the Threshold overlay when adjusted (when Threshold dialog is visible and
segmentation method is set to Auto Bright or Auto-Dark).
The absolute Bias value is also reported in the tooltip over Bright or Dark segmentation
buttons:

Grow or Shrink Object

The Grow or Shrink option can be used to grow or shrink segmented objects auto-
matically. The option defines the size increment (in pixels) of segmented objects that is
applied after a Count operation using dilation or erosion of the segmented regions. If the
value is negative, the objects shrink. If the value is positive, the objects grow. To use this
tool, follow the steps below:
1. Count the objects in your image.
2. Use the blue selection arrow to select multiple objects to grow or shrink.

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Choose the drop down arrow next to the blue selection arrow to Select All if you
want to grow or shrink all the objects.
3. Go to the Measure tab and choose the Grow or Shrink options from the Relative
Measurements group.

The drop-down arrow next to the Shrink icon lets you specify the incremental amount
to grow or shrink the object.
4. Adjust your image.

Open or Close Object

Use the Open and Close tools to manually combine neighboring objects when using auto-
matic segmentation and counting tools. To use these tools, follow these steps:
1. Count the objects in your image.
2. Use the selection arrow to select multiple objects to merge or close.
3. Go to the Measure tab and choose the Open or Close options from the Relative Meas-
urements group.

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 Open or Close Object

The Open tool will combine the two objects for your count results without changing
the actual area of the two objects. The drop-down arrow next to the Open icon lets
you specify the incremental amount to grow or shrink the object.
The Close option will use a morphological filter to close the space between the two
objects, thus changing the total area of the counted object.

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Smart Segmentation
The Count/Sizeribbon contains a group called Segmentation. It appears on the far left side
of the ribbon.

This feature provides an easy and intuitive method of image segmentation where you learn
to define the reference areas for background and objects. These areas are then used to cre-
ate segmentation masks to count and measure objects. Unlike the standard intensity-based
segmentation, Smart Segmentation allows you to include Color Channels, Background
Correction, and Morphological Filters when segmenting objects. When you select Smart
from the Segment group, the Smart Segmentation dialog appears. This panel contains the
tools and controls that you will use for image segmentation.

Use Smart Segmentation to segment images with colored objects, uneven backgrounds,
and textures. To use Smart Segmentation, follow these steps:
1. To start, go to the Count/Size ribbon.
2. Choose Smart in the Segment group. The Smart Segmentation dialog will open to
the right.

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 Select and Count

3. Draw a reference object and then define your background to start segmenting.
4. Click on the Select Objects tool to activate the object selection tool.
Use it to draw a circle on the object you wish to segment.
5. Click on the Background tool to activate the background selection tool. Draw on the
background area to activate the segmentation.

You can continue to add objects or background to perfect the segmentation.

Tip: To select measurements, choose the Types button in the Count/Size or Measure
ribbon to open the measurement selection tool. All available measurements are dis-
played on the left. Double click on a measurement to add it to your list on the right.
6. Select Count to measure your objects.

Select and Count

The Select and Count panel includes controls to create and edit elements that define back-
ground and reference objects.

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The background and reference objects can be created using one of the following tools:
Polygon, Ellipse, Circle, Rectangle, Line, Polyline, and Point. Select one of these shapes
by clicking the drop-down arrow under Select Objects or Background.

n Class defines the class of the reference elements. You can use this option to segment
objects of different classes. When objects are counted using the Count button, the
class information is passed to the Measurements module and can be displayed as
Object.Class measurement.
n Background defines the areas that are not objects. When the Show mask option is on,
the segmentation mask is created automatically as soon as a new reference object is cre-
ated or modified.
Note that the image must have at least one background and one reference object to do
the segmentation.
n Clicking the Count button measures the objects according to the segmentation mask.
Note that the same result can be achieved by clicking the Count button on Count/Size
tab.
The objects are counted using the selected measurement filters and other measurement
options defined in the Recipe Options.
n The Select and Delete All buttons can be used to edit existing reference objects. The
Load and Save buttons load reference objects from a file and save them to a file
respectively.

Smart Segmentation Training

You can train your smart segmentation recipes on multiple images to provide the best pos-
sible segmentation and count/size results.

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 Mask and Overlay

To use this feature, follow these steps:

1. Open multiple images of a similar sample.
2. Go to the Count/Size tab and select Smart from the Segment group.
3. Use the Smart Segmentation tools to segment your first image. Then select the Set
Global option under the Train and Save section.
4. Go to your next image and select Apply Global from the Train and Save selection.
Continue to adjust your smart segmentation. Then select the Set Global option again.
5. Continue to do this until you are satisfied with your results.
6. You can also save and reload your segmentation recipes for future use.

Mask and Overlay

The Mask and Overlay group contains buttons to control mask visibility, create output
images and count objects.

n The Show button show or hides the segmentation mask. The drop-down menu allows
you to set the degree of mask transparency:

n Clicking the Create Mask button creates an image with segmentation mask. The out-
put image is an 8-bit gray pseudo-color image where the intensity of pixels cor-
responds to the object class.

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A sample image displaying a multi-class mask is shown here:

n Clicking the Create Imagebutton creates a floating point image for the active
class.This image can be used for further processing if necessary.
The following image contains an example of a floating point output image:

Recipe Options
Object segmentation is done by creating a Recipe based on the reference objects and
Recipe options. Recipe options include four main groups of parameters that are all active
by default.

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 Recipe Options

By activating and deactivating various parameters, you can use different combinations of
object properties for segmentation. Note that the more properties you activate, the slower
the segmentation is performed.

Colors in RGB, YIQ and HSI color spaces: When this option is active, the image
is segmented using color information from the objects. The segmentation channels can be
toggled independently opening the button's drop-down menu:

The option can be disabled when colors should not be taken into account when seg-
menting objects.

Background correction: With this option, the image is segmented using back-
ground correction.

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This option can be useful on images with uneven backgrounds.

Morphological operations are applied to the image in order to find the best seg-
mentation between object and background. The set of morphological filters includes Low
Pass, Edge, Erode, Dilate, Max, Min and Range. The size of the filter is defined in the
drop-down menu and should approximately correspond to the object size.

The pixel parameters of the background and object areas are analyzed using all active oper-
ations defined by the options and the segmentation recipe is created. The segmentation
recipe includes the transformations with maximum degree of difference between the
objects and background. The recipe can be saved using the Save button or loaded to a
new image using the Load button. Loading a recipe to a new image automatically per-
forms image segmentation according to the recipe.
When Auto Weight is on, the weight of the channels based on the significance calculated
from reference objects. When the option is off, all channels have the same weight. Switch
this option off if the objects have to be segmented using discontinuous range.
When the Top option is off (default) all enabled channels are included into the seg-
mentation process. You can limit the number of channels activating the Top option and
selecting the number of most significant channels from the top of the list.
The Accumulated checkbox define how multiple reference objects in one class are treated.
When the option is on, the recipe is created based on the accumulated value calculated
from all objects in the given class (mean, deviation). When off, every object is used as a
target to segmentation. Use this option if multiple small objects are used, which will
define accumulated ranges. Switch it off to create segmentation where single class can
include discontinued range of measures, for example when the background is gray and the
object can be either bright or dark. Switch it on, if segmentation should be done based on
average value of many reference objects. (Default is off)

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You can set the active recipe to Global using the Set global button.

The global recipe can be applied to the active image using Apply global button. A
global recipe can be used for segmenting similar images using the same recipe. A Seg-
mentation recipe loaded from a file or applied from the global recipe can be adjusted by
adding new reference objects on the current image.
Note that you cannot add new recipe channels to the loaded recipe; you can only use exist-
ing channels and add reference objects to them.

You can use the Clear button to reset the accumulated objects loaded from a global
recipe. After these objects are cleared you can add new recipe channels if necessary.
The recipe can be saved using the Save button or loaded to a new image using the Load
button. Loading a recipe to a new image automatically performs image segmentation
according to the recipe.

You can use the Record button during macro recording to record the recipe contents
to a macro. The Record feature can be used to create macros based on smart segmentation
without using external recipe files. The recipe command then can be adjusted in the
designer window of the scripting workbench.

The All button adds all available recipe channels to the recipe. Reset sets the
default recipe channels, and resets the recipe and segmentation classes.
The tooltip for the Save button shows the weights of the transformations in the seg-
mentation recipe. These indicate which object properties are most significant for seg-
mentation:

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The Reference objects line shows the total number of reference objects in the recipe. The
number in brackets shows the number accumulated objects (objects loaded from external
recipe and not present on the current image).
The example below shows how segmentation can be used to segment different color and
textures in the same image.
Add reference objects to the image, perform the segmentation, and the results can be used
for measurements:

Before segmentation After segmentation

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 Recipe Summary

Recipe Summary
Recipe Summary shows the overview of the recipe parameters and the recipe description
(optional).

The summary shows the number of reference objects, the number of segmentation classes,
and the list of significant segmentation channels.
The values in the brackets show the mean intensity values for the given channel. When the
Accumulated option is off, the values for every Reference object are shown. When the
Accumulated option is on, the average value for every segmentationclass is shown. The
class is shown in the square brackets - [1], [2].
[Bg] indicates the background objects. The right-most value is the weight of the channel.
In case of recipes with user-defined channels, the summary shows the commands, inputs,
and parameters of user-defined channels. If the inputs are also user-defined channels, they
get expanded up to the simple channels, as shown next:

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The example above shows the recipe with one active channel Difference, which is cal-
culated as a Subtract operation between InverseGray and Histogram Mean where
InverseGray is the NOT operation of Gray and Histogram Mean is the image of Mean
histogram intensity of InverseGray.

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 Classifying Objects

Classifying Objects
The Classify group includes controls to classify objects by single or multiple meas-
urements and define colors of classification palette.

Single Variable Classification

The Single button opens the dialog for single variable classification.

The classification measurement is defined in the top combo-box. The Bin ranges table con-
tains the list of start positions of every classification bin following by the end value of the
last bin. By default the range is spread according to the data values on the current image.
The values are editable.

You can define the number of bins, set the first and the last value and click the Spread

button to divide the range evenly by bins. The ranges can be reset using the Reset
button. The ranges can be divided linearly or using logarithmic scale base on the state of

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the Log button. Note that logarithmic scale can be used only when min and max
range values are greater than zero.
Clicking the Ok button will assign classes to objects according to the bin ranges. This
function can be used to classify objects by any parameter, for example by the Diameter,
which can be used in metallurgy for quality reports:

Auto Classification
Multi-measurement auto classification can be applied using the Auto button.

The Number of Classes defines the maximum number of groups that can be created by
auto-classification. The list of Classifiers include all selected measurements of numerical
type.

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User can select any number of measurements. All measurements can be selected/deselected
clicking the column header button.
Clicking the Ok button applies hierarchical clustering to the objects using the selected
measurements and assigns classes based on the cluster created by cutting the clustering tree
where the number of branches correspond to the number of requested classes.
The following example shows automatic clustering of objects based on their X and Y pos-
ition.

The classification statistics are available in the measurement data table.

The colors used for automatic classification can be defined in the drop-down of the Colors
button:

Palette controls include start/end colors and spins that define the parameters for the color
palette used to classify the objects.

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Learning Classification
Learning classification can be used to classify objects using multiple parameters based on
reference objects. It can be especially useful when you are not certain which parameters to
use for classification, but you do have an idea of how the objects of the given should
appear.
The Learning Classification dialog can be opened when an image already has some meas-
urements. Usually this operation is performed after counting the objects.
The Learning classification dialog contains a Classification bar, a list of Classes and a list
of Classifiers (measurements):

The classification bar contains buttons to handle classification recipes:

Load loads a classification recipe from a file.

Save saves a classification recipe to a file.

Load Global - loads global classification recipe

Set Global - Sets current classification recipe as global.

Reset - resets classification recipe.

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New classes can be added to recipe clicking the Add Class column
header. The name and color of new classes can be modified. To use this feature, follow
these steps:
1. Manually segment objects.
2. Select Count to count objects.
3. Go to the Classify group and select Learning Classification.
4. You’ll see a range of measurement that you can use to classify your objects.
5. If you need additional measurements, go to the Measurements Types group to add
more measurements.

6. From the Learning Classification dialog, select the green class button to add a new
class.

Use the little blue arrow to activate your selection tool and then click on the type of
counted object (on your image) that you wish to classify. For example, you can click
2-3 brown stained objects.
7. Continue to add classes using this technique.
8. Select OK and your objects will be classified using your selected objects.

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How to View a Histogram of Classified objects.

To create a histogram view of classified objects, follow the steps below:
9. In Count/Size Types, select Object:Class and add it to the selected measurements as
shown.

10. Count the objects.

11. Set up and run a Classify operation such as Single Variable Class.
12. In the Data Histogram dialog box, use the drop down menu to select Object:Class:

You will now see a histogram of the classified objects based on the classification
scheme as shown here:

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Finding Shapes
The shape-finding tools appear on the Extras tab:

Find Shapes is used to locate and measure objects of some particular shape, such as a
straight line, a circle, or a search pattern.

Lines
Click the Lines drop-down arrow to see the following options for straight line detection:

Length defines the length of line in pixels. The value assumes that the lines on the ori-
ginal image have a maximum intensity corresponding to the image class (e.g. 255 for 8-bit
grayscale). If the lines have lower intensities, the value has to be increased proportionally.

Clicking the eye dropper button allows you to select the length right from the image.
When the Find line ends option is on, the application uses the caliper feature to find the
exact start and end positions of any detected lines. The longest line along the detected dir-
ection is extracted. When the option is off, the lines are drawn between image borders.
Angles control defines the number of angle steps in the Hough transform (width of Hough
image).
Filter size is the size of spike detecting filter. This parameter defines how close spikes on
the Hough image could be. If spikes are closer that the filter size, they will be detected as
one spike.

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 Finding Shapes

Clicking the Create Hough Image button will create a Hough image that can be used to
examine the transform using other tools. The Hough image is not displayed in the work-
space and only the resulting lines are created.
The Show Demo button opens the demo image and locates the lines. The resulting image
looks like this:

Circles
Clicking the Circles drop-down arrow displays these options:

Use the Prefilter to select the filter to extract circle edges. Use None if the image is
already processed and circles are extracted. (Circles must have bright outlines on a dark
background).
Min radius and Max radius define the minimum and maximum size of the circles. Click-

ing the eye dropper button allows you to select the radius directly from the image.

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Number of steps defines the number of Hough images to be created by detecting circles
from Min to Max radius. This parameter defines the granularity of detected circle radii.
The Completeness parameter defines how complete circles have to be to be located. Set
this value to 1 to locate only complete circles. If the value is set to 0.5, then half-circles
will be located. The values from 0.6 to 0.8 produce the best results. The Completeness
value assumes that the circles on the original image or after edge preprocessing have a max-
imum intensity corresponding to the image class (e.g. 255 for 8-bit grayscale). If the circles
have lower intensities, the value has to be increased proportionally.
Min c-c distance (minimum center to center distance) defines how close circles of the same
radius can be. The parameter is in minimal radii. The default value is 1.0, which means that
the circles of the same size cannot be closer than minimum radius (Min Radius). If the
objects on your image are not overlapping, the parameter should be set to a larger value
(e.g. 1.8).
Min radius diff defines the minimum difference in circle diameters when circles have the
same center point. The value is relative to the Radius range (Max Radius-Min Radius). The
default value is 0.3, which means that the circles can be located separately if their dia-
meters are different for more than 30 percent of the range. If there are no overlapping
circles on the image, so that only one circle can be located at the given position, the para-
meter should be set to 1.
Create Hough image creates image sequence of circular Hough transforms for all steps.
Show demo button demonstrates the functionality using a demo image:

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 Finding Shapes

Irregular Shapes
This button finds a selected pattern on the image. The number of found objects and the loc-
ations can be incorporated into a report. Note that search pattern must be set first.

To set the search pattern, draw an AOI around the object you want to find and click the
Set Search Pattern button. When the pattern is set, the button is checked and the tooltip
shows the current pattern:

The Full FFT checkbox indicates whether or not to use the Full FFT Transform (box is
checked) or Phase Only (box is unchecked).
The Objects control sets the maximum number of expected objects. Threshold options
define the matching threshold to find all objects.
The Create image button creates an image of cross-correlation between the search pattern
and active image.
Clicking the Show demo button demonstrates this feature using a demo image and dis-
plays the located cells on an image:

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 Creating a Voronoi Map

Creating a Voronoi Map

A Voronoi Map can be used to analyze the spatial distribution of objects. The map image
consists of isolated areas of pixels that are closest to the given object. In one case, a
Voroni map was used to pinpoint the source of contaminated water by mapping outbreaks
of water-borne illness.
1. Open your image with the counted objects.
2. Click on the Voronoi drop-down arrow in the Image group on the Count/Size ribbon.

When the Show Mask Objects is checked, the map includes the source objects or their
centers (depending on Map Type).
3. Click the Voronoi button to create a Voronoi map image.
Map Type defines how the areas between objects are divided; either Center to Center
or Edge to Edge. When Center to Center is selected, the areas are mapped by meas-
uring distances from the objects’ centers. When Edge to Edge is selected, the areas are
mapped using the distances from objects’ edges. The areas of the map are colorized
using the object classification from the Data Histogram.
The Measure option defines what type of measurement will be displayed on the out-
put map. The options are None, Area or Minimum Distance.
When Minimum Distance is selected, the image will include circular regions cor-
responding to the minimum distance from the given object to its closest neighbor. The
distance is reported as Region: Diameter.

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Min Distance Edge to Edge map Min Distance Center to Center map
When Area is selected, the cells are measured using the outlines of whole cells.
Objects with the cells are also included the measurements (they are not excluded as
holes).

Area Edge to Edge map Area Center to Center map

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 Tracking Objects

Tracking Objects
The Tracking feature in Image-Pro enables you to follow an object as it moves through
time and space. For example, a researcher could use it to follow the progress of a cell as it
moved through the capillaries of the heart.

Manual Tracking
1. Open the sample image Trackseq.seq from the Images/Tracking folder.
Open the entire sequence (all 8 frames).
The image workspace will look something like this:

2. Click the Play button on the Sequence toolbar to see how the objects move.
3. Click the Stop button.
n Select ObjectTracking from the 2D Measure tab. You will see the
MotionTracking ribbon.

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4. Click the Add button from the Manual Tracks group:

You will see a message asking you to click on the points to be tracked in the active
image.

5. Click on a point on the active image. Continue to click on it as the frames advance,
as shown here:

The Tracking module automatically draws a line showing the track you have selected.
This is now Track 1.
6. Click Close Track to stop adding points.
The Tracking module displays data and information about the track in the Tracking
Data Table, as shown here:

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 Manual Tracking

7. Click the Add Track Manually button again to add another track. This will be Track
2.
8. Click Close Track to stop adding points. The image in your workspace should look
something like this:

Each track on the active image is identified with a different color. You can change the

color of the tracks using the Tracking Options button .

The Tracking Data Table displays information about the tracks recorded from your
image, as shown here:

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9. To display the statistics for each track, click the Show Tracking Statistics but-
ton. The tracking statistics are shown in the bottom half of the Tracking Data Table, as
shown here:

The Tracking Data Table has the following tools:

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 Manual Tracking

10. Click the drop-down arrow on Types in the Measurements group on the Count/Size
ribbon:

You will see the Measurement Options dialog.

You can edit the list of measurements displayed in the Tracking Data Table by choos-
ing items from the Available Measurements list box, and adding them to the Selected
Measurements/Filters list.
11. Use your cursor to highlight the Or. Distance measurement. Then click the Add but-
ton. The Or. Distance will appear in the list of Selected Measurements.

12. Click OK.

When you next look at the Tracking Data Table, the Or. Distance will appear in
addition to the measurements already selected, as shown here:

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13. Use your cursor to select Track 2.

14. Click the Hide Tracks button to hide Track 1.

Only Track 2 will be displayed on the image.

15. Click the Show All Tracks button.

Now both tracks will appear on the image.

16. Click the Save Tracks button.

You will see the Save Tracks dialog box.

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17. Enter the file name as Mytracks.trc.

18. Click the Save button.
Your tracking information is now saved in the Tracking folder.
19. Go to the Data Views group on the Tracking ribbon.

20. Click the Show Graph button .

The graph displays all the tracks found on the image.

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The pull-down list displays the different graphing options.

Automatic Tracking
In this part of the tracking exercise, you will learn to use the automatic tracking feature in
Image-Pro. This feature uses current segmentation settings defined in the groups Seg-
mentation Options, Threshold, and Measure, groups to detect objects on all frames. You
should define all segmentation parameters to identify the tracking objects before this oper-
ation. You can adjust threshold level, set measurements filters to eliminate unwanted
objects, like dust, from tracking etc.

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 Automatic Tracking

1. Open the sample image polyp.seq.

2. Open the entire sequence.
3. Select Object Tracking from the Measure tab

You will see the MotionTracking ribbon:

4. Click the Auto All button in the Segmentation Tracks group.

5. Click Yes when Image-Pro asks if the outlines are correct.

You will see a message indicating the number of tracks found on the image:

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6. Click OK.
You will see the tracks appear on the image:

7. From the Tracking Options group, click the Histogram button .

8. Use the pull-down to display a different measurement, such as the Or. Distance.
You will see a histogram of the selected tracking data.

Intensity Tracking
The Intensity Tracking feature operates on objects of interest that have been captured
over time in an image sequence. This feature allows you to measure the objects'

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 Intensity Tracking

intensity changes over time.

When you click on the Intensity tool on the Measure ribbon bar, the Intensity Track-
ing ribbon bar is displayed:

To create an intensity track for a sequence:

1. Open the sequence that contains the object(s) of interest.
2. Using one of the drawing tools in the Select group on the Intensity Tracking ribbon
bar, draw an ROI around the object(s) of interest.
3. Click on the Types tool to select the kinds of measurements you want reported on.
4. Click on the Data Table button to view the intensity track data results.

Select Tools
The Select groups allows you to select existing tracks, draw regions on the image to create
intensity tracks, toggle visibility of tracking overlay, show/hide selected tracks, delete
selected track and remove all tracks.

The Draw button allows you to select various tools to create regions for intensity track-
ing:

Your region can be a polygon, ellipse, circle or rectangle. The most recently selected tool
becomes the default tool.

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Intensity Measurements
Click on the Types tool to display the Measurements dialog box:

This dialog box shows a sortable list of measurements available for selection in the left-
most panel (titled "Available Measurements"). Measurements can be selected by adding
them to the list in the right-most panel (titled "Selected Measurements").
For example, in the above image, six measurements have been selected: Regions: Intensity,
Mean; Regions: Intensity, Max; Regions: Intensity, Min; Regions: Intensity, Std Dev;
Regions: Integrated OD, and Regions: Area.
Use the two pull-down list boxes at the top of the Available Measurements panel to filter
the list of available measurements. The left-most pull-down list box (the one that shows
"All Types" in the image above) allows you to filter the available measurements list to
show only those measurements applicable to a certain kind of feature. Not all meas-
urements are applicable for all features. In the left-most pull-down list box, select the type
of feature that you want to select measurements for. If you want to see all measurements,
select "All Types."
The right-most pull-down list box (the one that shows "All Categories" in the image
above) allows you to filter the available measurements list to show only those meas-
urements belonging to a selected category. For example, you can limit the list to only
those measurements related to Density analysis, Shape analysis, or Holes analysis. If you
want to see all measurements, select "All Categories."
A description of each measurement can be accessed using the Image-Pro tooltips feature.
Simply position your mouse over the name of the measurement in the Available Meas-
urements panel, and view the tooltip for that measurement.

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Intensity Tracking Options

The options dialog can be opened using the Options button:

The options can be saved, loaded, or reset to default.

The Options dialog by default is opened in the right side panel:

The panel includes controls for changing the Font, Labels, and Outlines.

Intensity Tracking Data Views

The options in this group allow you to view different displays of measurement data.
Data Table: Opens the Tracking Table and populates it with the intensity tracking data
for the tracks in the active sequence. The measurement data for each track in the sequence
is displayed in the data table. A sample tracking table is shown here:

The Name column is editable, so user can give meaningful names to the tracks if neces-
sary. Clicking the column headers will sort the data in the table.
The tracking toolbar shows the following tools:

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Any track can be set as the Reference track by selecting the Set as reference track
button.
When reference track is set, all track parameters are calculated relative to the reference
track. Reference tracks can be used to:
n Correct geometrical position (velocity, acceleration) of test tracks assuming that the
positions of objects on the reference tracks are static. The correction is done sub-
tracting position of reference object on every frame from all test objects. It can be
used to correct camera shifts during sequence acquisition.
n Correct optical density (or any Regions parameter), where the reference track is
placed on the background area and all density related parameters of test tracks will
be corrected by the intensity on the reference track. It can be used for background
intensity correction.

Clicking the Reference track button a second time resets the reference track.
Any track can be set as the Normalization track by selecting the Set as

Normalizationtrack" button. It can be used for normalizing optical density (e.g.

Bleaching normalization), where the normalization track will be placed on the reference
intensity area and all density related parameters of test tracks will be normalized by the
change intensity on the track.

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Note that the values of the normalization track are singles, because they are normalized

individually. Clicking the Normalization track button a second time resets the nor-
malization track.
If more than one track is selected as a Reference or Normalization track, the average value
of the marked tracks is used.
Clicking the right mouse button over the table opens a context menu relates to the track
under the cursor:

If the Scroll to Object option is on, selecting the object in the table will make visible on
the image scrolling the window to the object position. It can be used when the image is
zoomed and some objects are outside the visible area.
The Color control allows you to change the color of the track.
Data Histogram: Opens the Tracking Histogram which allows you to view distribution
data for selected measurements. A sample histogram is shown here:

Object Table: Opens the Track Table and populates it with the measurement data for each
intensity measurement of the active track. A sample object table appears here:

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The Index spin box specifies the ID of the active track. For example, in the above table,
the Index is "1" indicating that the active track is "R1," and the data for "R1" is shown in
the data table. To see the data for a different track, simply change the Index number.
Data Graph: Opens the Tracking Graph which allows you to view a graphical rep-
resentation of the data in the Tracking Table.

The Tracking Graph shows a graph of the selected measurement value across all frames of
the sequence file. For example, the graph above shows the mean intensity values for the

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objects of interest over time. Note that the Tracking Graph is interactive with the Track-
ing Table. Tracks selected in one place are automatically selected in the other.

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Features Manager
The Features Manager in Image-Pro allows you to create collections of predefined items
called Feature Collections. Once features are in the collection, they can be converted to
other types of features, or added as-is to other images. The Features Manager handles fea-
tures from your active Annotations, ROIs, Measurements, Line Profiles, or Grids.
Each feature definition included in a collection can be applied repeatedly to a single
image or can be applied to different images. Feature collections can also be saved so that
they are available across sessions, and so that you can manage your collections in different
files. Using the Features Manager, you can:
l Define feature collections by adding features from your open images, or deleting fea-
tures from the current collection
l Apply any feature from a collection to the current image
l Convert features from one type of feature to any of the other supported types
l Save the current collection to a file
l Load a previously saved collection

NOTE: The Feature Manager tool is available via the Features group on the Select tab.
By default, the Features group is hidden. If you don't see it, right click somewhere on the
Select ribbon to display the list of available groups, and click on Features.
In addition to the instructions below, you can read more about the Features Manager
earlier in this manual. " Features Manager " on page 281

To Add a Feature to the Current Collection

Feature collections are created by adding existing features (that is, features already defined
in the active image) to the list of ‘remembered’ items through the Features Manager dialog
box. You can add a single feature definition, or you can add a group of definitions under a
single name.
1. Use any of the supported application tools to create features. Annotations, ROIs, Meas-
urements, Line Profiles, and Grids are all supported.
2. Click on the Add button. You will see a list of whatever types of features are avail-
able on the active image:

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 Features Manager

If you have not previously created a collection, use the New collection menu item to cre-
ate one. The new collection definition is added to the list box under a heading for the act-
ive image:

The new group of features can be renamed by clicking in the name field:

Once one or more collections are available in the Feature Manager, the Add list will give
you a choice of where to add the new feature(s):

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Features can be added to the original image, or any other open image, using the Set Col-
lection To Image button ( ).

Converting Features
You can convert features from one type to another by changing the selected type before
adding the feature to the image. The types of features that you can convert any given fea-
ture to depend on the feature type. As an example, to convert a group of ROIs into a group
of measured regions, change the feature type to Measurements:

Then click the button to set the converted measurements to the active image.
Note that if you set the converted features back to the original image, the new features are
added to the image. The original features will still be there until and unless you choose to

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 Features Manager

delete them. The original and new features will be overlaid on top of each other, which
may make it difficult to see one or the other.

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Data Collector
Data Collector provides a place where you can accumulate, summarize, and store inform-
ation from the analysis of multiple images.
Unlike most tools in Image-Pro which show data one image at a time, Data Collector
provides a place where you can collect analysis data from multiple images. Tools such as
Count/Size, Histogram, and Manual Measurements are set up to respond to collection
requests from Data Collector. To check whether a given tool supports Data Collector,
look for its name in the Types drop-down menu in the Data Collector group.
Data can be collected in two ways: Automatically, every time new data become available
from one of the data sources, or at specific times that you can specify by pressing the Col-
lect button. Each new collection updates the statistical summary available on the Statistics
page. One way for Data Collector to be aware of the various data items that can be col-
lected is to first invoke the tools that supply them. It is also possible to specify a list of col-
lected items via macro before or after any data source is invoked. In this case though, data
will not be collected until the tools are invoked and supply the data.
You can find the Data Collector group on the Measure ribbon:

Click the drop-down arrow to see the Data Collector options:

n Collect: Clicking the Collect button will collect data from the active document.
n Collect from All Frames: Use this option to collect data from an image sequence or
movie file.
n Buffer size: Use the spin buttons to define the size of the rolling buffer used for col-
lecting data from live images. The buffer size defines the number of data blocks (col-
lection events) to be retained in the data collector. When the limit is reached, the data
blocks collected at the beginning are deleted and new blocks are added to the most

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 Data Types

recent part. If buffer size is set to 0, no limit will be used.

Types: The list of collected data types is defined in the Types drop-down menu.
These are discussed in the next section.

Automatic Collection: This button turns automatic collection on or off. With Auto-
matic Collection on, the collection process is triggered each time one of the tools gen-
erates new data. When Automatic Collection is off, no new data is collected until the
Collect button is pressed. This feature is useful when collecting intensity data from live
preview workspace.

Delete Last: Click this button to delete the last collected data block.

Delete All: Click this button to delete the whole collection. Only data stored in Data
Collector will be deleted. This does not affect any of the data stored by the data sources or
data collected prior to that and saved to disk.

Save: This button saves the list of measurements to a file.

Load: This button loads the list of measurements from a file.

Reset: Clicking the Reset button will reset the defaults.

The new data table, block, graph, histogram, scatterplot, and 3D windows will be dis-
cussed in subsequent sections.
Note: In order for measurements to be collected automatically from the live preview work-
space a density or luminance measurement type must be selected in the Types list in the
Measurements group.

Data Types
The list of available data providers and data types can be selected in the Types drop-down
dialog:

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The Available Measurements panel shows the list of available data types/measurement
from the selected data providers, such as Count/Size or line measurements. Data providers
may have multiple tables of measurements. Selected measurements are those that meas-
urements you choose to collect.
The Selected Measurements list contains two columns: Name and Visible. The visibility
of the measurement can be switched off by clicking the corresponding checkbox. This fea-
ture can be used with derived and user-defined measurements. In this case, all measurement
columns that are included in the expression of the derived measurement can be hidden in
the output table and only the resulting measurement is shown.
The tooltip over the measurements in both tables provides extended information about that
measurement, including Caption, Name, Data Type, Description and Expression (some
properties may be empty if not available).

Refresh: Clicking the Refresh button will add new properties from the active doc-
ument to the list.
Some measurements may be extended with new tables and measurement information col-
lected from the active image, as shown here:

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 Data Types

Reset: Clicking the Reset button loads the default list of available measurements for
the selected data providers.
The list of available measurements can be very extensive, so finding the necessary meas-
urement can be difficult. If you know the name of the measurement you can use the quick
search option. You can type a part of the word in the "Find..." textbox and only meas-
urements with the given sub-string in the name will be included in the list:
Sync will add all statistics and sub-measurements for a given type to the available meas-
urements list.
The list below shows only measurements that include "size”:

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Any selected measurement can be added to the selected list by double-clicking on the

name of the measurement and clicking the Add button . Note that if a group
header is selected, all the measurements in that group are added. Clicking the Add all but-
ton will add all available measurements to the visible list.

New user-defined measurements can be added clicking the New button .

It will open the following dialog:

You can select the table where you want to add the measurement, measurement type,
define a name or caption, and create an Expression. An Expression can include any exist-
ing measurements that are listed in the Variables list, and may include arithmetical and
logical operations between them. Clicking the Insert button will insert the selected
variable into the Expression line.

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 Data Types

It is possible to include variables from other tables. The variables can be created using par-
ent-child relations. Tables from the Image data provider can be used as parents for all other
providers.
Available variable names are generated automatically and added to the Variables list:

The example above shows how to add an Image Name column from the Image table to
the Measurements table.
Tables with parent status (tables from Image data provider) can include measurements from
child tables (e.g. Measurements). Since one row of the parent table corresponds to multiple
rows of the child tables, only aggregate functions can be used.
An example is shown here:

In this example, new measurements are added to the ActiveFrame table that corresponds
to the maximum line length of all measurements on the given image.
Note that by default, the Variable list inserts variable with Avg aggregate. You will need
to replace it with the required aggregate type. Available aggregates are:
Sum (Sum)

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Avg (Average)
Min (Minimum)
Max (Maximum)
Count (Count)
StDev (Statistical standard deviation)
Var (Statistical variance).
The complete list of rules creating Expressions can be found by clicking the Help button
.

Any existing measurement can be modified by clicking the Edit button :

In Edit mode, the table, name and type cannot be modified. Double-clicking on the name
of any measurement will open this dialog also.

The list of measurements can be saved to and loaded from a file using the Save and/or
Load buttons:

Clicking the Reset button will set the defaults back to their original states.

Data Windows
Data Collector has 6 data windows, which can be opened using any of the following con-
trols:

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 Data Windows

Data Table
The Data Table shows data collected from all data sources. The table has a multiple tab
layout where every tab represents a separate table:

Statistics: This panel can be activated using the Show statistics button.
Data Collector data can be saved and loaded from a file, which allows you to analyze data
collected earlier, or continue to collect data in the next session. The data is saved with
"adoxml" extension.

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The data can also be exported to Excel™ as a tab-delimited file.

Viewing a Data Table

The Data Table displays measurement values created by manual measurements or Coun-
t/Size operations. The data table appears as part of the View group on the Measure ribbon:

When you select the Data Table, it appear at the bottom part of the application window.

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 Data Windows

The table contains measurements values taken from the active image.
The data table will generate measurement names using the abbreviations shown below for
the measurements.
Abbre- Meas- Abbre- Meas- Abbre- Meas-
viation urement viation urement viation urement
Name Name Name
AN Angle AB Angle BFA Best-fit
between Arc
Lines
BFC Best-fit BFL Best-Fit
Circle Line
C Circle CCD Center to CPT Center
Center dis- Point
tance
E Ellipse FPT First Point L Line
LPT Last point MMD Minimum MXD Maximum
distance Distance
P Polygon PL Polygone PR Count/Size
Line Polygon
region
PPL Point to PWL Pitch with

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Point dis- Line

tance
R Rectange T Tag

You can manually rename any features in the data table.

The header includes measurement names and units (if the Units checkbox is active in the
Labels ribbon bar of the Count/Size tab). The measurements can be sorted by clicking on
the column header. The Feature Name can be edited.
Data rows can be selected and this selection is reflected on all data windows and the
image itself.
The Data Table toolbar includes:

Note that are statistics is calculated only from visible measurements. Clicking the Select
measurement button opens a dialog where you can select measurements to view. Clicking
Create Mask creates a new image with a binary mask of the objects in the selected region.
Clicking the right mouse button above any cell will open a context menu, similar to one
shown here:

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 Data Windows

Selecting Set measurement as default will set the selected measurement as label for the
given type of features. The default measurement column color is violet.
If you want to define a new origin and direction for the X axis of the calibration, then
select Set line as X axis item and the beginning of the line will become the origin and the
direction as the offset angle of the newly created spatial calibration. Note that pixel size in
X and Y directions remain unchanged from the previous calibration.
If you want to create a calibration based on the size of known features, use Calibrate by
feature item. It will open the dialog shown here:

Using this dialog, you can define the feature size, units and calibration name. Clicking Ok
will create a new spatial calibration based on this feature and apply the calibration to the
image.

Data Blocks
Data can be grouped to get statistical values per block or by any other key or combination
of keys.

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The Totals control defines the type of aggregate function calculated for every group.

Data Graph
Clicking the Data Graph button opens a window similar to this one. The Data Graph
shows selected data values in a chart:

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 Data Windows

Y axis and X Axis combo boxes select data to display, while the mode buttons

define other options.

Show All: When Show All button is selected, the data are plotted for all data sources.
When it's off, only the information from last data block is plotted.

Per Collection: The Per Collection(Block) button plots each block in a separate line
on the graph. When the option is off, all blocs are on the same line.

Per Item: This option plots the data per item in each block. The number of lines cor-
responds to the number of items in a block. It can be used to plot intensity data from dif-
ferent objects on a live image.

Data Histogram
The Histogram displays binned data for the selected measurements:

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Bins:The Bins control defines the number of bins. The number of Bins defines the gran-
ularity of histogram. When either Per Collection or Per Item is selected, the data are
grouped and each group is displayed using a different color.

Scatterplot
A Scatterplot displays the data distribution of two measurements. This feature is limited to
displaying only measurement data from the same table on one chart.
The Scatterplot appears something like this:

You can also create a 3D scatterplot, as shown here:

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 Data Windows

When Per Collection or Per Item are selected for either scatterplot, the data are grouped
and each group is displayed using a different color.

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Chapter 9 : Customizing the User Interface and Automating Processes

Customizing the User Interface and Automating Pro-

cesses
This chapter provides instructions for the many ways you can customize the
Image-Pro application interface. You can use a Japanese version of the user interface.
Image-Pro allows you to set up the interface in the most efficient way for your work. You
can also use macros, apps, and batch processing to speed up various tasks.

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Using the Quick Access Toolbar

The Quick Access Toolbar appears at the top left of the Image-Pro interface.

The Quick Access Toolbar allows you to display icons that, when clicked, execute a sys-
tem-supplied or user-defined command. In this way, the Quick Access Toolbar provides a
shortcut to commonly used functions.

To Add a Shortcut to the Quick Access Toolbar

1. Click the drop-down button next to the Quick Access Toolbar.
The following menu appears.

2. Click on More Commands in the drop-down menu.

The Customize Quick Access dialog box appears.

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 Using the Quick Access Toolbar

The list box on the left displays all the available commands and icons. The list box on
the right displays those commands and icons currently displayed in the Quick Access
Toolbar.
The Choose commands from...drop-down list displays different groups of available
commands, as shown here:

3. Select a group of commands to display in the list box.

4. In the list box, highlight the command you want to add to the Quick Access Toolbar.
For example, if you regularly use the Paste command in Image-Pro, you can make a
shortcut that performs a paste by adding a Paste command and icon to the Quick
Access Toolbar.

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Chapter 8

5. Click the Add button and the command appears in the right hand list box.
6. Click on the OK button.
The shortcut is added to the Quick Access Toolbar.

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 Customizing the Image Strip

Customizing the Image Strip

The Image Strip displays thumbnails of all opened images in the application and provides
an easy way of activating and deleting images. The Image Strip controls are located in the
Workspace group on the View tab’s ribbon:

Image-Pro opens the image strip automatically when you launch the application. It
appears on the left side of the application window.

If you have several images open, the image strip will display thumbnails of all of them:

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Chapter 8

The layout of the images can be selected from the context menu.
n Click the right mouse button on the image thumbnail to see the context menu.

n Auto Fill: all thumbnails are fitted to the strip window.

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 Customizing the Image Strip

n Single row/column: when this mode is activated the thumbnails are resized to fit the
width or height of the window. Scrollbars become visible if the number of images
exceeds the size of the window.

n Fixed Small: thumbnails are 64x64 pixels

n Fixed Medium: thumbnails are 128x128 pixels
n Fixed Large: thumbnails are 256x256 pixels
n Lock/Unlock selected windows: Select this option to lock the image windows for the
currently selected thumbnail(s). The lock will disallow panning, zooming, or bringing
other image windows to the foreground, depending on what is selected for Lock
Options.
Lock options: Allows you to select what you want locked when the Lock/Unlock selected
windows option is activated.

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l Pan Scroll : Synchronizes image pan and scroll positions

l Zoom: Synchronizes magnification of images
l Active Frame: Synchronizes active frame index on all locked images
l Auto Arrange: When this option is on the locked images will be arranged on the in
the application workspace to be visible. For example if you lock 4 images, they will
be arranged in 2 rows and 2 columns. The option arranges only images to be locked. If
the workspace contains more images, other images will be placed in the last pane
below the last locked window, as shown here:

Viewing Tooltips
Tooltips show the basic image information:

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 Activating and Closing

n To view an image tooltip, move your mouse cursor over the image thumbnail.
The zoom and pan controls allow you to resize the image or a portion of the image.
When the active image is zoomed and doesn’t fit in the viewer, the viewing area is shown
on the image strip thumbnail as a semitransparent orange overlay.
n Click and drag the center of the viewing area on the thumbnail to control pan
and scroll of the image.

Activating and Closing

1. Click on a thumbnail in the image strip to activate the corresponding window.
If the thumbnail represent a surface plot or 3D view (or other viewer) clicking will
also update the thumbnail.
2. To close the image, click the Close menu item from the context menu, or click the X
button:

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Chapter 8

To remove an image from the image strip, select that image and click the <Ctrl> or
<Shift> keys. More than one image can be removed at the same time using this method.
Using the interface customization capabilities provided by Image-Pro, you can customize
the Image-Pro toolbars by:
n Moving, docking, and undocking toolbars
n Showing/hiding toolbars
n Creating new toolbars
n Adding/deleting buttons from existing toolbars.

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 Using the Multiple Document Interface (MDI)

Using the Multiple Document Interface (MDI)

The Switch to MDI button will toggle the workspace windows to MDI windows or back
to tabbed windows. This feature allows you to view more than one image at a time, and
move the images and panels to different locations in the application.
This feature is located in the Workspace group on the View tab’s ribbon:

Normally, when the workspace windows are set to Docking, the image appears like this:

When you switch to the MDI view, several images can be viewed at once, as shown here
(these are shown in the Tiled layout):

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Chapter 8

The pull-down MDI menu allows you to select window display:

This version shows image windows in the Cascade layout:

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 Floating Panels

Floating Panels
One of the unique features of Image-Pro is the “floating” or “dockable” panels. The image
strip, details panel, mini-histogram panel, or other workspace areas can be moved inde-
pendently from the main image workspace. This may be particularly useful when working
with a dual-monitor system, as you may want to display the image on one monitor and the
details on the other. An example of floating or “undocked” panels appears here:

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 Tools and Layouts

Tools and Layouts

The View tab contains specialized tools and graphs to display information about the active
image or image set. Some of these tools allow you to see different aspects of the active
image also.

The Tools in the Workspace group allows you to load and save custom tool sets and lay-
outs for your specific situation. Click the drop-down arrow to see these features

Redock All Tools: Resets any open moveable panels that are undocked to their default
docked position.
Reset Tools Layout: Resets the application to its factory default tools layout.
Load Layout: This tool allows you to load a previously-saved tools layout. When you
click on this tool, the following dialog is displayed:

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This dialog shows all available tool layouts to be loaded. The Recent area shows a list of
the last loaded layouts. The Custom area shows a list of the custom layouts currently
stored in the Configuration Files folder. The Default area shows a list of the available pre-
defined layouts from installation directory \Resources folder.
SaveLayout: Click this tool to save the current layout of tools to a file that can be loaded
at a later time to restore the layouts to its current configuration. When you click this
option, you are prompted to give the layouts configuration file a name. Type a name for
the layout configuration file, then click Save. You can use the Load Layout tool
(described above) to restore the tools layout to its current configuration at a later time.

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 Creating a Custom Tab

Creating a Custom Tab

One useful feature of Image-Pro is the ability to create a Custom tab on the ribbon. It can
contain any of the features you use most frequently, saved in one place.
By default, the Custom tab appears on the far right of the ribbon, but you can control its
placement in the application Options. The default placement is shown here:

The first time you open it, the Custom tab’s ribbon will be empty. You can add any group
or tool to it, as described here:
1. Decide which tools or group you want to add to the Custom tab. For example, Annot-
ate:

2. Right-click the mouse button in the Annotate group. You will see the context menu:

3. Choose Add to Custom Tab.

The Annotate group now appears in the Custom tab’s ribbon:

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Chapter 8

You can drag the groups into different positions on the ribbon. The image below
shows that the Calibration and Filter groups have changed position:

4. To remove an item from the Custom tab, click Remove from Custom tab in the con-
text menu:

Rename and Make your Custom Tab the First Tab

If you want to give the Custom tab a new name, and make it the first tab that a user sees,
use the following steps:
1. Click on the File button in the top right corner of Image-Pro:

2. Choose the Options button at the bottom of the menu.

3. From the Options dialog, click on the Display section.

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 Rename and Make your Custom Tab the First Tab

4. In the Ribbon Tabs area, select First Custom Tab to display your custom tab as the
first tab.
5. Type in a new name for your tab in the Name text box

6. Press OK at the bottom of the Options dialog to apply the changes.

Your new tab should appear first in the tab list with its new, custom name.
In the example below, we have created a new tab, named Jacob’s Analysis which is
displayed first in the ribbon.

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Chapter 8

Hide or Show Tabs

Image-Pro allows you to hide or show the tabs to display only the tools you need. To use
this feature:
n Right click on any of the tabs to see the Show or Hide Tabs dialog.

Tabs with an active check mark will be displayed. Those without will be hidden from
view
For some situations, you may find it most effective to hide all of the other
Image-Pro tabs and focus only on the Custom tab or a specific App. To hide the tabs, use
the following steps:
1. Click on the File button in the top right corner of Image-Pro.

2. Choose the Options button at the bottom of the menu.

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 Hide or Show Tabs

3. From the Options dialog, click on the Display section.

4. Under the Ribbon Tabs section, select the Hide Tabs option and make sure Show Cus-
tom Tab is checked.

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5. Now you will see that just the Custom tab is shown. If you happen to have an image
opened, you will also see the contextual Image tab, which is displayed when an image
is opened.

Showing or Hiding Groups

You can also hide or showImage-Pro tool groups to only display the tools that you need
for your image analysis work. To use this feature, follow these steps:
1. Right click on the open space in the ribbon bar.

Tip: Click on the far right side of the workspace. In the example below, you would
click in the grey area to the right of the “Movie” button.

2. You will see the Show or Hide Groups dialog. Groups that are highlighted will be
displayed. Toggle this feature on or off to show/hide groups.

Expanding and Collapsing

Sometimes, you may need to resize the Image-Pro workspace to make it smaller. When
you do, you will notice that the groups on the ribbon collapse and some of the tools are
hidden. In the example below, the Calibration, Intensity, and Quick Save groups on the
right side of the Capture tab’s ribbon are collapsed:

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 Hide or Show Tabs

To expand and see the group’s controls, click the drop-down arrow, as shown here:

Users are encouraged to fully expand the Image-Pro workspace when at all possible, so
that all the tools and features are available.

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Image-Pro in Japanese
Image-Pro is also available with a Japanese-language user interface. It is installed along
with the English-language version from your electronic download or product CD.
To use the Japanese version, follow these steps:
1. Go to your Start menu and selectImage-Pro:

2. Choose Image-Pro 9.3 Japanese.

After a moment or two, you will see the Japanese version of the application.

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 Working with Macros

Working with Macros

As you become proficient with Image-Pro, you may find that you want to automate
routine procedures or tailor its interface to your specific needs. For example, you may
want to automate a series of steps that are performed daily, or perform certain steps only
when certain conditions exist. You might also want to call Image-Pro functions from a
program of your own creation. These levels of customization can be achieved with the
macro scripting facility provided with Image-Pro.
Macros allow you to translate a sequence of actions into a set of written instructions that
can be recalled and “played back” whenever they are needed. The scripting facility also
lets you add variable definition and flow control statements (e.g., looping and branching)
to these instructions, so that you can specify when and how often the actions are per-
formed.
The macro scripting features are found in the Macros group on the Automate tab’s ribbon
(this feature may not be available to all users):

Use the Record button to record a series of commands as you perform them. When Record
Macro is enabled, Image-Pro begins saving your commands to a script file using the script-
ing facility. It continues to record your actions until you instruct it to stop by clicking the
Pause Recording button.

Project Explorer
The Project Explorer panel displays the minimal scripting dialog. From here, you can find
and load existing scripts, as shown here:

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The Project Explorer appears as a dockable panel on the right side of the application
window. Macros and scripts are organized by modules and projects. Modules are files that
can contain multiple macros (or scripts). Projects are used to group related modules
together. The state of all these elements is maintained between sessions so that branches
that are not used can be collapsed, and only those scripts that are used be made visible.
The Project Explorer has a built-in search feature, which will highlight (and expand)
those scripts whose name or description contains the specified string.

The drop-down menu in the search box gives you access to search and browse tools.
Search My Macros lets you search for a saved macro containing a specific text string.
The App Center search will use a built-in web browser to connect to the App Center web
site where macros can be searched, rated and downloaded.
Browse Macros lets you search saved macros without specifying a text string.
You will find more information about macros and scripting in the Macro Scripting help
file provided with your Image-Pro installation.

Project Workbench
The Project Workbench is the place where you create and edit scripts and macros in
Image-Pro. You can find it on the Automate tab in the Macros group, as shown here:

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 Project Workbench

When you first open the Project Workbench, you will see the following workspace:

It covers the Image-Pro workspace, and can be moved and minimized without closing or
exiting.
The Project Workbench has two tabbed ribbons: Script and Edit. Both of them will be
described in the next section.

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The Macro Script Ribbon

The Script ribbon contains those tools for creating new projects, apps, or modules, or
recording, running, and debugging macros:

The Create New group contains the tools for adding a new project, app, module, or report:

Adding a project, app, or module is discussed later in this chapter. Reports are described in
Chapter 10 of this manual.
The Project group contains the basic operations for creating a macro:

Record Macro: Use the Record Macro command to record a series of Image-Procom-
mands as you perform them. When Record Macro is enabled, Image-Probegins saving
your commands to a script file using the Auto-Pro scripting facility. It continues to record
your actions until you instruct it to stop by clicking the Stop Recording or Pause Record-
ing button.
Load Macro: Clicking this button loads the selected macro script and switches to the
Run mode to run the macro.
Edit Macro: Clicking this button switches to the Edit mode so that you can modify the
macro. This action will invoke the built-in Macro Editor so that you can modify the mac-
ros as needed. When you have finished your edits, remember to save the file.

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 Project Workbench

References: Click this button to review or edit the list of active project references. A
sample list appears here:

License Generator: This new feature in Image-Pro allows linking your apps and scripts to
a specific Image-Pro license. More information about this feature appears later in this
chapter.
The View group offers you two different ways of viewing your macro script:

Designer view lets you edit macros and projects graphically. Switch to the Code view to
edit macros and project code directly.
The Run group is fairly self-explanatory:

Run executes the selected macro. Pause will stop it midway through. End stops the macro
execution.
Debug: For debugging purposes, there are occasions when it is useful to run your macro in
“debug mode.” Debug mode lets you execute the macro in step-by-step fashion, allowing
you to see the effect of each command. It can help you quickly identify the specific state-
ment that is causing a macro failure.

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The Debug function uses the following buttons:

Break: Turns a break point in the current line on or off.
Watch: Lets you watch the value of the current code selection.
Step Into: Execute the current line. If the current line is a subroutine or function call, stop
on the first line of that subroutine or function. (If the macro is not active, start it.)
Step Over: Execute to the next line. If the current line is a subroutine or function call,
execute that subroutine or function completely.
Step Out: Step out of the current subroutine or function call.

The Macro Edit Ribbon

Use the Edit Macro ribbon to edit an existing macro.

The Clipboard group contains the familiar Cut, Copy, and Paste commands.
The Search group lets you find specific macros or lines of code.

The Find command searches for a specific sting of code. Replace replaces one string of
code with another.
My Macros looks for a specific macro among all the macros stored on your computer.

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 Project Properties

Clicking the App Center button takes you to the Thermo Fisher App Center. This is a
searchable on-line database of macros, device drivers, plug-in modules, application pro-
grams and development consultants that support Thermo Fisher image analysis products.
Here, you can look for macros and other helpful information.
The Comment group lets you add or remove comments on specific lines of code.

The Dependencies group lets you import and export macro information and references.

The Windows group lets you navigate through multiple windows in the workspace.

Project Properties
All the settings affecting your projects are gathered on the Project Settings page in the cen-
ter of the Project Workbench. They can be displayed by double-clicking the project’s node
in the Project Explorer when you are working in design mode (in code view, the project’s
source code is displayed).

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Chapter 8

l Additional Files are only applicable to packaged projects. These properties allow you
to designate additional files that will be included in the package. When the files ori-
ginate from a known location (Images or Configuration Files folders for example) they
are installed in the same location on the target system.
l Compiled is used to switch between interpreted (the default) and compiled mode. Com-
piled mode is fully compatible with VB.NET and faster, but does not allow debugging
or macro design.
l Documentation allows you to browse for a documentation file (.PDF, .DOC…) that
will be included in the package. It will be highlighted in the Project Explorer with a
small question mark icon used to launch it.
l Filename is the name of the project file.
l Hidden enables you to hide this project so that it’s not shown in the Project Explorer.
l Imports lists all the project level “imports” which are used to select the libraries that
are directly available for coding in the global namespace.
l Packaged is true for packaged projects. Project packages are self-contained compressed
archives including all the files needed by the project.

Project Workbench Options

Project Workbench options are displayed in the Properties panel when “My Projects” is
selected, it can also be accessed with the Options button of the Workbench’s File menu.

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 Project Properties

These options gather all global settings used by the Project Workbench including edit-
ing tool configuration and display selections.

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All about Apps

Apps are little applications that run inside Image-Pro. Like mini-programs, they have their
own user interfaces and they automate complex tasks in and through Image-Pro.
When you first open Image-Pro, the Apps group of the Apps ribbon will be empty. When
an app has been loaded (as described in the next section), it appears on the Apps group, as
shown here:

Loading an App
Image-Pro allows you to load pre-recorded apps, projects, and macros that you may find
useful. To add an existing App (project or macro), follow these steps:
1. Go to the Search group on the Apps tab in Image-Pro.

2. If you want to load one of the pre-recorded apps provided by Thermo Fisher, click the
App Center button.
The browser will take you directly to the Apps portion of the website:

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 Loading an App

You will see a list of available apps.

3. Click the name of the app you want to use.
You will see a description of the app in the browser window.
4. Scroll to the bottom of the browser window, and click the Download button.

5. You will be asked to accept the license agreement. Click I understand and accept the
user agreement.

Apps and projects are stored in the Scripts folder of Image-Pro. Apps have the exten-
sion *.ipx.
6. Click Save.
You will see the Open Project dialog.

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7. Select the app you want to load, and click Open.

Your app appears in the Apps tab:

Running Apps
Once a project is loaded, its app appears in the Apps ribbon.

It can be started by clicking the appropriate button. To run the sample app, follow these
steps.
1. Open the image you want to process in the workspace. This example shows
tumor.tif.

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 Running Apps

2. Click the name of the app you want to run. This sample app is called
White Balance.
The contents of the app appear in the right-side panel:

3. Click Measure White Balance.

The first step in the app appears.

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4. Follow the instructions, and click OK.

5. Click Correct Image.
A new, corrected image appears in the workspace.

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 Using the Project Explorer

Using the Project Explorer

The Project Explorer appears panel on the right side of the workspace. It contains a
list of current projects.

1. Use the Search Macros drop-down to find a list of current projects:

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2. Click Browse Macros. You will see the Select Project dialog box.

3. Click the name of the project you want to open. It should appear in the Project Work-
bench project area.

4. Click the Edit icon to make changes to your item. When you are finished, click the
Load icon again to reload and run your app or macro.

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 Creating a New Project, Module or App

Creating a New Project, Module or App

Projects, apps, modules, and reports can be created using the Create New group on the
Script tab of the Project Workbench, shown here:

l Add New Project creates a new scripting project where you can store apps, mod-
ules and reports.
l Add New App creates a brand-new app in the current project.
l Add New Module creates a new scripting module in the current project.
l Add New Report creates a new report.

Adding a New Project

1. To add a new project, click the Add Project button. You will see the New Project dia-
log:

2. Give your new project a name and click OK.

1) To change the location where the project is stored, click the Browse…button. You
will see the Browse for Folder dialog.

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2) Select a location and click OK. To store your project in a new folder, click Make
New Folder.
3) To cancel without saving your project, click Cancel.
3. Indicate what items your project will contain: a module, app, or report:

4. Click OK. The framework for your new module appears in the Scripting Workbench.

Adding a New Module

A module is a file where you can store apps and macros. To add a new module to the cur-
rent project, follow these steps:
1. Click the Add Module button. You will see the New Module dialog:

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 Creating a New Project, Module or App

2. Assign a name to your new module, and click Save.

Your module will automatically be stored in the current project.

Adding a New App

To create a simple app, follow the steps below:
1. Go to the Macro group on the Automate ribbon.
2. Choose Project Workbench.

3. Click App in the Create New group.

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4. Give your app a name and click Save. You will see the App Form appear in the
Designer workspace:

5. Drag and drop the actions and features for your app from the Toolbox into the
Designer Workspace. They will appear as individual buttons:

The toolbox on the left-hand side contains expandable lists of actions and features that
you can include in your app.
6. From the Properties panel on the right, add properties to your app, such as a descrip-
tion or caption:

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 Designing Apps

Note: When cutting and pasting controls from one area of an App to another, or from
one App to another, the code attached to these controls is left as is and is not trans-
ferred to the new location.
7. When your app is complete, use the Save button to save it.

This app will appear on the Apps ribbon.

Designing Apps
Apps and Forms have special designer tools which are displayed when loading them in the
Project Workbench. Double-click on the App to see the Designer window:

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In this mode, the Project Workbench displays several tools which are useful for the design
of Apps.
l The Toolbox on the left side displays the controls that can be used to build your App.
Once selected, these tools are activated and enable you to create the control in the
App design area. These controls can also be dragged and dropped into the App
designer.
l The Toolbox also lists macros and commands that provide a convenient way to create
preconfigured buttons that will call selected commands and macros.
l The central area is the App or Form designer and is where the Apps are designed. Con-
trols can be moved around, aligned, copied, or pasted.
l The Property Panel on the right side displays the properties of the selected control or
of the App itself, including App Settings which are used to document and control the
App visibility in Image-Pro. The control and App properties can be modified in the
Properties Panel, and their new values reflected immediately in the design area.\
They can be edited, modified, or linked to a URL.

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 Designing Apps

These properties are displayed in the App’s label:

You can choose an icon from the icon library, shown below, or create one of your own.

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If you create an original icon,Image-Pro supports *.PNG, *.BMP, and *.JPG

formats. The standard size is 32 x 32 pixels.
The icon appears with the app name in the ribbon:

Finally, the visibility of the apps and their icons depends on the App Settings where you
can hide or show branding and contact information, as shown here:

Saving Your Project

1. To save your new project (app or module) go to the File menu on the Scripting Work-
bench:

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 Designing Apps

2. Choose Save.

3. Choose a format to save your project:

l File saves the active code module in the current *.ipx file.
l File As saves the active code module as a new *.ipx file.
l Project saves all the current project’s files.
l Project As saves all the current project components in a new project file.

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4. Click Save. Your project file will be saved in the selected location.

Packaging a Project
In order to share your new app or project, you will need to package it. Follow the steps
below.
1. Go to the File menu on the Scripting Workbench.

2. Choose Package.

3. Select a format for your project package:

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 Designing Apps

l Project Package is an unprotected format that contains the active project and all
its related components (macros, modules, and apps) in a single file.
l Read-Only Package stores your project and its components in a file that can be
read but not edited.
l Encrypted Package stores your project in an encrypted file where all the macros
will be hidden. This file can be read but not edited.

4. Choose Save. Your project package is saved in the selected location.

Licensing and Encryption

Licensing associates a particular app, project, or macro with a specific copy of Image-Pro.
Encryption protects your work against unauthorized use or modification. To use this fea-
ture, follow the steps below:
1. Go to the File menu on the Scripting Workbench.

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2. Choose Package.

3. Choose Encrypted Package.

Before assigning a name to your package you will be prompted to choose one of 3
encryption options:

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 Designing Apps

l Basic Encryption is the same as the current method where the project package is
protected by a secret key, and its code is never revealed. You will not be able to
see the code but can run the macros or apps without restrictions.
l Password Protected is a new option that may be of interest for some (to keep some
projects private in a multi-user environment for instance). In this case the project is
protected by a unique private password, which is required in order to load the pro-
ject. Once the project is loaded, the code is visible but read-only. Note that if the
password is lost, the project can’t be loaded.
l Licensed is the option that associates the project license with an Image-Pro serial
number. The first time such a project will be loaded, you will be prompted to enter
a license key, which will then be remembered if valid. The license key as well as
the project’s encryption password is also dependent on a secret key that the project
developer is the only one to know. A checkbox allows to start the Project License
Generator automatically once the project is packaged.
l Upload to App Center let you send your package to the App Center automatically.

4. Choose your encryption option and click OK.

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Project License Generator

The license key required for licensed projects is created by a tool accessible from within
the Scripting Workbench or from the Automate tab, as shown here:

1. Click License Generator. You will see the Licensing dialog:

This tool prompts for the project package, the secret key defined when the project was
packaged, an optional serial number as well as an optional expiration date. The com-
bination of all this information results in a 25 characters string which is similar to the
Microsoft product keys.

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 Designing Apps

2. Enter the requested information and click Generate.

Changing any of the information will result in a totally different license key, and as
many license keys as desired for a given project can be created using the tool.
Time limited projects are highlighted in the macro explorer so that users are aware of
the availability of their projects.

Expired projects will fail to load.

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Batch Processing
Batch Processing allows you to run a series of tasks in a predefined order. Scripts can be
grouped into batches. The Batch Processing feature appears in the Tasks group on the
Automate tab’s ribbon:

You will need to check Show Automate Tab in the Ribbon in the Popular options to see
and use this feature.
The Batch Processing panel appears on the right side of the application workspace.
It is divided into 3 sections that allow you to:
1. Select the macros that will be used for the batch.
2. Select the images, documents, files and folders to process.
3. Start the batch and control its options.

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 Selecting Macros

Selecting Macros
Step one in the process is to select the macros that will control the batch processing.
The Select Macros section provides three drop down lists that expose all the loaded mac-
ros.

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l The Run Before macro runs once when you press the Start button. Its optional
parameter is the list of all the data that will processed.
l The Loop On macro runs for each document selected in section 2 (or loaded from
a selected file or folder). Its optional parameter is the document being processed.
A sample list of available macros is shown here:

l The Run After macro runs once after all the selected documents have been pro-
cessed.
Macros selected for batch processing may define parameters to handle the documents
loaded during the operation. Otherwise, they’ll use the active document. These macros are
analyzed to assess their ability to run in background threads and enable multithreaded
batch processing.

Macros containing commands interacting with the user interface (identified by the sym-
bol) are not suitable for background processing. Compatible macros show the green symbol
instead.
When you have located the macro you want to run, click OK.

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 Select Images

Select Images
In step two, the Select Images section is where you select the data to be processed by the
batch. There are three types of data that can be specified, the details of each are presented
in a tooltip.

The three types of data are:

l Image Documents loaded in the application
l Single image files
l File folders.
Note that the selection list can be expanded by resizing the panel vertically. Selected doc-
uments can be removed individually or cleared using the 2 right buttons.

Images selected in the Image Strip can be copied by pressing the Image button, or loaded
images can be selected individually from its drop-down list. The Add AllDocuments item
will add all loaded documents in one click. Images can also be dragged from the Image
Strip and dropped into the batch panel.

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The Add Folder button allows you to select a folder containing images to be processed
using a standard Browse for Folder dialog.

The type of image file to be processed is defined in a drop down entry of the same button,
which precedes the list of the most recently used folders. Subfolders can be processed
when this option is selected in that same menu

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 Process the Batch

Folders (as well as files) can also be dragged from Windows Explorer into the batch pro-
cessing panel.

Process the Batch

In step 3, the Process button is then used to start the batch processing, or change one of its
options.

l Choose Process Subfolders to process subdirectories of the selected folder.

l Check Display Task Manager to display the Task Manager when starting the batch.
l Select Display Documents to display the documents while they are processed.
l Choose Save Modified Documents to automatically save the processed documents.
l Choose Close Documents to close them after they are processed.
l Choose Loop On Set Locations to run the "Loop On" macro on all set locations.

Grouping and Sorting

The Grouping and Sorting feature allows you to select and process groups of files
together instead of individually. The documents loaded from the files are loaded together
and received by the Loop macro as an array. When the Display Documents option is
enabled, workspaces are created for all documents in the group.

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Grouping is controlled by the last toolbar button. This button turns grouping on or off,
and also provides several basic grouping schemes, as shown here:

When files are grouped, you can control the order of groups and the order within each
group by using the column headers.

Advanced Batch Processing Options

When basic grouping is not sufficient, the Advanced Option menu item, as well as the
expandable splitter just below the toolbar, allows you to display an additional panel with
grouping and sorting parameters:

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 Process the Batch

Files and documents can be sorted using the full path or by using some of the characters in
name. This name part can then be interpreted either as a character string or as a number.
In the example below, files are sorted by pattern.

Similarly, grouping is performed based on the specified part of the name, interpreted either
alphabetically or numerically. You can create groups of fixed size if the grouping pattern
is a simple number.

Some examples of grouping patterns are:

l Group by folder:^(.*)\\[^\\]* As Strings
l Group by extension:\.([^\.]*)$As Strings
l Group by index:(\d+)[^\\]*$As Numbers
l Group by “channel”:([^\d\.\\]+)[^\\]*$As Strings
l Group by “experiment”:_(\d+)As Numbers

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Grouping Macros

When grouping is used, the Loop Macro is intended to process an entire group of files
or documents together instead of individually. Therefore, it should be written in such a
way that it will process all the files in the group.
This can be accomplished in two ways. The first approach relies on selecting both the Dis-
play Documents and Close Documents options, and then processing all the loaded work-
spaces in the macro.
The second approach does not require displaying the documents but instead uses a macro
parameter to access each item in the list as in the following example.

PublicSub ListMacro(list)
ForEach doc AsObjectIn list
ThisApplication.Output.PrintMessage doc.DisplayName
Next
ThisApplication.Output.PrintMessage "----------"
EndSub

Task Management
The Task Manager is accessed through the Tasks group on the Automate tab.

It can also be displayed by double-clicking the circular progress control that appears
in the status bar while tasks are being processed.

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 Process the Batch

The Task Manager is where the status of the various tasks (commands and macros) run-
ning in the application is being displayed and where they can be controlled.
Clicking the Task Manager button displays the Task Manager panel. This example
shows an empty window:

When filled, the Task Manager displays a hierarchical table of all the tasks that have
been scheduled. It includes their descriptions and status of each task. It also allows you to
control these tasks (Priority, Cancel, Clear...) using a context menu.

n The Input column describes the input data for each task, including the frame selec-
tion if available.
n The Progress column is updated incrementally while tasks are being processed.
n The End Time and Duration columns are estimated when the command starts and ini-
tially appears in light gray.
n The Priority column which can be changed with the context menu or in place controls
the sequencing of commands.
n The Engine and Thread columns indicate where commands are processed and by
which thread (commands are initially issued in the main engine then moved to back-
ground engines for processing).

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n The Email column, which is only visible when an email address and HTTP server
have been configured in the Task Scheduling options, allows you to select those
tasks which will notify of their completion by an email similar to this.
Green is used to indicate the commands that have completed successfully. Errors are dis-
played in red. Double-clicking will bring up a detailed error dialog.

A context menu helps you control the Task Manager settings:

n Run starts a task that has not been executed yet immediately.
n Schedule configures an idle and unprocessed task for batch processing.
n The Set Priority submenu lets you change the priority of the various tasks.
n Cancel allows you to cancel a background task during its execution.
n Delete and Clear All Processed are used to remove unwanted tasks from the
Task Manager.
n The Select Columns item brings up a floating window to and from where
columns can be moved to configure the Task Manager display.

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 Task Scheduling Options

Task Scheduling Options

Options related to batch processing are divided in two categories. One is displayed in its
own option group and the other one appears in the Advanced group. The Task Scheduling
tab of the Options dialog contains the most common settings:

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General
Batch Process All: Process all compatible operations in the background tasks using the
task manager.
Email Results To: Email address where to send notifications
SMTP Server Address: Emails are sent using this server. Note that some servers do not
support this feature.
Email All Batched Tasks Results: Send email for all tasks.

Task Manager
Expand All: By default, commands are not expanded when they have sub-commands res-
ulting of a processing split, this option allows to auto-expand all commands.
Display Hidden tasks: Some commands are used internally and would confuse the display
(filter preview, guard band processing) so by default they are not displayed in the task man-
ager.
Remove Processed Tasks: Clear tasks from the Task Manager once they are done.
Number Of Processed Tasks To Retain: When clearing tasks, keep that many.

Advanced Options
The Task Scheduling tab of the Options dialog is where all the various scheduling para-
meters are defined:

Split Large Sequences: Allows Image-Pro to split processing of sequences.

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 Task Scheduling Options

Minimum Frames per Engine: Displays the minimum allowable number of frames for
splitting sequences
Split Large Single Frames: Allows Image-Pro to split processing for large frames when
possible.
Minimum Region Size per Engine: Indicates the minimum allowable region size for split-
ting large frames.
Number of Background Engines: This setting defaults to the number of cores minus 1
minus the number of priority engines
Number of Priority Engines: Displays the number of engines.
Threads Per Core: Indicates the number of threads allowed for each physical core.
Threads Os Factor: Displays a number between 0 and 1 that defines if and how to mod-
ulate the number of threads based on CPU utilization. 0 ignores the CPU utilization, 1
decreases the Threads Total Count with the CPU usage ratio.
Threads Queue Factor: Displays a number between 0 and 1 that defines how much pri-
ority to give to the first commands in the task queue as opposed to distributing resources
amongst all queued commands.
Threads Task Factor: Displays a number between 0 and 1 that defines how to distribute
resources between background engine main threads and operator sub-threads. 1 means that
all free threads are made available to task scheduling, 0 means that all free threads are
made available to sub-thread allocation.
In case the application is closed while background tasks are being processed, a small icon
appears in the Status Bar notification area. Double-clicking this icon will restore the
application window, and a context menu also allows you to cancel all remaining tasks. If
they are not cancelled, all the tasks will be processed and the application will exit after
that.

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Chapter 10 : Generating and Printing Reports

Generating and Printing Reports

This chapter explains how to create and print reports in Image-Pro.
The Report tools are located on the Review tab’s ribbon:

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Creating and Printing Reports

Image-Pro allows you to create reports from images and data saved in your application.
The Reporting feature uses an interface that is similar to Microsoft Word. You can display
your data in any of several predefined layouts, and then send them to an output window,
print device, or a file. To begin creating your report, go to the Report group on the Review
tab’s ribbon:

1. To begin creating a report, click the Images button.

The red number superimposed on the Images button indicates the number of active
images. You will see the drop-down menu with all the available images on it. Instead
limiting you to just the document open in the workspace, this feature allows you to
select any document loaded in the software.

Images with a check next to them are available to include in a report. When the Use
Image StripSelection option is checked, this selection will track and match the
changes to the images in the image strip. The Select All option will ensure that all
entries in this list are selected. Using the Select All option or adjusting the checkmark
will disable the Use Image Strip Selection option and visa-versa.
2. Next, click the Templates drop-down arrow to select your layout .

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 Creating and Printing Reports

3. Several predefined report layouts are included with Image-Pro:

4. Choose a format.
TheImage-Pro reports are loosely designed for an A4 or letter paper output size. The
sizing of the content and the naming of the reports are based on the output page size.
Once you have selected your documents and report template , you have the following
options:
n Preview the report. In this case the resulting report is read-only and can only be
saved (in Word, PDF or HTML format) or printed. The edit controls are disabled.
n Edit the report. The resulting document is loaded and contextual ribbons can be
used to customize it further before saving or printing. The editing controls are sim-
ilar to those available in Microsoft Word.
n Edit the report template in theProject Workbench. A copy of the report template
itself is loaded for editing the report content.
5. Click Preview Report.

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The hourglass icon will appear, indicating that your report is being created.
When finished, your report will appear in the application’s workspace:

6. If you are satisfied with the appearance of the report, go to the File group on the
Report ribbon.

7. Select Save As to save your report as a PDF. Choose Quick Print or Print to print a
paper copy.
8. To edit your report, close the sample report.

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 Creating and Printing Reports

9. Click Edit Report.

You will return to the Report ribbon with the editing tools activated:

From here, you can use the Report, Insert, and Layout ribbons to adjust the content
and appearance of your report.

Report Tools
Once a report is created, you have the option to add specific content without leaving
Image-Pro. The report tools use the familiar ribbon tabbed layout.
The Report tab’s ribbon contains controls for saving, printing, and formatting text.

The Insert ribbon allows you to add pictures, illustrations, page numbers, and hyper
links to the body of your report. You can also add and edit headers, footers, and text
boxes using these controls.

The Layout tab’s ribbon lets you adjust the page size, color, margins, and text ori-
entation.

The Contents ribbon lets you add a table of contents to your report. You can also add
captions to tables and figures using these controls.

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Reporting in the Project Workbench

An alternative to the Review tab is the Project Workbench which appears on the Automate
tab. You can use the image strip thumbnails to select images for your report. To create the
report, double-click on the image or use the Project Workbench context menu, or select
Edit Template on the Review tab.

The reporting features available in the Project Workbench are detailed below:
1. The Report Tools in the ribbon.
2. The Project Explorer, where reports can be loaded and created.

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 Reporting in the Project Workbench

3. The Report Designer, to lay out the various report components as in an MSWord doc-
ument. A context menu is also provided.
4. The Report Data and Report Fields, similar to the Toolbox allow selecting the report
data and dragging data items to the report designer.
5. The Properties Panel is to configure reports and their components.
These features are explained in more detail in the next section.

The Report Tools in the Project Workbench

The ribbon’s Report Tools in the project workbench are similar to those in the main
Image-Proribbon with the addition of a few ribbon tabs.
The Home tab contains the basic tools for creating and editing reports.

The Insert tab lets you add tables, symbols, dates and other specialized details to your
report:

The Layout tab controls table cells, columns and rows. You can adjust the background
color here also.

The View tab controls the aspect of the report template as well as the visibility of report
fields.

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The References tab lets you add a table of contents, table of figures, or captions to your
report.

Project Workbench
The Project Workbench now has a section called My Reports where known reports are lis-
ted (those appearing on the Review tab).

Right-click on the My Reports folder to create new reports or load existing ones.
When you double-click on one of the reports, a report designer is loaded in the workspace
area together with Apps and macros.

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 Reporting in the Project Workbench

Reports as App/Project Components

In addition to being listed in the My Reports section, reports can also be added to pro-
jects. When projects are packaged, embedded reports become part of the package and are
available for the App and macro code using simple commands.

The Report Data and Report Fields panels

The Report Fields panel is displayed on the left-hand side of the Project Workbench, since
it will be used most often. It reflects the actual content of existing reports.

To add data to your report, click Start Here. You will see the Select Report Data dialog:

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The Report Data panel is used to select and configure the data that will be available for
the report and displayed in the Report Fields panel. You can select the individual fields
using the Add, Remove, and Clear buttons.
The following example screenshot shows a sample report displayed at the end of the
Wound Healing App:

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Reporting in the Project Workbench

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Chapter 9

541
Chapter 11 : Managing Multiple Users

Managing Multiple Users

This chapter provides information for managing passwords and identities when several
people are using one installation of Image-Pro.
Multiple Windows Users
The most high-security method of managing multiple users accessing a single installation
of Image-Pro is to use Windows logins. Each Windows user on a system will automatically
have their own set of settings and private data files, as well as access to shared demo files
and templates. So if your users have different Windows user names and logins, then your
system is already fully configured to support multiple users.

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Chapter 10

Managing Identities
When more than one person is using a particular machine or sharing a copy of
Image-Pro, access can be controlled by setting up different identities for each person. To
manage identities, follow the steps below:
1. Multiple users are enabled in the application's Options dialog. You access the Options
dialog through the Application menu, by clicking the File button in the top-left corner
of the application:

2. Go to the Popular Options page and check the EnableApplication Configuration

through User Logon at startup option. This enables prompting at login for the iden-
tity to use. Identities can optionally be password protected, which will be discussed in
the next section.

3. Enabling user logon at startup will not affect your current session. The next time that
you start Image-Pro, you will see the login dialog, in which you can select an identity
to log into, and also manage the list of available identities:

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 Managing Identities

Note that the default identity used by Image-Pro is based on the Windows user name
of the user who is logged in when Image-Pro is running.
4. If you are not one of the listed users, you can click New to create a new identity. You
will be prompted to enter the new identity name:

5. Enter the name of your new identity and click OK.

The new identity will be added to the list of valid identities:
n If you have password-protected Image-Pro login, then identity/user management
is done by the Administrator. Please refer to the Password Protection section that
follows.

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Password Protection
Password Protection is a new feature of Image-Pro. It enables you to safeguard your work
by requiring a password to log into your application. You can password protect the single
login that you have by default without managing multiple users, but you will most often
use Password Protection in conjunction with enabling multiple user logins.
Password protection can be enabled in the application's Options dialog. You access the
Options dialog through the Application menu, by clicking the File button in the top-left
corner of the application:

1. Go to the Popular Options page and check the Enable Application Configuration
through User Logon at startup option.

2. At any time after selecting Enable Application Configuration through User Logon at
startup, check the Require Password to log into Identity option. Doing so will dis-
play the Administrator Password Required dialog:

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 Password Protection

Important Note: The Administrator identity is unique in Image-Pro, and is created for
you when you select the option to require identity passwords. Make sure to keep the
Administrator password that you specify in this dialog in a safe place, because you
will need to contact Customer Service to remove password protection if you forget it.
3. Once you check this box, you will see the Enter Password dialog:

4. Enter your selected password (twice) and click OK.

The next time that you log into Image-Pro, you will see the Password Required dia-
log:

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5. Enter your password and click OK.

Image-Pro should start normally.

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Chapter 12 : Using the 3D Features

Using the 3D Features

Image-Pro with 3D Module includes the advanced 3D Viewer volume rendering, 3D
Measurements, and 3D Animation features. Volume rendering enables you to view an
image stack, or a portion of an image stack, from any angle. You can also adjust the trans-
parency and shading of the image. Animation allows you to create a moving image
sequence. The measurement tools allow you to make manual and automatic measurements
of object dimensions as 3D volumes.
In volume rendering, light emission and light absorption parameters are assigned to each
point of the volume. Each of these points is called a voxel; that is, a 3-dimensional version
of a pixel. Voxels have height, width, and depth, as well as intensity characteristics. Sim-
ulating the transmission of light through the volume makes it possible to display your data
from any view in any direction.
The 3D Viewer window allows you to examine an image stack in a variety of different
ways: by volume, in slices, and as an animated display. In the 3D Viewer, all geometric
objects are shown in 3D space. It can be regarded as a virtual camera which can be moved
to an arbitrary position within or around the 3D scene.

Note: This chapter discusses the advanced 3D features of Image-Pro with 3D Mod-
ule that are not included in the basic Image-Pro product. The 3D features of Image-
Pro are discussed in Chapter 5, Working with Images.

Image-Pro with 3D Module requires a 64-bit computer and operating system, and
also places greater demand on your operating system. Please see the Image-Pro with
3D Module system requirements in Chapter 1, Installing Image-Pro.

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The Image-Pro with 3D Module Workspace

Once you have installed the Image-Pro with 3D Module software and opened an image
sequence or image set containing on or more Z-stacks the workspace will look something
like this:

If your image sequence does not include Z-position information, it may not load into the
3D Viewer automatically. In that case, you can use the 3D view button on the image con-
text menu or in the status bar to switch to the 3D view.
When you open an image, image set, or image sequence in Image-Pro with 3D Module, an
icon that indicates which view you have open appears next to the file name:

By default, a new volume is loaded in View mode, which allows you to click and drag
anywhere in the 3D workspace to move the camera, effectively rotating the volume in the
3D space. The X/Y/Z axes that may be displayed in the bottom-left hand corner of the

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 The Image-Pro with 3D Module Workspace

workspace will show the current orientation of the camera with respect to the X, Y and Z
axes of the volume. Use the controls described on the following pages to manipulate and
analyze the image.

Use the <F> key to check the performance of your 3D viewer on your graphics
hardware. It may be difficult to manipulate the 3D view with less than 3-5 FPS
(frames per second). Please refer to the system requirements in Chapter 1 for addi-
tional information.
Image-Pro with 3D Module includes three more tabs in addition to the tabs included in
Image-Pro.
When the active view is using the 3D Viewer, the 3D View context tab will be displayed,
which lets you load and save 3D rendering settings, and adjust a wide variety of 3D ren-
dering settings that affect the display of your volumes:

3D Measure contains the tools and features you will need to make volume measurements:

3D Animation contains the tools for creating advanced animations and movies of your
volume. The Animation feature allows you to create, record, and play animation files
(*.anm) in the Image-Pro with 3D Moduleworkspace

When you start Image-Pro with 3D Module, you will notice that the status bar has been
rearranged:

The icons for the 3D viewer and the Slicer views have moved to the start of the list of
views on the image context ribbon, and in the tools portion of the status bar.

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Chapter 11

The 3D View Ribbon

The 3D View ribbon contains the following controls to view your image or sequence:

The Volume Display group lets you load, save, and refresh the settings for your 3D
Viewer:

The Reload button allows you to reload the volume with different subsampling. Clicking
the drop-down arrow displays these choices:

The X, Y, and Z indicators refer to the scaling factor use to subsample the display of the
volume. If subsampling is used with this volume, you will see a tooltip similar to this:

The Load, Save, and Reset buttons in the Volume Display group control the various ren-
dering options. Rendering options include the current visual representation of all elements.
The options can be saved/loaded using the REN file format.
The complete experiments including references to the source image and all rendering
options can be saved to Image-Pro with 3D ModuleExperiment (.s3d) files.

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 Navigate Group

These experiment files can be opened directly in the application's File Open dialog by
selecting the Image-Pro with 3D Module Experiment type.
Reload opens the following dialog:

You can also define voxel size and subsampling manually. When the Auto checkbox is
active, the volume is subsampled automatically. With typically large volumes, (larger than
128 x128 with 128 or fewer Z-planes) subsampling allows faster processing and smother
volume handling, at a cost of reduced resolution in the display. Subsampling for display
purposes does not affect the accuracy of any measurements taken.
When Large Volume license is available, user can activate the Large Volume Display
handling to display the volume at variable resolution. The large volume options can be
configured clicking the Options button.
Reload when Imagechanged indicates that the volume should be reloaded if the under-
lying image or image set is changed. For example, after filtering the image.

Load 3D settings lets you open and load saved 3D Viewer settings files from a *.ren or
*.S3D file. The *.ren file format saves only the 3D settings, which allows you to apply
those settings to another volume. The *.S3D file format saves those settings and also
saves a reference to the image or image set file to open. After saving an S3D file, you can
use it to re-open the same image file with the same settings from the File:Open dialog

Save 3D settings lets you save your current settings to a *.ren or *.SD3 file.

Clicking the Reset button sets the subsampling to 1x1x1.

Navigate Group
The Navigate group allows you to select and view the 3D volume from different positions.

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Select: Use this button to select object manipulation (and deselect View mode). The cursor
shape will change to an arrow. In this mode, you are manipulating and holding objects in
the Viewer window. Using the <Ctrl> key while clicking the mouse will allow normal
multiple selection or deselection of objects.
View: Use this button to select camera or viewer mode (and deselect object manipulation
mode). The cursor shape will change to a hand icon. In this mode, you are moving the cam-
era in 3D space, which changes the perspective of the rendering in the Viewer window.
When View mode is selected, use the mouse to control the camera position of the viewer.
In this mode, you can rotate, move (using <Shift>+drag), or zoom objects (using the mouse
wheel).
The View/Select modes can also be activated from the image context menu by clicking the
right mouse button:

The AdvancedOptions button contains the advanced options for visualization and ren-
dering. These features are discussed later in this chapter.
TIP: You can switch between Select and View modes by holding down the <V> key.

Voxel Size Group

The Voxel Size group controls the size and location of 3-dimensional pixels:

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 Decoration Group

Voxel size: This group box defines the dimensions of one voxel (a pixel in 3 dimensions).
The values displayed are the last ones used. If the image does not have a spatial cal-
ibration, and no system calibration has been set, the values are 1, 1, and 1. If the image has
a spatial calibration, the X and Y units per pixel values and the calibration units are taken
from the calibration. If the active sequence or stack includes information about the Z frame
position, the Z size is set from the distance between first and second frame in the sequence.
If Z information is absent, Z voxel size is set to the average value of X and Y sizes. These
values can be modified to achieve the desired volume aspect ratio, but doing so will affect
all 3D measurements taken on the volume. For accuracy of your measurements, we
strongly recommend that you do not modify calibrated values. Note that you can change
the subsampling in the Load dialog on the previous page without affecting 3D meas-
urements.

Decoration Group
The Decoration group contains useful features for viewing and enhancing your 3D image:

The ShowAxes button hides or shows the volume axes. This option display axes that
show the orientation of the volume (Red for the X axis, Green for the Y axis, Blue for the
Z axis). The origin of the axes is at the 0, 0, 0 location.

Show Small Axes hides or shows a pair of small axes in the bottom left corner of the
view. Show small axes is the default selection.

The Bounding box button hides or shows the bounding box. Select this button to dis-
play a bounding box that encloses the entire volume. Clicking the drop-down arrow lets
you choose between the volume frame and bounding box views:

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Chapter 11

Show Grid superimposes a grid on top of the image.

Show Labels displays the volume labels around the outside of the image.
Font scale determines the size of the labels.
Tick Frequency controls the placement of tick marks.

The following example shows both grid and labels:

The Scale bar button hides or show the calculation markers on the view. Clicking the
drop-down arrow lets you turn the marker on or off (Toggle Marker) or display the marker
options. Calibration is discussed in greater detail in Chapter 5.

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 The View Style Group

Note that the scale bar shows the size in the central part of the volume.

View All sets the camera to view all visible objects and decorations in the scene.

Stereo View displays the Stereo Mode dialog:

From this dialog, you can turn the stereo mode on or off, and change the stereo type.

The View Style Group

The ViewStyle group contains controls for volume visualization.

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Chapter 11

Transparency: This slider controls the degree of transparency of

the volume. Moving the slider to the left makes the entire volume progressively more trans-
parent.
Interpolation activates voxel view smoothing. The pop-out menu displays your choices:

l Linear interpolation does bilinear approximation between voxels, which is fast but
less accurate.
l When Cubic interpolation is activated, a cubic interpolation of neighboring voxels is
used. This option produces smooth volumes, but is expensive in terms of computing
resources.
l The Pre-Integrated option can be used to increase the quality of the volume view
with a smaller number of slices. It is also useful when the transfer function has a lot of
fast variations, i.e., the color changes very rapidly.
An example appears here:

Pre-Integrated Off Pre-Integrated On

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 The View Style Group

l The Voxelize options lets you visualize each non-transparent voxel by rendering
each voxel as a cube of that voxel’s volume and intensity. It can be used to look
at the volumes at the voxel level. The picture below shows the volume with the
voxelize option on:

Voxelize on

Boundary Opacity increases or decrease the opacity of object edges, where gradi-
ents are high:

Boundary opacity off Boundary opacity on (thr. =2.5,

intens.= 8.5)

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Chapter 11

The pop-out menu lets you change the threshold or intensity:

l The Threshold parameter defines the sensitivity of boundary opacity. Gradi-

ents with a length less than the Threshold values are ignored during the light-
ing computation. This avoids doing lighting computations on noise in the
volume while still lighting important data. In the screenshot, setting a
threshold value of 2.5 removed lighting on the noise surrounding the pollen.
l The Intensity parameters define the darkness of the edges.
Note that using Boundary Opacity can significantly increase rendering time.

The Edge Coloring changes the color based on the gradient direction (normal).
Areas where the normal direction (computed from the gradient) is facing the camera will
have an unmodified color, whereas areas where the normal direction is more perpendicular
to the view direction will tend towards Edge Color.
Edge Color lets you select a color from the palette. Edges are highlighted with the color
specified in Edge Color.
In the example below, the coloring is on and the color is white:

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 The View Style Group

l Background: Use the color picker to select a background color for your volume
display.

l When the Gradient Light option is on, the color gradient is drawn as a back-
ground for a scene. The Background color appears at the top of the window and the
second color appears at the bottom of the window.

l When the Show Image option is on, an image will be displayed on the background.
The position of the image is controlled in the Style drop-down box and the image file
can be selected using the Browse... button. The following image formats are sup-
ported: BMP.

l The Light button activates directional light and volume lighting. The drop-down
menu contains additional lighting and volume appearance options:

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Chapter 11

These options only affect volumes displayed in Blended composition.

l Camera Light turns a light coming from approximately the same location as your cam-
era on or off.
l Directional Light turns on or off a light that you can position relative to the
volume itself. Use the Edit Directional Light menu item below to edit that light’s
position and intensity.
l Edit Directional Light: Clicking this button activates the directional light editor.
Note that this option is disabled when the Directional Light is off. Choosing this
option displays the Light Direction Editor, shown here:

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 The View Style Group

Use the handle to manipulate the light direction. The Edit menu allows you to
change the color using a color editor. The options are set to the default, always on
top.
l The Show Shadows option activates shadows on volume and scene elements.
Note that when the light is off, the shadow is cast from the headlight, which fol-
lows the camera. When the directional light is on, the shadow is cast by the cam-
era light, the position of which can be controlled using the Edit Directional
Light dialog.

Shadow OFF Shadows ON

l Shadow intensity and smoothness can be controlled using the corresponding con-
trols. Intensity is given in range from 0 (no shadow) to 1 (dark shadow). Higher
values of smoothness give smoother shadows.

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Chapter 11

l Shadow screen can be used to show dropped shadows. You can control the
places where the shadow screens are shown (Y, Y, Z). You can also control the
color and the screen offset from the edges of the volume. Offset is defined in per-
cent of the volume size in the given direction.
l Pseudo Surface is a lighting effect that looks like a virtual surface created at the
threshold level of volume opacity. It gives a better result when the volume data
contain regional boundaries with relatively sharp gradients. Threshold defines the
opacity level for the Pseudo Surface in a range from 0 to 1. The default is 0.1.
l Material groups defines material properties of pseudo surface. The color of the sur-
face is calculated from a combination of 4 color components, each component is
in range from 0 to 1:
l Ambient - the color of an object where it is in shadow. This color is what the
object reflects when illuminated by ambient light rather than direct light.
l Diffuse - represents direct light hitting a surface. The Diffuse Light contribution is
dependent on the incident angle.
l Specular - the white highlight reflection seen on smooth, shiny objects. Specular
light is dependent on the direction of the light, the surface normal and the viewer
location. Specular component makes the object look like metal or glass. The reflec-
tion property is also defined by Shininess, with values close to 1 the reflections
will be very sharp as on polished metals, with lower values the radius of reflection
increases to make it look as brushed metal.
l Emissive - the self-illumination component of the color, has neon effect.

Pseudo Surface OFF Pseudo Surface ON

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 Composition Group

Here are the examples of the Pseudo Surface mode with different Material properties con-
figurations:

Use the <Q> key to switch between rendering modes. There are two rendering methods
that support trade-offs between rendering speed and rendering quality:
l Sorted Blended Transparency
The default rendering mode, this features fast rendering, but may not accurately render
semi-transparent surfaces.
l Delayed Sorted Pixels Blended Transparency
A more complex rendering mode for complex scenes with semi-transparent objects.
Also more demanding of graphics resources.

Composition Group
The buttons in the Composition group define the image composition method.

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Chapter 11

Blend: Blending averages the values of the voxels in a straight line through the
volume of the object.

Max:Maximum Intensity Projection takes the value of the brightest voxel in the
same straight line as used by the Blend option

Sum: This method displays the sum of the value of the voxels in a straight line. It is
most useful for dark images.

Min: Minimum Intensity Projection takes the value of the dimmest voxel in the same
straight line as used by the Blend option.
Some examples are shown here:

Blend Sum Max

Number of slices: This slider controls the number of slices drawn

for 3D and 2D multi-texture map rendering. Zero automatically selects the number of
slices. Rendering fewer slices will generally improve performance
(with reduced image quality).

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 Geometry Group

Geometry Group
The Geometry group defines the camera parameters:

Activates the perspective view.

Activates the orthogonal view.

In Perspective projection view, the parts of the volume closest to the viewer will look big-
ger; those that are further away will appear smaller. Switch the projection type to Ortho-
gonal to retain a cubic shape without perspective.

Clicking the Projection button sets the view to the last-selected projection, from
one of the following:

Sets the camera perspective to an XY projection.

Sets the camera perspective to a ZY projection.

Sets the camera perspective to an XZ projection.

The drop-down menu also includes a new feature: Wheels. Wheels shows or hides the in-
frame control for rotation wheels at the bottom and right side of the projection view. Use
the wheels to rotate the display along a single axis.

The bottom slider in the Geometry group is the View Angle slider:

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Chapter 11

This slider adjusts characteristics of the camera’s virtual lens, which changes the angle that
defines the field of view of the camera. Changes to the field of view can indirectly make
your scene look larger or smaller. Changes to the viewing angle can also cause lens effects
like a “fish-eye” view of your scene if you are using the Perspective setting in the Geo-
metry group. The following illustrations may help to understand how the view angle
affects the rendering:

Note that the volume appears much smaller with the larger view angle. The following illus-
tration may explain why this happens:

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 Volume of Interest Group

Viewing Angle: 45 ° Viewing Angle: 120 °

You can see with the wider field of view, from the greater viewing angle, that the same
size box looks much smaller, even though it is the same distance from the camera. You can
adjust for the change in the size of the scene by using the Zoom control to move the cam-
era closer to the volume. In that case, you can clearly see the “fish-eye” effect of the wide
angle lens:

Volume of Interest Group

The Volume of Interest group modifies the visible portion of the data volume in the Viewer
window. It contains the following controls:

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Chapter 11

The Show VOI Axes button turns the VOI axes on or off.

Show VOI handles shows or hides the "dragger" associated with the VOI. The drag-
ger is used to move and resize the VOI.
To use the dragger tool:
1. First make sure the 3D Viewer window has focus (click in it if necessary).
2. Then make sure the Viewer is in selection mode (cursor is an arrow shape). If neces-
sary, click the Select icon in the Navigate group to switch to selection mode.
3. Click on the Show VOI handles button to activate the VOI adjustment handles

To Resize the VOI:

1. Move the cursor onto one of the green handles
2. Press the left mouse button and drag. The VOI cannot be larger than the data volume.

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 Volume of Interest Group

To Move the VOI:

1. Move the cursor onto one of the sides of the dragger box. The side of the VOI that
you click on will determine the axes along which the VOI can be moved. If you find
that you cannot move the VOI in the direction that you want to move it, try clicking
on an adjacent side of the VOI cube
2. Press the left mouse button and drag. The VOI cannot move outside the data volume.

The size of the VOI (XYZ) and the volume are given in spatially calibrated units. Volume
fraction shows the ratio between total volume of the image and the VOI.
You are able to copy and paste VOI extents from one 3D view to another using the Copy
and Paste buttons. You can also crop the volume to the VOI extents using the

Crop button.

The Reset button will reset the VOI to the full size of the data volume.
The sub-volume information is shown in the Reset button’s drop-down panel which also
has controls to set the VOI parameters by numbers. You can define start and size values for
X, Y and Z axis and then click the Set by Numbers button. When the option In Calibrated
Units is selected, the values are in calibrated units, otherwise in voxels.
The position and size of a VOI can be displayed in the status bar when the ROI extents
and ROI size are selected in the Status Bar options:

Sub-Volume: Choose a shape for the VOI (Volume of Interest) from the drop-down list.

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Chapter 11

l Cross: The VOI will be displayed as a cross-shaped volume.

l Fence: Displays a fence-shaped VOI.
l Exclusion box: The VOI can also be an exclusion box which specifies a region of the
volume that will not be displayed. This is very useful for cutting away part of the
volume.
Examples of the VOI shapes are shown here. The gray sections indicate which parts will
be visible in the Viewer:

Sub -Volume Exclusion box

Cross Fence

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 The 3D Measure Ribbon

The 3D Measure Ribbon

The 3D Measure ribbon contains a variety of volume measurement tools for 3D analysis.

You may already be familiar with some of them.

Navigate Group
The Navigate group lets you choose between Selection and Viewing mode.

When View mode is selected, the mouse controls the camera position of the viewer. In this
mode, you can rotate, move, or zoom objects. In Select mode, you can select objects in 3D
view (slices, measurements (volumetric and manual), draggers,) and create manual meas-
urements. Using the <Ctrl> key while clicking the mouse will deselect any object.

TIP: You can switch between Select and View modes by holding down the <V> key.

Count Group
The Count feature lets you count specified volumes within the 3D image. Clicking the
Count Button executes volume segmentation and measurements on one of the volume
channels.

If the Count control is active, the 3D objects in an isosurface are remeasured every time
the threshold levels or isosurface parameters change.

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Chapter 11

When you click the count button, you will see the following dialog:

Add isosurface: Here you can select which channel of a color image will be used to create
the isosurface. Your choices will depend on the volume that you have open:
l For monochrome volumes from a sequence and single-channel sets, your only choice
is a Gray channel from the monochrome data.
l For color volumes, you will have Red, Green and Blue channels that correspond to the
color components of the image.
l For single and multi-channel sets with channel information, there will be one volume
channel per set channel, using whatever channel names and tinting information is
available from the set metadata.
Sub sampling: The Subsampling group in this dialog is very important to understand, as it
affects the isosurfaces that are created. You can use subsampling to smooth out the isosur-
face that is generated, which will allow it to render and animate more quickly. However,
excessive subsampling can also result in removing smaller objects that you may wish to
visualize or measure, and subsampling will always affect the volumes of the objects that
may be measured later. For maximum accuracy, you will want to use Reset to make sure
that there is no isosurface subsampling.
Auto: Selecting the Auto checkbox activates automatic subsampling mode. In this mode,
the subsampling is calculated based on the size of the image. This provides faster loading
and rendering of volume images. By default this option is on. Advanced users can switch
this option off and set subsampling manually.
Reset: Clicking the Reset button clears all your volume settings and sets 1:1:1 sub-
sampling. The Reset button is disabled when Auto is on.
Filters: Filters can be used to smooth the surface. Choose a filter from the drop-down list.
Excessive smoothing using Filters can also affect the accuracy of any 3D measurements
that you make, so use the minimum amount of filtering needed to get a representative
isosurface.
Threshold Type: The initial threshold level can be selected automatically when Threshold
type is defined as Auto Bright or Auto Dark.
Close edges: Select this check box close the surfaces of the objects where they touch the
bounding box of the volume.

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 Count Group

When Close edges is turned on, the surface is closed on the sides that touch edges regard-
less their intensity value. (The edges must be closed to measure volumes.)
Execute Count: Check this box to execute the object segmentation count when an isosur-
face is created.
Auto-Split: Check to this box to split objects that are touching in two.
Ridge Size: When the Ridge size option is active, only narrow junctions with diameters
below the given size are split. Note that the split is also applied after adjusting the
threshold levels of the segmentation.
Your image will look something like this:

This example shows that the green channel was selected for the count.
Information about the isosurfaces created by the Count operation appear in the options
panel on the right of the screen:

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Counting the volume created an isosurface, which is represented by the Volume Surface
Green section of the 3D View options panel. When Execute Count is turned off, only one
isosurface will be created. Then you can adjust the Threshold levels interactively, and
execute the count by checking the Count check box:

The thresholds can be adjusted by clicking and/or dragging the start or end of the
threshold range on the histogram. There are a number of controls here that you can use to
fine-tune the segmentation of the isosurface, which are discussed in detail in the Add
Isosurface section later in this chapter. You can also edit it in the From or To controls.
When the Count option is turned off, the color of isosurface can be adjusted in the
Advanced Color drop-down:

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 Volume Measurements Group

Note that when Count is on, the colors of individual objects are controlled by the Col-
oring parameter in the 3D Measurement options. When Coloring is set to Parent the seg-
mented objects have the color of the isosurface. When the Coloring is set to Random, then
the color of every segmented object is selected randomly, but the transparency is taken
from the isosurface.
The results of the count operation are displayed in the Count Results group:

Volume Measurements Group

The Volume Measurements group provides a variety of measurements for volume analysis.

The measurement types can be defined in the Types drop-down list.

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Available measurements are grouped by types:

l Lines, either manually created or derived line or point measurements.
l Volumes are automatically segmented volume objects
l Object class, which can be applied to both Lines and Volume measurement objects.
Clicking the Add button adds one of the available measurements to the Selected
Measurements / Filters list.
Clicking the Add All button adds all of the available measurements to the Selected
Measurements / Filters list.
Clicking the Label button uses the name of the selected measurement as a label for a
given group of objects.
Clicking the Remove button deletes a specific measurement from the list of Selected Meas-
urements/Filters.
Clicking the Clear button deletes all the measurements from the list of Selected Meas-
urements/Filters.
Clicking the Options button displays the Orientation Options dialog. Orientation meas-
urements are described later in this chapter.
Edit Range lets you make adjustments to the measurement ranges:

Min and Max filter values can be adjusted by dragging the corresponding markers on the
histogram or editing their numerical values. The number of objects in range/total objects
and percentage are shown below the histogram.

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Ranges sets the filter range that can be applied to each measurement. If a filter range is
set, objects with parameters below the minimum or above the maximum values are
excluded from the view. The filter ranges are used only when the Ranges option is active.

The VolumeMeasurements list includes the following measurements:

Volume indicates the volume of an object in calibrated units.

Surface area indicates the surface area of object in calibrated units.

Width shows the size of a bounding box in the X direction.

Height shows the size of a bounding box in the Y direction.

Depth displays the size of a bounding box in the Z direction.

Center X indicates the X coordinate of the center of an object.

Center Y indicates the Y coordinate of the center of an object.

Center Z indicates the Z coordinate of the center of an object.

Center of mass X indicates the X coordinate of the center of mass of the object.

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Center of mass Y indicates the Y coordinate of the center of mass of the object.

Center of mass Z indicates the Z coordinate of the center of mass of the

object.

Box Volume displays the volume of an object's bounding box:

(V = W * H * D).

Box ratio displays the ratio between the maximum and minimum sizes of the
bounding box: R = Max / Min.

Volume fraction displays the ratio of an object's volume to the box

volume. R = Vobj / Vbox

Diameter displays the equivalent diameter of an object.

Sphericity displays the sphericity of object, calculated as 6 volumes of an object

divided by the equivalent diameter and surface area of the object. For spherical objects,
this parameter equals 1; for all other shapes it is less than 1.

Density (mean) indicates the mean density of an object.

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Density (min) indicates the minimum density of an object

Density (max) indicates the maximum density of an object.

IOD displays the Integrated Optical Density of an object.

Density (st.dev.) displays the standard deviation of the optical density of an

object.

Radius (max) indicates the maximum distance between an object's centroid and sur-
face.

Radius (min) displays the minimum distance between an object's centroid and
surface

Radius Ratio displays the ratio between the minimum and maximum radii.

Class shows the class to which an object belongs. The classification can be done
using the Histogram window.

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Feret (max) indicates the maximum distance between two parallel planes
enclosing an object.

Feret (min) indicates the minimum distance between two parallel planes
enclosing an object.

Feret Ratio displays the ratio between Feret (max) and Feret (min).

Alpha angle indicates the angle between the reference vector and surface normal
(in degrees).

Gamma angle indicates the angle between the Direction vector (Dir) and the
projection of the normal (Proj) to the plane created by the direction vector (Dir) and the
vector (Perp) perpendicular to Ref-Dir plane. The angle can range from -180 to 180
degrees.

Theta angle indicates the angle between the Reference vector (Ref) and the pro-
jection of the normal (Proj2) to the plane perpendicular to the Direction vector (Dir). The
angle can range from -180 to 180 degrees.

Surface deviation is calculated as the deviation of the end-points of triangular

normal. The calculations are done as follows: all triangular normal vectors of the surface
are normalized (length set to 1) and the average distance from the mean positions of the
end-points to all other vectors is calculated. A uniform surface will have deviation of 0.
The maximum deviation of 1.336 will have a sphere.

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, Angle X, Y and Z indicate the angle between longest object

line (max feret) and the X, Y and Z axes in degrees.
The longest object line vectors can be visualized using the corresponding option in the
OrientationMeasurements Options dialog.

Holes and Negative Volumes

The current version of Image-Pro with 3D Module handles holes as if they are independent
objects. Holes are identified by negativeVolume measurement. By default, holes are
filtered out by the measurement filters when Volume limits are set from 0 to 1E9. You can
disable or adjust the filters to display the holes.
Object measurements ignore holes. It is equivalent to using the Fill Holes options in the
2D measurements. If it’s important to exclude hole volumes from the parent objects, you

can do it by manually selecting the parent object and hole(s) and clicking the Merge
button. This will merge the outlines of the parent object with those of the holes which
will result in measurements that exclude the negative volumes of holes.

3D Measurement Type Options

Orientation measurement options can be set clicking the Options button on the Measure
Types dialog:

Orientation in 3D space can be defined using at least 2 angles from perpendicular vectors.
These angles are called Alpha and Gamma. Alpha is measured relatively to the Reference
vector and Gamma relatively to Direction vector.

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The Reference vector is defined by the X, Y and Z components. The default vector of 0, 0,
1 is parallel to the Z axis. The alpha angle is calculated between this vector and the sur-
face normal.
The Direction vector is defined by the X, Y and Z components. The default vector of 0, 1,
0 is parallel to the Y axis. The gamma angle is calculated between this vector and the pro-
jection of the normal to the plane of the direction vector, and the vector perpendicular to
the plane is defined by reference and direction vectors.
Maximum angle between triangle normal and reference vector defines which areas (tri-
angles) of the object surface will be taken into account when calculating surface normal.
Every surface consists of triangles; every triangle has its own normal vector. If the angle
between triangle’s normal and the reference vector exceeds the maximum angle range, the
triangle is excluded from the surface normal calculation. The default value is 80 degrees,
which excludes triangles on the sides of the object (angles around 90 degrees) and tri-
angles on the opposite side of the object (angles greater than 90 degrees). The maximum
value for this parameter is 180.
There are two methods provided to calculate the normal vector of surface:
l Average vector, which is calculated as area weighted average normal vector of all sur-
face triangles (within angle range). This method is relatively fast in calculation, but
sensitive to deviations on the surface orientation. Small bumps and pits on the surface
can slightly affect the vector.
l Median vector, where median vector from all triangles are selected as surface normal.
Median vector is the vector that has the minimum sum of distances to all other vec-
tors. This methods provides very stable results; as small bumps and pits on the surface
do not affect the result. The vector represents the normal direction of the surface which
covers most of the object. This method is very computation intensive, and can be
much slower than the Average method for large objects (if each object contains more
than 10000 triangles).
The Show Surface Normal vectors option hides or shows the surface normals on the
image.
The Show longest object line vectors option hides or shows the longest object lines that
are used to measure Angle X,Y and Z.
An example appears here:

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The colors of the normal vectors can be changed using the Selection color.

3D Options Panel
The 3D Options panel can be opened by clicking the 3D Options button in the Volume
Measurements group:

The 3D Options panel is shown here:

Appearance options
The Appearance options control Labels and Outlines.
Volume Text sets the appearance of measurement labels displayed on the 3d volume.
Manual Text sets the appearance of the manual measurement labels.
Both text combo-boxes indicates the type of labels. The labels can be:

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l None: No labels are displayed.

l Name: The measurement name is displayed.
l Measurement: The value of the first selected measurement will be displayed as a label
(you can drag any column in the data table to the fist position to be displayed as a
label).
Font sets the label font size.
Decimal defines the number of digits after the decimal point that will be displayed in the
data table and labels.
The Outlines group contains display options for the measurement elements.

Line width defines the width of lines for the manual measurements
Arrow Size defines the size of arrow line ends.
Sphere Size defines the size of sphere line ends for point measurements. When a manual
measurements feature (point, line, polyline) is selected, the points of the polyline are
shown as spheres in the selected color with boxes around them. If the viewer is in the
selection mode, and the selection mode of the manual measurements is active, the pos-
itions of the spheres can be edited by dragging the points to new positions.
Color and Selection define colors for normal and selected state of the elements.
The Spheres and Dragging boxes are switched off when the feature is deselected.

The Volume Objects group provides options for coloring.

The Coloring control defines what color the objects will have. Two choices are available:
Parent and Random. If Parent is selected, the objects inherit the color from the parent
isosurface/volume measurements. If Random is selected, the colors for objects are generated
randomly, so every object has different color.

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Measurements Group

The Measurements group shows, hides, or deletes the various manual measurements on the
image.

The Show button shows or hides any manual measurement objects on image. The
Show All group lets you show or hide selected measurement objects.

The object can be deleted using the Deleteselected or Delete Al l buttons.

Active Tool Group

This group defines the active tool that can be used to create manual measurements:

The available tools are:

Select measurements allows you to select measurements on the image and also edit
the positions of external objects.

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Add pointmeasurement activates the point tool.

Add line measurement activates the line tool. A line is added after 2 clicks.

Add polyline measurement activates the polyline tool. The line is added after you
click the right mouse button.

Add polygon measurement activates the polygon tool. Click on several points to
create the polygon. Click the right mouse button to close the shape.

Add anglemeasurements activates angle measurements. An angle is added after 3

clicks.

Add surface line along object surface connecting 2 points creates a poly-line feature
that connects 2 points going along the surface in the plane that passes through the selected
two points and the camera position.

Add an outline of object crossed by a plane creates a closed poly-line feature that is
an outline of a plane crossing an object. To add an outline, click twice on the imaginary
line along which you want to create the outline of the object. The outline plane passes
through the selected two points and through the position of viewing camera, so the outline
is always created in the plane perpendicular to the screen plane.

Split objects activates the split mode. To split a 3D object into 2 objects, click twice
on the imaginary splitting line of the object. The splitting plane passes through the selec-
ted two points and through the position of viewing camera, so the object is always split
with a plane perpendicular to the screen plane.

The Magic Wand tool enables you to draw polygons on slices. Clicking the
drop-down arrow displays this menu:

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l Range indicates the number of gray levels used for thresholding (combination of gray
ranges depends from type of active image and selected interpretation).
l Smoothing indicates the degree of post-filtering of the outline (0 – no filtering, 9 –
high degree smoothing).

Relative Measurements Group

Various other measurements can be created based on the selected measurements

Getcenterof the object creates a point measurement of the center of selected objects.

Measure distance between centers creates a manual measurement line that connects
centers of two selected objects.

Measure perpendicular distance between point and line creates a line measurement
between a point and a line as a perpendicular line. Note that the function is active only
when 1 line and 1 or more points are selected

Measure angle between lines creates an angle measurement between two lines. Note
that the function is active only when 2 lines are selected.

Measure distance between point and surface of volume object creates a line
between the selected point and a selected 3D object. The 3D object can be selected either
in the Volume measurements data table or on the image itself, using the Select meas-
urements/select objects mode.

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Measure distance between surfaces creates a manual measurement line between the
surfaces of two selected objects. This line represents the shortest distance between two
objects.

Split polyline into lines creates a new line measurement from every segment of the
selected poly-lines/tracks. Analyzing the length of every line provides information related
to velocity of the object moving along the track.

Create a polyline including points of all selected measurements creates a poly-line

that includes all points of the selected measurements. For example, if 2 lines were selected
the new poly-line would include 4 points.

Extract points from lines/polylines creates a new point measurement for every
coordinate of the selected poly-lines/tracks. It can provide information about object
coordinates on different time points (the same information can be obtained in the Volume
measurements data table).

Merge selected objects merges selected objects into one. It can be used if seg-
mentation does not allow connecting all the pieces that belong to one object.

Undo split/merge undoes the last split or merge operation. The number of undo
levels is practically unlimited.

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Guided Segmentation
Guided Segmentation is designed to allow 3D segmentation of complex objects that can-
not be easily segmented using threshold segmentation. Guided segmentation involves
defining the object outlines on a 2D view and then generating 3D volume objects from
those outlines. To activate this feature, click the Guided Segmentation button on the 3D
Measure ribbon:

The Guided Segmentation panel appears on the right hand side of the workspace:

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The panel contains four sub-panels:

l Collections
l 2D Segmentation
l Z-propagation
l Isosurface
To use Guided Segmentation, follow these steps:
1. Open a sequence in the workspace.
2. On the 3D Measure ribbon, press Guided to open the Guided Segmentation dialog.
Note that in the Collections sub-panel, a first object collection will already have been
created. If the volume that you want to segment opens in the 3D view, Image-Pro will
switch to the Image (2D) view for the process of drawing object outlines. If you
already had the Guided Segmentation panel open when you opened your sequence,

use the Image button ( ) in the status bar or Image Context menu, or the 2D but-
ton in the Collections sub-panel to switch to the 2D image view.
3. Navigate to any frame (or Z-plane) in the volume where the first object of interest is
visible. You can start at any plane in the Z-stack.
4. Use any tool in 2D Segmentation sub-panel to create one or more object outlines for
that object. See the documentation of the 2D Segmentation sub-panel for more details
about the tools that can be used to create the object outlines.
5. Once you have defined your outlines, use the Validate button to set those outlines as
the objects' ROIs.
6. In the Z-propagation sub-panel, choose a method to copy your outlines to other
frames in the z-stack.
7. Press the forward and/or back buttons in the Z-propagation sub-panel to extend the
object ROIs through the stack.
8. Lock the object collection by checking the box next to its name in the Collections
sub-panel. You may continue to add object collections by pressing the Add button in
the Collections sub-panel. Note that adding a new collection will automatically lock
the previous collection(s).
9. Repeat steps 4-9 for each collection to be added.
10. To view the collections in 3D, use one of the selections in the Isosurface sub-panel to
turn your collections into isosurfaces in the 3D Viewer.

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More information about each of these steps appears in the following pages.

Collections Sub-Panel
The Collections sub-panel of the Guided Segmentation panel contains tools to control col-
lections. It allows you to add and define certain properties, and to visualize segmented out-
lines interactively.

Segmentation collections are defined by a set of outlines on multiple frames. Multiple out-
lines can be defined for a single collection on one frame. A collection is identified by its
name and color, which can be edited in the table shown above.

Depending on your images, you may be able to define all your object outlines in a
single collection. Image-Pro with 3D Module includes support for multiple collections to
allow you to separate outlines that would otherwise cause incorrectly segmented objects.
This can be a problem if separate objects touch or intersect anywhere in your volume.
When the outlines are analyzed to create isosurfaces, separate outlines may be merged
into a single isosurface if in any frame of the sequence, the outline for an object overlaps
multiple outlines on an adjacent frame. This is necessary to be able to connect up Y-
shaped or branching objects. However, if separate objects touch, they may both be over-
lapped by the outline in the adjacent frame, and incorrectly merged. If this occurs, then
make a new collection and put the outlines for either of the objects in the new collection.

A new collection can be added clicking the Add button. The collection can be

deleted or reset . It can also be saved or loaded from a file. You can

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use the Delete All button to remove all the existing collections and start over. Note
that when all the collections are deleted, a new, default collection R1 is automatically
added.
The Lock option applies only to the active collection. It allows you to review the seg-
mentation without applying the active propagation method when scrolling through the
image sequence. It also controls whether editing tools are available, or if the collections
are locked. All collections except the active one will always show a locked state, and the
active row can be locked or unlocked by clicking the check box. Checking the Lock
check box freezes the selected collection and prevents additional editing until the col-
lection is unlocked. Note that the 2D Segmentation and Z-propagation sub-panels will
include the name of the active collection.
Multiple collections can be displayed on the image when the Show All Collections box is
checked. When this option is off, only the active object ROI will be displayed.
Display of the collections’ labels is controlled by the Show labels check box. Note that
the size and font of the labels can be controlled from the measurement options panel.
The Frames column shows the number of frames in the sequence.
The View buttons are used to display the view(s) that you would like to have visible
while defining the object outlines and isosurfaces. The 2D button will open a new view of
your volume in the Image view if there is not already an Image view open, and it will
make that view the active workspace view. The 3D button will open a new view of your
volume in the 3D view if necessary, and it will make that view the active workspace view.
The 2D / 3D button will make sure that both views are open, and display them in a split
workspace layout with the 2D view on the left and the 3D view on the right.
Outline color allows you to change the color of the object’s outline using the color
selector. When this option is off, only the outlines for the active collection will be dis-
played.

Volume objects can also be exported as outlines from the 3D Data Table to Guided
Segmentation using the Export Outlines menu item from the context menu over the 3D
Data Table object row.

2D Segmentation Sub-panel
The 2D Segmentation sub-panel offers 3 main modes of operation that enable different
methods that you can use for defining the object outlines:

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The three modes are:

l Wand, where you will primarily use the Magic Wand tool to define the outlines by
outlining an area based on the color similarities or difference of intensity ranges
between the pixel under your cursor, plus or minus a specified tolerance interval.

l Smart, where you will use the Smart Segmentation tools to define the outlines by
defining reference areas for background versus objects.

l ROI, where you will primarily use manual tools to draw the object outlines.

These modes will be discussed in detail in upcoming sections of this chapter. In all three
modes, there are tools available to define and refine the object outlines:

The 2D Segmentation sub-panel provides the following tools for all modes:

Use the Selection Tool arrow to select an ROI on the image.

The Polygon Nudge tool allows you to adjust the size and shape of irregular ROIs
and measurements

The Polygon tool allows you define a polygon-shaped or freeform ROI in the
image.

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Use the Delete buttons to delete the selected outline(s), or all outlines. Note that
you can also use the Delete key to delete the selected outline(s).

Magic Wand Mode

You can define object outlines by selecting the Wand tab and clicking on the object to
segment.

After the first click on the image, the threshold range will be displayed in the Histogram
control. You can adjust intensity range of the Magic Wand using the From and To con-
trols or by dragging the range indicators on the histogram. You can also use additional
magic wand controls to adjust the automatic outlining: Range, Filter, Smoothing and Fill
Holes.
l Ranges. Indicates the lower and upper levels used for thresholding.
l Filter: Defines the minimum object area in square pixels.
l Smoothing: Indicates the degree of smoothing that is applied to the object outline (0
= no smoothing, 9 = maximum smoothing).
l Fill Holes: Check this box to automatically fill any holes in the outline.
When you are satisfied with the outlines and the range, click the Validate button. That
will convert the magic wand outline to an ROI and add it to the active object. Note that
the defined intensity range will be then used to create outlines on other frames with the
Auto-Range propagation method.
Smart Segmentation Mode

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Use the Smart Segmentation tab to create objects. This method can be used on noisy
objects where the Magic Wand has difficulties creating outlines.

Select the Smart tab to define the segmentation recipe parameters and draw reference
objects and background areas. When you enter Smart Segmentation mode, the Smart Seg-
mentation sub-panel will be displayed, along with a balloon message:

This message balloon will stay active while you set up the Smart Segmentation recipe in
the Smart Segmentation panel. (More information about Smart Segmentation appears in
Chapter 8 of this manual and in the in-product Help.)
When you click OK, whichever objects have been identified by the Select Object tool will
be added to the collection of outlines.
Filter defines the minimum object area in square pixels.
Smoothing is applied to the object outline (0 = no smoothing, 9 = maximum smoothing).
Note that if you want to modify the recipe going through frames, use the Modify Recipe
button.
You can also add new outlines to the frame in Smart mode by following these steps:
1. Make sure that the Auto-Smart propagation mode is selected in the Z-Propagation
sub-panel.

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2. Select the Polygon drawing tool and draw one or more ROIs in areas that should be
identified as objects. These ROIs can be very rough as they only need to overlap the
area detected by the Smart Segmentation.
3. Click the Validate button. One new object will be created for each ROI that overlaps
the smart segmentation mask.

ROI Mode
If you prefer creating object outlines manually, you can switch to the ROI tab. When you
are in ROI mode, clicking the Validate button reads the intensity range within the
region’s outlines, which can be used for other frames when you choose the Auto-Range
propagation method.

The following options will be applied when detecting regions using the Auto-Range
propagation:
l Filter defines the minimum object area in square pixels.
l Smoothing is applied to the object outline (0 = no smoothing, 9 = maximum
smoothing).

Z-Propagation
The Z-Propagation sub-panel offers you options for propagating the 2D objects from a par-
ticular frame across other frames in Z-space. The available options depend on the 2D seg-
mentation type. Options that are not available for a particular 2D segmentation type are
shown as inactive:

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Use the arrow buttons to navigate through the image stack. The spin controls indicate how
many frames you advance each time. The following methods are used to create outlines on
new frames:
l None: Advancing to a new frame will not add any new outlines to the next frame, or
change any existing outlines. Use this method to review the outlines that you’ve
defined so far, and also when the object outlines on neighboring frames are very dif-
ferent, and you want to draw outlines on every frame manually.
l Duplicate: Changing the frame will duplicate object outlines from the previously act-
ive frame.
l Auto-Range: Detects an object on a new frame that overlaps with the outline on the
previous frame, using the same intensity threshold range. This propagation method is
available when you are using the Wand or ROI modes.
l Auto-Smart: Detects an object on new frame using global smart segmentation recipe.
It will identify any overlapping outlines. This propagation method is available when
you are using Smart mode.
You can use any tool from the 2D Segmentation sub-panel on any frame to redefine intens-
ity ranges, smart segmentation recipe or modify the outlines. Any change of method will
be repeated throughout the stack.

Use the Forward and Back button to go through the stack by one step at a
time. If you use the Auto-Range or Auto-Smart method, and are going through the stack

to create correct outlines, you can automate this process using the Detect Forward

and/or Detect Back button to go through the all frames in the stack automatically.
The detection will stop automatically when no more overlapping objects are detected. You
can also stop the auto-detection process manually pressing the <Esc> key.

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Object outlines will be automatically detected using the last segmentation recipe. If the
object detection is not satisfactory on some frames, the outlines on those frames can be
adjusted or re-drawn with the 2D Segmentation tools.
If you are using Smart segmentation, try adjusting the recipe by adding new reference or
background objects and click OK.
Note that when you draw outlines manually or edit them, the intensity range within the
object’s outlines will be used for other frames when you choose the Auto-Range propaga-
tion method. After adjusting the outline manually, click the Validate button to accept the
changes. Note that the outline is automatically validated clicking by the Forward or Back
button

Isosurface
The Isosurface sub-panel allows you to create isosurfaces on selected objects by con-
necting the 2D segmented areas across the Z-planes. When all object outlines are created,
use the Isosurface group to add 3D objects as isosurfaces to the 3D view.

You can add only the active collection using the Add Active button (which contains the
name of the active collection, for instance Add R1 in the illustration above). Clicking the
Add All button will add isosurfaces for all of the defined collections. You can set the sub-
sampling and smoothing to use during isosurface creation (see the more detailed discussion
of these settings in the Add Isosurface section later in this chapter).

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Every object in each collection will be segmented and measured. The values appear in the
data table.
If you have an existing isosurface node and the Create new isosurface option is not
checked, the new objects will be added to that existing isosurface. If there are no existing
isosurface nodes, or the Create new isosurface option is checked, then a new isosurface
named Guided Segmentation will be created with the new objects.
The outlines can be viewed in the Slicer by view activating the Show Outlines option:

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 View Group

View Group
The View group allows you to see the 3D data in different formats:

These tables contain the same features and functions as the tables and graphs described in
Chapters 4 and 8 of this manual.
The Data Table collects all the information from the measurements taken on your image.
The 3D data table shows measurement values for manual and segmented 3D objects.

Load Data Table Measurements: This tool allows you to load meas-
urements saved in a previous session from a *.ipm file.
Save Data Table Measurements: This tool allows you to save your
measurements in a *.ipm file for use at another time.

Send Measurements to File: This tool allows you to save your meas-
urement data to a file or export it to an Excel spreadsheet. The pull-
down menu lets you choose one of the following:

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Send to Report: This tool allows you to save your measurement data
to Image-Pro report. The pull-down menu lets you create a new report
or choose an existing one.
Show all Statistics: This tool allows you to hide or show the statistics
panel for the data table measurements. A sample statistics panel is
shown here:

The drop-down option Statistics per Group defines what is displayed

in the statistical pane. When this option is off, the statistics of all vis-
ible features is shown. When the option is on, the statistical values are
shown per group. This option has affect only when Grouping is active
Setup Group Statistics display the statistics by group. It will activate
grouping by class, add Percent Area Parent to the list of measurements
and activate statistics per group
Grouping: Click this button to see options for grouping your data in
the table. You can choose one of the following:

Show All, Show Selected Measurements, Hide Selected

Measurements: These tools let you choose to display or hide some of the meas-
urement statistics.

3D Mask: Click this button to create a mask of volume objects.

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The 3D Data Histogram illustrates, in graph form, the distribution of 3D measurements for
the selected measurement:

The Object Window allows you to view information about a specific object in an image.
The example below shows complete data for one object:

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3D Options
The 3D Options panel on the right-hand side of the workspace contains the following con-
trols:

All of the Add buttons add nodes to the 3D view that contribute to the rendering of your
volume.
Add Isosurface/Volume measurement: Isosurfaces provide a very quick, yet often suf-
ficient, method for reconstructing polygonal surface models. By correlating transparency
with the local orientation of the surface relative to the viewing direction, complex spatial
structures can be understood much more easily.

Delete: Click this button to remove the selected node.

Add Ortho Slice: Clicking this button adds individual ortho slices to the ND projection.
Ortho Slices are used to display a slice through the X, Y, or Z axis orientation in the 3D
Viewer. Voxels on the plane of the slice will appear as a different color.
Each slice can have a different color outline. This can be useful for identifying slices on
complex scenes with multiple elements (oblique, ortho slices). The visibility and the color
of the outline can be set using the Outline control.
Add Oblique Slice: Oblique slices are used to display slices of the volume which are not
precisely aligned with the X, Y, or Z axes.
Add External Object: External objects are isosurface coordinate links that can be imported
into the 3D Viewer.
Expand/Collapse all nodes: Click the plus sign to expand the channels and nodes. Click
the minus sign to collapse them.
The rest of the 3D View panel displays the rendering nodes that currently exist in your 3D
visualization:

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Color Image: All channels are loaded into an indexed color volume (The sequence is con-
verted to a palette class using a palette containing the most popular colors in the
sequence).
Channels: The channels node represents the visualization of the volume intensities. The
drop-down box will only be enabled for multi-channel volumes, and controls the way that
the channels are combined in the volume rendering. Clicking the drop-down arrow dis-
plays the following options:
l Max: Maximum Intensity Projection takes the value of the brightest voxel in the
same straight line as used by the Blend option.
l Blend: Blending averages the values of the voxels in a straight line through the
volume of the object.
l Min: Minimum Intensity Projection takes the value of the dimmest voxel in the
same straight line as used by the Blend option.
Each channel of your volume will have a sub-node for the channel, with a name derived
from the channel name (if the volume is a multi-channel set), the color component (of a
color image), or “Gray” for monochrome images. Each channel or color component’s ren-
dering can be adjusted with the following control panel:

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The Show button hides or shows the selected element. Every node in the 3D View
panel has this button, which lets you select which nodes are visible, and are included in
the 3D rendering.
Every channel is shown using a palette that is scaled from 0 to 255 covering the whole
dynamic range of the channel data. The All Channel Display controls will change
PaletteSpread and Opacity range on all channels.

The Blinking icon control activates the blinking mode of the channel.
The Palette can be one of the following:
l Wavelength allows you to display wavelength in nanometers.
l Color lets you select any color using the color picker.
l Palette allows you to select one of predefined palettes displaying the channel.
l Custom
Selecting Custom palette allows creating user-defined color combinations:

Palette points can be added by clicking on the histogram area. Clicking the
existing point makes this point active and allows selecting the color using
the color-picker button. Positions of palette points can be adjusted by mouse.
The Y position of the point defines opacity of the color, higher position –
higher opacity. Ctrl-Click on the existing point will delete the point. Custom
palettes can be saved to a file (pal3D format) and loaded (iqpal, pal3D) using
the Save and Load buttons.
Histogram: Activating the Histogram option allows you to set the Spread and Opaque
levels on a volume histogram. The Spread limits are controlled by the top controls and the
Opaque limits are controlled by the bottom ones.</p>

Activating the Histogram option allows you to set the Spread and Opaque levels on
a volume histogram. Spread defines the start and end positions for palette spread and the

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Opaque control defines the range of intensities that are visible. The values outside the
Opaque range are transparent. The Spread limits are controlled by the top controls and the
Opaque limits are controlled by the bottom ones.

The Histogram view is the default view for the Spread and Opacity controls, but you can
turn this view off to edit the limits directly,

The Best-Fit button ( ) can be used to set the Spread and Opacity values to a best-fit
display of the volume.
The Reset button ( ) can be used to reset the Spread and Opacity values to the min-
imum and maximum intensities.

Add Isosurface
Isosurfaces/volume measurements provide a very quick, yet often sufficient, method for
reconstructing polygonal surface models. By correlating transparency with the local

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orientation of the surface relative to the viewing direction, complex spatial structures can
be understood much more easily. If you are making volumetric measurements, you will
need to define at least one isosurface/volume measurement.
Automatic segmentation of 3D objects can be done using 2 different tools:

- Add Volume Measurements

- Add Iso-Surface
The Add Iso-Surface segmentation is based on iso-surface, which is generated using the
Marching cubes algorithm with consequent labeling of mesh triangles (we will call it Iso-
Surface segmentation). The Add Volume Measurements uses segmentation creating
objects directly based on voxel connectivity data, without generation of iso-surfaces (we
will call it Direct Volume Segmentation).
Both methods have their advantages, which will be compared here:
Advantages of Direct Volume Segmentation over Iso-Surface segmentation:
1. Lower memory usage. Since no iso-surface is generated, the memory requirements for dir-
ect segmentation is much lower than for the iso-surface segmentation, which allows seg-
menting larger volumes.
2. Faster segmentation. The segmentation is applied to a 3D volume directly, the
algorithm uses a single pass labeling, which significantly speeds up the segmentation, up
to 100 times on complex volumes.
3. Holes handling. Direct segmentation algorithm handles holes according to the Fill
Holes option, which allows user to decide if they want to include or exclude the holes
from segmented objects. Generated Hole measurements (Hole Count, Hole Volume and
Hole Volume Ratio) are available only for directly segmented objects. The iso-surface seg-
mentation handles holes differently, the holes are always included into the surface object
measurements, no Hole measurements are generated.
Drawbacks of Direct Volume Segmentation over Iso-Surface segmentation.
When Direct Volume Segmentation is used no iso-surface is generated, which leads to
some drawbacks.
1. The segmented objects cannot be saved as 3D models to IV, WRL or STL files. If you
need to have a 3D model file, you should use iso-surtface segmentation.
2. Derived measurements that use volume objects, such as Distance between surfaces,
Point to Surface are not available for Direct Volume Segmentation.

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3. Pixelated visualization. Visualization of 3D objects are done using shader rendering on

GPU, so it misses some smoothing effects of triangular mesh iso-surface rendering. You can
change material appearance of segmented objects using Pseudo Surface tools, such as
pseudo-surface material or using the Voxelize option in the Interpolation drop-down. If
object visualization is important, you should use the iso-surface segmentation.
4. Clipping planes created by ortho and oblique slices can clip iso-surface objects and
volume independently, depending on the position of the slice relatively to the iso-surface
element in the options tree, so volume can be clipped, but the surface not. With Direct
Volume Segmentation both Volume Channels and Segmented objects are clipped in the
same manner.

Multiple isosurfaces can be added clicking the Add Isosurface button in the options
panel.

Note that the same action can be performed from the 3D Measure tab by clicking the
Count button.
When you add a volume measurement, you will see this dialog:

Here you can select which channel of the volume will be used to create the isosurface.
You can select the desired subsampling. The Auto subsampling is selected by default.
Filters can be used to smooth the surface. A filter can be selected from the following list:
l None
l LoPass 3x3x3
l LoPass 5x5x5,
l LoPass 7x7x7
l LoPass 9x9x9
l Gauss 5x5x5
l Gauss 7x7x7
l Gauss 9x9x9.
The initial threshold level can be selected automatically when the Thresholdtype is
defined as Auto Bright or Auto Dark.
Close Edges will close surface edges on the volume boundary.

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Fill Holes option is on, the holes are included into the measurements of the segmented
objects.
If Clean borders is on selected the objects that touch the bounding box of the volume are
excluded from the analyzing.
The segmentation with default options has advantages on memory consumption and speed
over iso-surface segmentation, and if user wants to get even better performance measuring
the volume, there are additional options available clicking the Advanced button.

The Activate pseudo surface options check-box makes the best visualization options for
direct segmentation surfaces active automatically. When this option is on, the volume visu-
alization is automatically switches to the Pseudo Surface mode.
The option Create Preview volume defines creation of the visualization volume for
threshold adjustment preview. You can switch this option off to save RAM, in that case
threshold preview will be generated on the original volume channel and may look slightly
different from the segmented volume as they may have different sub-sampling and filter
options.
When the Create segmentation volume is on, the segmented objects are shown in dif-
ferent colors on the volume. If user just want to have measurements and the visualization is
not important, this option can be switched off to save more RAM. Note, that all meas-
urements will still be generated properly and object volume boxes will be displayed for
selected objects. When this option is off user can show equivalent spheres instead of the
proper outline by activating the Show object equivalent spheres option, spheres display
takes less RAM then the segmentation volume and can be used to handle large volumes
when Create preview volume and Create segmentation volume options are off.
Execute Count will automatically execute the object segmentation count when an isosur-
face is created. When Execute Count is off, only the isosurface will be created, so you can
then adjust the Threshold levels interactively. Start the count by checking the Count but-
ton:

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The thresholds can be adjusted by clicking and dragging at the start or end of the
threshold range on the histogram, or editing it in the From or To controls.
When Count is turned off, the color of the isosurface can be adjusted in the Color drop-
down:

Note that when Count is on, the colors of individual objects are controlled by the Col-
oring parameter of the 3D Measurement options. When Coloring is set to
Parent the segmented objects are the same color as the isosurface. When the Coloring is
set to Random, then the color of every segmented object is selected randomly, but the
transparency is taken from the isosurface.

Clicking the Options button allows you to edit the surface parameters, subsampling
and filtering the auto-threshold:

Using the Add Iso-Surface button, the iso-surface based segmentation can be created.

The Precision control defines whether surface outlines of 3D object are created with Sub-
voxel or Voxel granularity. The default option is Sub-voxel. This option allows creating
smooth surface outlines that can be created in sub-voxel space and the position of this out-
line depends how close the threshold value is to the intensity value of the voxel.

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When the Voxel option is active the surface outlines and all volumetric measurements are
build from voxel cubes. Voxels with intensity above or equal the threshold level are com-
pletely included into the object.
The following element added clicking the Ok button:

The visible volume measurement objects can be exported to an IV file by clicking the
Export button.
It is possible to import volume objects using the Import button. This option can be used,
for example, when you have segmented objects on one channel and want to measure
intensities within these outlines on another channel.
When the outline is imported, the channel mode changes to outline where the Count
option is always active:

To return to the interactive segmentation mode, click the Reset Outline button.

Add Colocalization
Colocalization measurements with independent threshold levels can be added using the
Add 3D Colocalization Measurements and Add 3D Colocalization Iso-Surface buttons.
Both actions will create colocalization measurements, the difference is the same as for
volume measurements and iso-surface, the first one creates colocalization using direct
volume segmentation and the second - using iso-surface segmentation.
3D Colocalization in Image-Pro with 3D Module requires a separate license. If you do
not have a license for this option, you will see the following message:

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Please contact your Thermo Fisher representative for additional information.

Clicking the button shows the dialog where you can define the colocalization pair and
also the initial auto-threshold method:

The default method is Auto-Bright.

Clicking the Ok button creates a new Colocalization measurement and opens the coloc-
alization panel with colocalization scatterplot and the table with colocalization para-
meters. The histogram window shows the intensity distribution for two channels.

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Colocalization thresholds can be adjusted for each channel independently using Channel 1
and Channel 2 numeric controls, dragging threshold levels on histogram or clicking on
scatterplot. The colocalization objects and measures are updated automatically.
It is also possible to use Free ROI tools to define colocalization areas on the scatterplot,
the colocalization objects get updated after creation or adjusting of ROIs. Note, that adjust-
ing thresholds on histogram switches colocalization mode to “Thresholds”.
3D view may include multiple colocalization pairs. The colocalization panel shows results
for the active colocalization pair. To activate the pair click the Colocalization button.
When the Count option is active, colocalization measurements can be reported for every
object. To add colocalization to the list of selected measurements, click the Add Measures
button. The measures will be added to the list and shown in the data table.

Add Ortho Slices

Ortho Slices are used to display a slice through the X, Y, or Z plane orientation in the 3D
Viewer. Voxels on the plane of the slice will appear as a different color.
You can add multiple ortho-slices by clicking the Add Ortho Slice button.

If the Show control is on for Ortho Slice, an orthogonal slice is shown on the volume.

Orientation selects one of three ortho slices, aligned with the X, Y or Z axis.
Clipping enables the clipping plane associated with the slice. The clipping plane will clip
off rendering of the volume channels on one side or the other of the clipping plane. The

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clipping side can be set to either Front or Back. The clipping plane has no effect if the
slice is not visible.
The Handles controls visibility of the "dragger" associated with the ortho slice. The drag-
ger is a 3D user interface gadget that can be used to drag the slice through the data
volume. To use the dragger make sure the viewer is in selection mode (cursor is an arrow
shape). Move the cursor onto the white cylinder, press the left mouse button, and drag to
move the slice.
You can also drag slices just by clicking and dragging the slice itself.

Slice composition is set by the dropdown and can be Opaque, Bin-

ary or Transparent. Binary means that voxels with non-zero alpha values are completely
opaque and voxels with zero-alpha values are completely transparent. In addition the drop-
down contains control to define Transparency of the slice affects the slice display in
Opaque and Binary modes. In addition the drop-down contains the Transparency % con-
trol to define the Transparency of the slice, which also affects the slice display in Opaque
and Binary modes.
Slice Number shows the slice number where the slice is currently positioned. It can also
be used to drag the slice through the data volume. The slice is moved dynamically as the
slider moves. You may also type a slice number in the edit box.
Smooth switches on linear interpolation on the slice, thus increasing the visible resolution.

Add Oblique Slices

Oblique slices are used to display slices of the volume which are not precisely aligned
with the X, Y, or Z plane. Multiple oblique slices can be added by clicking the
Add Oblique Slice button in the panel.

The program can display an "oblique" (arbitrary orientation) slice through the data volume.

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Slice position controls the slice position along the normal vector of the slice plane.
Alpha and Beta angles control orientation of the oblique slice plane, which can also be
adjusted interactively using the slice handles (see the following illustration):

The dragger is a 3D user interface feature that can be used to drag the slice through the
data volume and change the orientation of the slice. To use the dragger make sure the
viewer is in selection mode (cursor is an arrow shape). Move the cursor onto the white cyl-
inder, press the left mouse button and drag to move the slice. Move the cursor onto one of
the white lines, press the left mouse button, and drag to rotate the slice.
You can also drag slices just by clicking and dragging the slice itself.
Clipping enables the clipping plane associated with the slice. The clipping plane has no
effect if the slice is not visible.
Slice Number shows the slice number where the slice is currently positioned. It can also
be used to drag the slice through the data volume. The slice is moved dynamically as the
slider moves. You may also type a slice number in the edit box.
Smooth switches on linear interpolation on the slice, thus increasing the visible resolution.

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Slice composition is set by the dropdown and can be Opaque, Bin-

ary or Transparent. Binary means that voxels with non-zero alpha values are completely
opaque and voxels with zero-alpha values are completely transparent. In addition the drop-
down contains control to define Transparency of the slice affects the slice display in
Opaque and Binary modes. In addition the drop-down contains the Transparency % con-
trol to define the Transparency of the slice, which also affects the slice display in Opaque
and Binary modes.
Each slice can have a different color outline. This is useful for identifying slices on com-
plex scene with multiple elements (oblique, ortho slices). The visibility and the color of
the outline can be set using the Outline control.

Add External Objects

External objects are isosurface coordinate links that can be imported into the 3D Viewer.
Multiple external objects can be loaded from files by selecting the Add External Object
button in the panel.

The loaded external object can be edited by clicking on it in Select mode. Use the dragger
to scale, rotate and move the object.

Tip: Holding down <Ctrl>+<Shift> allows you to adjust the dimensions inde-
pendently.
Clicking the object again will hide the draggers.

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The 3D Slicer Ribbon

Image-Pro with 3D Module includes an advanced Slicer view in the available toolbars. To
use the Slicer tools, click the Slice viewer button on the Image tab. The Slice viewer
allows you to add up to 3 additional views of the volume from different orthogonal dir-
ections. A Slicer tab appears on the ribbon.
The 3D Slicer ribbon looks like this:

The View group shows the volume using three different modes (thin slices, thick slices or
projections), volume object outlines, and manual measurements
An example of the Slicer view with XY, YZ, XZ and 3D views all displayed appears on
here:

You can control the layout of the Slice Viewer using the tools in the Layout drop-down
menu in the View group:

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 The 3D Slicer Ribbon

The layout can include one, two, or three of the volumes and projections. For example, if
you select the XZ, 3D and XYZ, 3Dviews, the workspace will look something like this:

XZ 3D Projection XYZ 3D Projection

The XZ3D projection is on the left, while the XYZ 3D projection is on the right.

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In addition to the layout selections, the View ribbon group contains other controls:

l Cross turns the crosshairs on the slicer views on or off. The color of the cross can be
selected using the color-picker control.
l Outlines turns the Volume Object outlines on or off. The option can be used to see out-
lines of volume objects on 3D slices. Note that only the outlines for Counted objects
are shown (execute Count to see the outlines). The thickness of the outlines is defined
by Line width parameter of 3D measurement options.
l Sliders turns the slider controls for each of 3D slicer views on or off (the default is
on). These controls appear on the sides of the panels in the workspace. The control can
be used to adjust a displayed slice position in the corresponding view. Note that the
slice position can also be adjusted by using the Slicer Position controls on the Slicer
ribbon, or by using the Navigate tools and clicking on any of the slicer views.
Slicer views can be zoomed using the mouse wheel or <Alt>+mousemove and translated
using the <Alt>+< Shift > + mouse move combination. The views can be reset to the ori-

ginal state (best-fit) using the Reset views button.

The drop-down menu of the Reset button contains controls that set the slice transparency.

Setting slice transparency to non-zero values allows the program to display manual meas-
urement elements behind the slice in thin slice mode.
The Slicer Position group contains three Extract buttons that allow you to extract the
XY, ZY and XZ views into a new image.
The Thickness group defines the display mode for 3D slicer views:

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l Thin: The view is displayed as a thin orthogonal slice. The slice can be semi-trans-
parent based on the Thin slice transparency option.
l Thick: The view is displayed as thick slice including multiple slices. The thickness of
the slices is defined by the corresponded numerical controls. The transparency of thick
slices is defined by the main transparency control (on 3D view tab).
l Projections: Projections of all slices are shown. The view uses current composition
and can be shown as maximum or minimum intensity projections (as well as blend and
sum).
l X, Y, Z: You can set the thickness of the slices by using the spin buttons.
Use <Ctrl>+ mouse click to add manual measurements on the slicer views. Clicking the
mouse button without the <Ctrl> key will change the slicer position.
Measurements created on a 3D view are also displayed on slicer views.
Note that all added 3D elements (slices, isosurfaces) can be rearranged by dragging and
dropping them to a new position. It can be used to define proper clipping of one element
by another, as slices can only clip isosurfaces and other slices located below the given
slice in the scene.

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The 3D Animation Ribbon

The Animation tab contains the tools for creating simple animations and movie files. The
Animation feature allows you to create, record, and play animation files (*.anm) in the
Image-Pro3D workspace.

The animation tools are described in the following pages.

Timeline Group
The Timeline feature lets you synch your animation in time and space. The timeline panel
shows the current animation with thumbnails of the key frames, and allows you to play
and edit the animation sequence.

The timeline panel will open automatically at the bottom of the page when you activate
the 3D Animation ribbon.

The individual frames in your animated image are synched to the locations along the time
line. The x-axis of the timeline represents the time of each key frame in the overall dur-
ation of the movie. The following controls are displayed in the status bar.

The X axis can be labeled as Frames or Time (s).

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Clicking on any position in the timeline will change the current position in the timeline,
and will activate the given camera position in 3D view. The current position is indicated
by a dotted line, as you can see at frame 20 of the Animation timeline in the previous fig-
ure. Note that the current position is very important when adding key frames to the anim-
ation. The timeline can be zoomed in or out using the zoom controls or the mouse wheel.
The number of key frames can be adjusted by dragging the key frame separators.
Timeline editing is done by adding or deleting key frames. Each key frame is indicated by
a blue pointer at the top of the timeline. Multiple key frames can be selected by clicking
on or before the first key frame, and then using <Shift>+<Click> on the last key frame.
Multiple key frames can be selected using <Shift> +<Click>. The selected range can be
then deleted by clicking the Delete button.

Animation Path Group

The Animation Path group lets you insert camera animations into your timeline with a
single click.

l The Spin button allows you to add a sequence of frames rotating the camera around
the screen axis (Vertical-Y, Horizontal-X, and Perpendicular to the screen – Z). The
drop-down menu contains buttons to rotate the camera around 3 axes in both dir-
ections by the given angle defined by 90,180 and 360 radio-buttons. The number of
intermediate frames for a 360-degree spin is defined by the Circular Frames option in
the Options group.
l The Fly-Around button allows you to add a series of frames by moving the camera in
an evenly spaced, circular path parallel to the Viewer window, and half-way between
the two sides of the window. The Fly Around rotations are created around the world
axis. World axes are X, Y and Z and can be visualized by activating the Axes button
on 3D View tab. Fly Around animation gives the effect of helicopter flight.

l The center options let you choose amount of rotation for both the Spin and Fly-
Around selections. To go completely around your screen or volume one full turn, use
360 degrees, or 90 degrees to go only ¼ of the way around.

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l Rocking or “see-saw” animation. This animation rocks the image around the vertical
axis back and forth by approximately 30 degrees. Clicking this button displays the fol-
lowing dialog:

The Rotation axis group defines the screen rotation axis. The Sweep angle and Num-
ber of steps controls define the amplitude of rocking and the number of intermediate
frames.
l Custom lets you design a custom animation by selecting time points and camera pos-
itions. You will see this dialog:

Clicking the Screen radio button allows you to create fast transitions by rotating the
camera around the screen axis (Vertical-Y, Horizontal-X, and Perpendicular to the
screen – Z).

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 Frames Group

Clicking the World axis radio button let you create animation by activating the Axes
button on 3D View tab. World animation gives effect of helicopter flight.
The Sweep angle and Number of steps controls define the amplitude of the animation
sweeps and the number of intermediate frames.
You can rotate your animation around the X, Y, or Z-axis, by clicking the drop-down
menu button and displaying these items

To save an animation path, click the Save button and select path and name for the
animation file. Saved animations can be loaded using the Load button. The Copy but-
ton does not work directly with you animation timeline, but will copy the current 3D
view of the volume to the clipboard as a picture that can be inserted into a present-
ation or document.

Frames Group
The Frames group gives you tools for managing the key frames in your animation, as well
as spacing between the individual frames.

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Clicking the Add Camera Position button adds the current camera position (and
any changes to the view) as a new key frame at the current position within the animation
timeline.

Replace camera position replaces the current key frame with the current camera pos-
ition (and any changes to the view).
The Key spin box indicates the current key frame number.
The Inter spin button indicates the number of intermediate fames and camera positions in
the key frame. You can adjust or edit the number of intermediate frames by using the spin
button, or by dragging the key frame handle on the timeline.

Click the Delete button to delete the active key frame and its intermediate frames.

Click the Clear button to delete all key frames and clear the animation timeline.

Click this button to double the number of intermediate frames at all camera positions.

Click this button to halve the number of intermediate frames at all camera positions.

Playback Group
The Playback group lets you play your animation forward and backward. It also controls
the playback rate in Frames Per Second (FPS).

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The toolbar has the following buttons:

Go to first frame

Go back one frame

Play animation forward

Go forward one frame

Go to last frame

Options Group
The Animation options group contains some additional controls:

The Record Rendering Settings option allows you to use different rendering settings for
every camera position in the animation. You can change any rendering parameter, such as
transparency or palette, and turn visibility of slices, channels, and volume measurements
on or off. The changes will be applied when the corresponding camera position and fol-
lowing intermediate frames are rendered. Note that only existing rendering elements can be
used here. You will need to add all necessary elements, such as ortho and oblique slices,
isosurfaces, and volume measurements before starting to record an animation. The vis-

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ibility and parameters of all rendering elements can be changed during recording. This
option should be on unless you are animating only simple views of the basic volume.
Connect Start and End: Check this option to have the final intermediate frames inter-
polate between the last key frame of the animation, and the first one. Note that any option
that has an on/off setting, for instance visibility of an element, cannot be interpolated. For
this reason, the final key frame of any movie that should be looped should have the same
on/off settings as the first frame.
Synch with Time: If the loaded image set has multiple time points, use this option to syn-
chronize the time points with the camera positions.
Additional options are shown when you click the Options drop-down button.

Hide or Show Animation cameras: Click this button to turn the display of the camera pos-
itions on or off.
Circular Frames defines how many frames will be generated when you create a new cir-
cular animation path using the Spin or Fly-Around features in the Animation Path group.
The Inter control defines the default number of frames that will be added with each key
frame. The actual number for any key frame can be adjusted later, as previously discussed.
Antialiasing increases the visibility of edges on created images. When the Antialiasing
option is on, the multipass antialiasing is activated during creation of animation images.
Every image is rendered x number of passes from slightly different camera positions, aver-
aging the results.

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 Output Group

a. b)
Image a) was created without antialiasing. Image b) was created with 8-pass antialiasing.
l Passes: The multi-pass antialiasing is activated during the creation of animation
images. Every image is rendered according to the number of passes from slightly dif-
ferent camera positions, averaging the results.
Use fixed image size option allows you to define the size of the generated animation or
image. Enter the values for Width and Heights (in pixels) to set the size. When this option
is off, the current window size is used. If you will be making your movie for display on an
HD monitor, you will usually want to use the Use fixed image size option, and set the
Width and Height to either 1280 and 720 (for 720p HD display), or 1920 and 1080.

Output Group
The Output group lets you create snapshots of your current view, or to render your com-
plete animation to a movie:

The Snapshot button will create a new image holding a snapshot of the view at the current
position in in the timeline. This image workspace can be saved to any file format sup-
ported by Image-Pro.

The Movie button will create a new image sequence of the complete animation. The new

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image workspace will have as many frames as is currently specified in the timeline, and
can be saved to any movie file format supported by Image-Pro.

Navigate Group
The Navigate group allows you to select and view the 3D animation from different pos-
itions.

Select Button: Use this button to select object manipulation (and deselect View mode).
The cursor shape will change to an arrow. In this mode, you are manipulating and holding
objects in the Viewer window. Using the <Ctrl> key while clicking the mouse will
deselect any object.
View Button: Use this button to select camera or viewer mode (and deselect object manip-
ulation mode). The cursor shape will change to a hand icon. In this mode, you are moving
the camera in 3D space, which changes the perspective of the rendering in the Viewer win-
dow.

Adding a New Animation

To add a new animation, follow these steps:
1. Make that the current animation is empty (click the Clear button if necessary).
2. Set the camera mode (Perspective or Orthogonal) in the viewer. Note that if you want
to create a Fly-through animation you have to select a perspective camera.
3. Using your mouse and controls for the viewer window, set the desired view of the
volume and click the Add camera position button.
Clicking the Add button adds the current camera position and also the given number
of intermediate frames between the previous and the current positions that produce
smooth transition from one position to another.
4. Move camera to the next view position and click Add again.
5. Record all camera positions you want to have in the animation.
Note that to create a circular animation you have to record at least two camera pos-
itions first and then click the Circular Animation button.

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A set of camera positions that go around the object pointing to the viewpoint of the
first camera position, and in the plane defined by the first two camera positions and
the center of view will be generated. The distance of the first camera to the focal point
defines the radius of circular animation.
If the current animation was empty, the animation will be rotated around the vertical
axis.
To create a seesaw animation, you should record the position of the first viewpoint
and then the last viewpoint. Click the Connect Start/End control.
If the current animation was empty, the animation will be rotated around the vertical
axis.
6. To play your animation, click the Play button. You can stop animation by clicking
on the image in the viewer window or clicking the Pause button.

To create an animation sequence in the application workspace, follow these steps:

1. Resize the volume viewer window to the size you need, or check the Use Fixed Size
option and specify the size.
2. Click the Movie button. This movie can then be saved to an AVI or WMV file.

The current view can be copied to the Windows clipboard using the Clipboard button or
can be turned into a new image using the Image button.

632
Appendix A
File Format Specifications

Image-Pro supports the following file formats. A brief description of each format will list
the name used by Image-Pro in the File:Open and File:Save As dialogs, the typical file
extensions that identify the file format, and any limitations such as formats that are sup-
ported for File:Open only.

AVI (*.AVI)
Image-Pro supports many of the video varieties of the MicrosoftAVI file format, which
will usually have the *.AVI file extension. The varieties that will be available in your
copy of Image-Pro depend on the AVI file compression support that has been installed by
the operating system and other multi-media components. You cannot annotate *.AVI files
in Image-Pro.

BMP (*.BMP, *.DIB, *.RLE)

Image-Pro supports all known varieties of the Microsoft BMP file format, which will
usually have the *.BMP file extension but may also have a *.DIB or *.RLE extension.
You cannot annotate *.BMP files in Image-Pro.

JPEG (*.JPG, *.JPEG, *.JP2, *.JPF)

JPEG files will have a *.JPG or JPEG file extension. Image-Pro supports all standard
varieties of the JPEG file format, including JPEG 2000.

Flex Image Transport System (*. FTS, *.FIT, *.FITS)

Image-Pro supports all standard formats of the Flex Image Transport System.

Thermo Fisher Experiments (*.MCE)

Image-Pro allows you to read and write Thermo Fisher experiment files with the extension
*.mce

633
Appendix A

Media Cybernetics Sets (*.MCS)

Image-Pro allows you to read and write Thermo Fisher set files with the extension *.mcs
.

MPEG-4 (*.MP4, *.MOV, *.M4V)

Image-Pro allows you to read and write MPEG-4 files with the extensions *.MP4,
*.MOV, and .M4V including H.264.

Portable Internet Graphics (*.PNG)

Portable Internet Graphics are a common format for all types of graphics. The extension is
*.PNG.Image-Pro supports all types of *.PNG files.

Picture (*.PCT, .PICT)

Image-Pro supports most types of picture files.

Sequence (*.SEQ)
Image-Pro supports multiple-image TIFF files with the .SEQ file extension. This format
allows you to save annotations without burning them into the image.

TIFF (*.TIF, *.TIFF)

Image-Pro supports all known varieties of the TIFF (the Tagged Image File
Format) file format, including most multiple-image varieties and PowerPoint TIFF files.
This format allows you to save annotations without burning them into the image.
Image-Pro can support TIFF files larger than 4GB to aid in the acquistion of large image
sequences. The ability to read calibration data from the following EM files has been
added:
l FEI
l Hitachi
l JEOL
l Zeiss

Image-Pro supports the following read-only file formats:

634
 File Formats

Aperio (*.svs)
AutoQuant (*.avz) (*.deb) (*.hdr) (*.xml)
BIGTIFF (*.btf)
DeltaVision (*.dv)
Gatan Digital Micrograph (*.dm3) (*.dm4)
Hamamatsu Nanozoomer
Image Cytometry Std (*.ics)
Image-Pro IQ Sets (*.iqs)
Leica (*.lei) (*.lif) (*.scn)
Micromanager (metadata.txt)
Molecular Devices Metamorph (*.nd) (*.stk)
Nikon Elements (*.nd2)
Olympus FV 1000 (*.oif) (*.oib)
OME-TIFF (.tiff)
PerkinElmer (*.tim)
Streampix (*.seq)
Windows Media Video (*.wmv)
Zeiss LSM (*.lsm)
Zeiss Widefield (*.zvi)
Zeiss CVI (*.cvi)

Flat file formats are now supported by Image-Pro. Please contact Media Cybernetics’ Cus-
tomer Support for assistance in setting up support for a specific flat file format.

635
Appendix A

Hitachi File Formats

There are a number of different types of image acquisitions that can be done with Hitachi
TEM, SEM and table-top SEM microscopes. These include:
l Single-frame acquisitions (one or several saved to same folder, which can generally be
saved in TIFF, BMP or JPEG file formats)
l Acquisitions of Z stacks or volumes
l Acquisitions of larger areas by moving the sample stage and taking additional images
(tiling)
l Acquisition of TEM images where the stage is tilted through a range of +60 to -60
degrees.
l Acquisitions by third-party hardware/software vendors like Gatan

Image-Pro with 3D Module should be able to open and analyze these images.
Image-Pro with 3D Module will also open Hitachi mosaic (tiled) images directly in to the
Tiling View. A mosaic image is one that contains multiple frames that are laid out in a reg-
ular X/Y grid in rows and columns.

636
Appendix B
Frequently Asked Questions
This section contains questions from our users, and answers from the Media Cybernetics
support team. Please feel free to submit additional questions to us, using the email address
[email protected].

637
Appendix B

Installation Questions
Q: What is the purpose of the Update Log in the product installation folder?
A: The Update log.log is a test file with trace information from the TrueUpdate pro-
gram. It is created every time the product runs or looks for updates. It is not needed for
ordinary daily use, but contains diagnostic information that will be helpful if there is an
installation or update problem.

Q: What are the Application and Data folders for?

A: The Data folder usually contains sample data other than images. The Application
folder is a system folder and is not visible by default in Windows Explorer. The Applic-
ation folder for the product can be safely deleted.

Q: What does the LastCapture.icq file in Application data folder do? What happens
if it is deleted?
A: This file saves the state of the capture settings when the application is closed. It is used
if the *None* capture settings file selection is selected. If it is deleted, the capture settings
will be set back to default and the file will be recreated when the application is shut down
again.

Q: How often does TrueUpdate look for updates?

A: Every time you start the application.

Q: If one person installs the product into “All Users” will a second Windows user have
access?
A: An “All Users” installation will install shortcuts, documents, and data to areas that are
ordinarily accessible to everyone. However, it is always possible, by customizing Win-
dows security to produce an environment where a second user may not have access.

Q: If the current Windows user installs the product will only that person be able to use the
files?

638
 Frequently Asked Questions

A: In the common case (i.e. with default Windows security) the DEFAULT locations doc-
uments and data will be restricted to the installing user. If that user is concerned about
access to their data by others, they need to be careful where they save their images and
data.

639
Appendix B

Application Questions
Q: What does Auto-Range in the Capture settings do, and how do you set it?
A: Auto-Range sets the black and white levels using the standard display features, so the
histogram B/W level bars, best-fit, reset, etc. are all able to set the auto-range functionality.
Dynamic auto-range uses best-fit to set the display range on every preview frame. The set-
tings for best fit apply, so you can set the low and high exclusion using the standard dis-
play controls on the ribbon. Also, reset and best-fit are now on the histogram window.

Q: In the Capture group, What does the Lock button do?

A: The Lock button locks the preview and capture controls together for that subpanel
group. The other selections are Capture and Preview that unlock the controls and allow the
user to specify them independently.

Q: If we have a 36-bit live preview and capture that image, then “save as” and choose
JPEG, what happens? Does it down-convert to a 24-bit image? If so, there is a bug that the
image has “36-bit” listed in the “info” table.
A: It down converts to 24-bits to save the file. The image, however, that is shown is the
original captured image, not the copy that’s on the disk. That’s why the info is showing
36-bit. If you open the image file that was saved, the image will be 24-bit.

Q: On different operating systems, what will the default paths for QuickSave be?
A: The default path is My Documents\Image-Pro 64-bit\9.3\Images
It is a subfolder of “My Documents” that is always writable.

Q: What are the JPEG Compression differences? What is Quality or Quality Factor? Is this
typical?
A: JPEG images are saved with Quality Factor = 95. This is the default, which produces
small images without much visual artifacts. You can also use PNG or TIF to save a com-
pressed file without losing quality (though the file size is bigger than JPEG).

640
Index

Index
2D Segmentation Sub-panel
* Guided Segmentation 593

*.AVI 633 3
*.BMP 633 3D Animation ribbon 623
*.DIB 633 3D Animation tab 92
*.FIT 633 3D Data Histogram 603
*.FITS 633 3D Data Table 602
*.FTS 633 3D Mask 353, 603
*.JP2 633 3D Measure ribbon 572
*.JPF 633 3D Measure tab 91, 550, 572
*.JPG 633, 640 3D Measurement Type Options 582
*.MCE 263, 633 3D options 584, 605
*.MCE Sets 263, 633 3D projections 620
*.MCS 634 3D scatterplot 459
*.MCS Sets 634 3D space 161, 548
*.PCT 634 3D View ribbon 551
*.PICT 634 3D View tab 91, 551
*.PNG 634 3D Viewer 161, 548
*.RLE 633 3D Viewer ribbon 162
*.Seq 634 3D volumes 620
*.TIF 634 A
2 Activating and Closing 469
2D Count/Size tab xxi Active Data Overlay 242
2D Measure tab xxi, 89, 424 Active frame 101

641
Index

Active Tool Group 586 Align Views 50

Add angle 587 Allow Multiple Regions tool 285
Add curve 587 Angle between lines 337
Add line 587 Angles
Add outline 587 adding 587
Add point 587 Animation 165
Add polyline 587 Animation options group 628
Add Track Manually 424, 426 Animation Path group 624
adding Animation tab 623
shortcuts to the Quick Access Annotate 477
Toolbar 462
Annotation group 277, 303-304, 309,
Adding 312-314, 316, 319
New App 503 Annotation shapes 308
New Form 501 Annotations 302
New Module 501-502 creating 304
New Project 501 deleting 314
new shape 282 grouping 313
Adjust tab 88, 99, 190, 288 moving 312
Adjusting brightness, contrast, or gamma ordering 314
using the mouse 190
resizing 313
Advanced display tab 99
rotating 312
Advanced tab 257
setting the appearance of 316
Align by Feature 196
Antialiasing 629
Align Images 195
Aperio 635
FFT 197
App Center 491
Manual 197
App Controls 506
User 197
App Settings 508
Align Tiled Images 210

642
 App tab – Auto Trace

App tab 505 Auto-threshold 394-395

App toolbox 506 best-fit minimum 395
Appearance bet-fit maximum 395
Annotations 319 mean value 395
ROIs 295 median histogram value 395
Appearance options 584 minimum histogram value 395
Application configuration 545 minimum variance 394
Application icon 543, 545 mode value 395
Application Options 23 Auto-trace tool 307
Application window components 45 Auto Classification 411
Applying an ROI from an ROI Auto Open 76
collection 283
Auto Range 152
Applying enhancements to an image 191
Auto Trace 346
Apps 494
Assume clockwise 348
adding 494
Auto accept 347
Designing 505
Auto start 347
Running 496
Disable 348
Apps tab 90, 494
Edge quality 347
Arranging annotations 312
Momentum 347
Aspect Ratio 125, 129, 133
Monochrome interpretation 348
Audit Trail 256
Noise reduction 347
Audit Trail Options 257
Range 348
Audit Trail tools 258
Remove redundant points 348
Auto-Range 97, 640
ROI bound 348
Auto-Smart
Smoothing 347
Guided segmentation 598
Tracing speed 347
Trim leading points 347

643
Index

Automate tab 90 Best focus frame 206

Automate Tab 516 BIGTIFF 635
Automatic Collection 446 Bitmap Analysis 166
Automatic subsampling 573, 610 Bitmap analysis ribbon 167
Automatic Tracking 431 Bitmap annotations 311
Automatic Update Check 24 Blend 565, 606
Automating Processes 461 Blending 249
AutoQuant 635 BMP file formats 633
AutoThreshold 327 Boundary Opacity 558
Available measurements Brightness, adjusting 190
Data Collector 446 Buttons
Available measurements list 324 Create circular animation 631
Average vector 583 Create rocking animation 625
AVI file formats 633 File 63, 65, 68, 75, 267

B Light 560

Background 401 Reset 573

Background correction 224, 404 Select/Pick 631

Background Correction 224 View 553, 631

Background group 224 C

Background Subtraction 224 Calibrate by feature 127, 133, 456

Background Tools 108 Calibration
Batch Processing 516 Export 138
Best-fit Arc 336 Import 138
Best-fit Circle 335 unlock 137
Best-fit Line 336 Calibration locking 137
Best-fit measurements 335 Calibration Marker 139
Best Fit 96, 190

644
 Calibrations – Color group

Calibrations Collect group

applying a calibration marker to the Data collector 117
image 139
collections
creating a new calibration
described 281
definition 126
saving 284
saving 136
Collections
Camera light. 561
adding a new shape 282
Camera views 566
loading 284
Canvas 177
Collections Sub-panel
Capture ribbon 115
Guided Segmentation 592
Capture tab xxi, 59, 87, 103
colocalization
Channels group 188, 191
3D measurements 605
Characteristics
Colocalization 358
Optical 122
object-based 365
Check for Updates 56
Colocalization measurements 364, 366
Circle tool 306
Colocalization methods 359, 363
Circle Tool 106
multichannel 364
Circular animation 624, 629, 631
Colocalization panel 361
Classes group 116
Colocalization plot 360, 362
Classification
Colocalization thresholds 363
Learning 413
Color
multiple variables 411
changing the color of an
single variable 410
annotation 316
Classifying Objects 410
changing the color of an ROI 293
Clean Borders 327
Color Channels 188
Clipboard group 490
Color Composite 182
Close object 397
Color group 179, 181
Collapse group 482

645
Index

Color group commands 181 Convert to Annotation 244

Color mode Converting Images 179
Surface Plot 150 Count 611
Color spaces 404 Count feature 572
commands, making shortcuts for 462 Count results group 576
Comment group 491 Count/Size 380, 401
Compare Tab 92 Count/Size tab 399
Comparing Images 218 Counting
Composite best focus 205 iso-suface 575
Composition group 164, 564 isosurfaces 611
Configuration results 576
Application 545 Volume objects 575
Connect Start and End 629 Counting objects
Connectivity 327 results 383
Content sort 385
types of 60 Counting Objects in Images 380
Context-sensitive tabs 91 Create a polyline 589
Context menu Create circular animation 629
Image set 54 Create Hough Image 418
Context Menu Create Image 403
Image 53 Create Mask 402, 455
Contrast, adjusting 190 Create New group 501
Contrast, adjusting with mouse 190 Create rocking animation 625
Controls Crop tool
Image sequence 239 Larege image tools 262
Image set 272 Cubic interpolation 557
Conventions used in this guide xxi

646
 Curves – Drawing Properties

Curves Defining
adding 587 a new spatial calibration 126
Custom animation 625 an ROI 279
Custom tab 90 Delete 307
creating 477 Line Profile feature 106
locating 478 Delete ROI 281
renaming 478 Deleting
Customizing an annotation 314
Image Strip 465 an ROI 291
Tabs 477 DeltaVision 635
Customizing the User Interface 461 Direct Measurements 330
Cut, copy , paste 307 Direction vector 583

D Directional light 561

Data Collector 97, 445 Display group 190

Data Blocks 456 Display group. 188

Data Graph 457 Display options 308, 478, 481

Data Histogram 355, 458 Display points for best-fit features 336

Data measurements 324 Display Range 189

filter values 326 Display Range feature, using 189

Data Overlay group 242 Displaying Individual Objects 385

Data Overlay Options 244 Distance between centers 337

Data Table 351, 452-453 Distance between objects 337

Measurement names 351, 453 Document Properties 55

Data Table toolbar 455 Dragger tool 569

Debug mode 489 Draw Mode

Decoration group 554 Surface Plot 148

Drawing Properties 294, 317

647
Index

E Extract button

EDF Test Strips 205 Large image tools 260

Edge Coloring 559 Extract command 181

Edge Detection Tools 110 Extract dimension 274-275

Edge Measurements 333 Extract points 589

Edge Settings 112 Extras tab 91

Edges 207 F
Edges group 115 Fast Fourier Transform 232
Edit light 561 Feature Collections 281, 301
Edit Macro 488 Features Manager 108-109, 281-285
Edit Ranges 326 Feedback 42
Ellipse tool 306 Feedback form 42
for defining ROI 280 FFT 197, 232
EllipseTool 106 FFT method 197
Emphasis Switch FFT Window
Large image tools 260 Forward Button 233
Enhancing an Image 174 FFTprint.tif 232
Exclusion box 571 File button 61, 63, 65, 68, 75, 267, 478
Expand group 482 File button described 61
Experiments 60, 263 File format 60
Mixed content 263 File Format Specifications 633
Export Calibration 138 File Formats 633
Export data 350 AVI 633
Export volume 613 Bitmap 633
Extended Depth of Field (EDF) 203 fts 633
External objects 618 JPEG 633
mce 263, 633

648
 File group – Grouping Macros

mcs 634 Frames group 626

Pictures 634 Frames Per Second 627
Portable Internet Graphics 634 Freeform ROI 280
TIFF 634 Frequency spectrum 233
File group 117 Frequently Asked Questions 637
File Options 76 FTS file formats 633
File Signature 257 Full FFT Transform 420
Fill Holes 327 G
Filter strip 228 Gallery view 155
Filters 226-227, 573, 610 Gamma, adjusting 190
Find Circles 418 Gamma, adjusting with mouse 190
Find Irregular shapes 420 Gatan Digital Micrograph 635
Find Lines 417 Geometry
Find Shapes 417 Surface Plot 153
Circles 418 Geometry group 164, 174-175, 566
Irregular 420 Get center 588
Lines 417 Gradient 560
Find Tracks Automatically 432 Graph button 116
Floating Panels 45, 473 Graphic Object Properties 293-294, 306,
Focus 317-318
Maximum contrast method 206 Graphical annotations 304
Maximum intensity method, 206 Grid Overlay 229
Minimum intensity method, 206 Group/Ungroup 307
Font group 117 Grouping 353-354, 521, 603
Font, changing in an annotation 316 Grouping and Sorting 521
Font, changing in an ROI 293 Grouping annotations 313
Forward Button 233 Grouping Macros 524

649
Index

Groups I
Expanding or collapsing 482 Icon library 507
Hide or show 482 Identities
Grow 327 Administrator 546
Grow object 397 create new 544
Grow or shrink objects 396 managing 543
Guided Segmentation 590 Image-Pro IQ 635
Guided Segmentation panel 590 Image Analysis Forum 56
H Image Compare 218

Hamamatsu Nanozoomer 635 Image Context Menu 53

Hardware Configuration Wizard 126 Image Cytometry Std 635

Help menu 29 Image Gallery 155

Hide Base Lines 340 Image Gallery ribbon 156

Hide group 482 Image histogram 95-96

Hide or Show Animation cameras 629 creating 96

Hide tab 480 Image Histogram 96

Hide Tab 481 Image Information 55

Hide Unselected Tracks 429 Image Math 220

Hide VOI axes 569 Image Navigator 173

Hide VOI handles 569 Image sequence controls 240

Hide/show data 353, 355, 603 Image Sequences 237

Hint windows 177 Image Set Controls 272

Histogram image sets 266

freeform 101 Image sets 60

Histogram button 99 defined 68, 266

Histogram display 99 Image Signature 256

Home tab xxi, 87 Image strip 531

650
 Image Strip – License

Image Strip 465 Invert ROI 281

Image tab 89, 170, 173 isosurface 572-573, 618
images Isosurface sub-panel
enhancing by limiting the display Guided Segmentation 599
range 189
Isosurfaces 608
Images 531
J
adjusting brightness, contrast, and
gamma 190 Japanese language 484

panning 172 JPEG File Format 640

resetting 191 JPG file formats 633

zooming 170 K
Import Calibration 138 Key, calibration 139
Importing volumes 613 L
Incremental distance
L*a*b* Color measurements and Color
measurement types 340 Correction 370
Incremental Distance 338-339 Label Properties 294, 318
Information Large Image 260
Document/Image 55 Large image map 261
Installation instructions 9 Large Image Tools 260
Installation requirements 1 Latest News tab 57
Intensity 562 Layout
Intensity Calibration 142 Custom 52, 476
Intensity Measurements 435 Load 51, 475
Intensity Tracking 433 Save 52, 476
Intensity Tracking Options 436 Learning classification 413
Intensity Tracking Tools 434 Leica 635
Interpolation 557 License
Cubic 557 Upgrading 39

651
Index

License Agreement. 9 Logarithms 101

light absorption 548 Look-Up Table 188
Light Button 560 Look For Updates 24, 36
Light emission 548 Look Up Table 100
line measurements 341 LUT Adjustment 188
Line profile 95, 103 M
Line Profile 103, 116, 118 Macro Edit Ribbon 490
Line Profile Options 96 Macro Script Ribbon 488
Line Profile Tools 104 Macro scripting 485
Line Tool 105 Macros 485
Lines Grouping 524
adding 587 macros, making shortcuts for 462
Live EDF options 247-248 Magic Wand 296
Live EDF, 247 defined 297
Live Extended Depth of Field (EDF) 247 Magic Wand tool
Live Overlay 301 Guided Segmentation 595
Live Tiling 251 maintenance agreement xxii
Live Tiling Options 253 Manual edit edge tool 110
Load Data Table Measurements 352-353, manual measurements 586
602
Manual Measurements
Load Macro 488
introduction to this feature 323
Local Zoom 171
Tools 329
Lock
Manual method 199
Calibration 137
Manual Tagging 349
Lock button 640
Manual Tracking 424
Lock Setting 114
Marker Options 243
Log group 117
Marker, calibration 139

652
 Mask – Merge objects

Mask 391 Best fit 335

Mask and Overlay 402 Circle 332
Material 563 Direct 330
Maximum Contrast (texture), 206 Edge 333
Maximum distance 337 Ellipse 332
Maximum Intensity Projection 565, 606 Line 331, 341
Maximum Intensity, 206 Manual 329
MCE file formats 263, 633 Manual Tag 330
MCS file formats 634 Orientation 582
Measure angle between lines 588 pitch with line 331
Measure distance between centers 588 Point to point 331
Measure distance between point and sur- Polygon 332
face 588
Polyline 331
Measure distance between surfaces 589
Rectangle 332
Measure perpendicular distance 588
Relative 336
Measure tab 358
selecting 586
Measurement Information 351
Measurements button 116
Measurement labels 343
Measurements group 116, 586
Measurement names 351, 453
Meaurement Options dialog 343
Measurement Options 326
Median vector 583
Measurement tools 548
Memory limit slider
Measurement types 324, 446, 576
Large image tools 260
Measurements
Memory usage bar
3D types options 582
Large image tools 261
Angle 332
Merge command 181
Auto-trace Polyline 331
Merge Files 193
Auto-trace Rectangle 332
Merge objects 397

653
Index

Merge Objects 338 Multiple ROIs 281

Merge selected objects 589 Multiple Users 542
Micromanager 635 N
Micrometer 132 Navigate group 552, 631
Minimum distance 337 Nikon Elements 635
Minimum Intensity 206 Normalize Illumination 206
Minimum Intensity Projection 565 nudge tool 299
Mixed content experiments 263 Nudging 299
Molecular Devices Metamorph 635 Number of slices 565
Morphological operations 405
O
Morphological Operations
Object class 401
measurements 341
Object window 385
Morphological operatios
Object Window 356, 604
Close 341
Objects
Grow 341
Classifying 410
Open 341
counting 380
Shrink 341
Counting 380-381
Motion Detection 195
Displaying 386
Mouse wheel, using to zoom 172
grow or shrink 396
Mouse, to adjust brightness, contrast, or
gamma using 190 open or close 397

moving Sorting 384

an ROI 287 splitting 382, 587

Moving Oblique slices 616

an annotation 312 Olympus FV 1000 635

multi-class mask 403 OME-TIFF 635

Multiple Document Interface 48, 471 Open or close objects 397

Open Saved Image 76

654
 Open Set From File – Polygon Nudge tool

Open Set From File 266 P

Opening an Image 63 Package contents 2
Opening an Image Set 67 Pan Tool 172
Optical Characteristics 122 Panning images 172
Optional tabs 91 Parent image
Options Colocalization 359, 363
Applications 23 Password
Display 478, 480 administrator 543
Image histogram 98 creating 546
Line Profile 96 Password protection 547
Live EDF 248 Password Protection 545
Live Tiling 253 Pattern matching 112
Popular 545 Pattern Matching 212
Project Workbench 492 PCT file formats 634
segmentation 326 Peaks 110
Options group 117 Pearson correlation 360
Ordering an annotation 314 PerkinElmer 635
Orientation Measurements 582 Pixel offset 111
Ortho slices 615 Pixels per Unit 125-126
Otsu algorithm 360, 394 Playback group 627
Outlines PNG file formats 634
adding 587 Point tool 307
Outlines group 585 Points 337
Output group 630 adding 587
Overlays 301 Polygon Nudge 299
Overview panel 45 Polygon Nudge tool
Guided segmentation 594

655
Index

Polygon tool 306 Quick Access Toolbar, using 462

for defining ROIs 280 Quick Calibration Method 127
Guided Segmentation 594 Quick Print 80
Polygon Tool 106 Quick Save 75
Polyline Tool 105 Quick Save for Analysis 76
Polylines Quick Save for Publication 76
adding 587 R
Popular Options XE 545 Ranges 578
Pre-Integrated 557 read-only file formats 634
Predictive calibration 134 Recent documents 62
Print 80 Recent Documents pane 121
Print Preview 81 Recent images 56
Printing an Image 80 Recipe Options 403
Process tab 88, 193, 226, 232 Record Macro 485
Project Explorer 485, 499 Record Macro command 488
Project group 488 Record Rendering Settings 628
Project Properties 491 Rectangle by Coordinates tool (for defin-
Project Settings 491 ing ROIs) 279
Project Workbench 486-487, 491, 500- Rectangle by Numbers tool (for defining
501, 503, 505-506, 537 ROIs) 280
Report Tools 536 Rectangle tool 305
Project Workbench Options 492 Rectangle Tool 106
Project Workbench tabs 536 Reference Calibration 126
Property Panel 504, 506 Reference Objects 400
Pseudo-Color 186 Reference vector 583

Q Region of Interest 101, 278

Regions of Interest 276
Quick Access Toolbar 82, 462-464

656
 Relative Measurements – ROIs

Relative Measurements 336 Reset Display button 191

Relative Measurements Group 588 Resetting images 191
Reload 79, 551 Resize 174
Reload 3D view 163, 551 Resizing
Reload when image changed 551 an annotation 313
Remove Periodic Noise 232 an ROI 287
Report 532-533 Resizing an Image 174
creating and printing 531 Resolution slider
Editing 533 Large image tools 260
File group 533 Rising Edges 110
Format 532 RMA
Preview 532 Return Materials Authorization 38
Printing 533 ROI Auto-Trace 107, 280
Saving 533 ROI Display Options 281, 293
Report Contents tab 534 ROI group 293, 295
Report data 538-539 ROI options 295
Report fields 538-539 ROI Options 281
Report Home tab 534 ROI Order 281
Report Insert tab 534 ROIs
Report Layout tab 534 Applying to image from an ROI col-
lection 283
Report tab 533
defining 279
Report tools 530
deleting 291
Report Tools 534
described 278
Reports 530
loading a collection 284
Requirements, hardware and software 3,
5 moving 287
Reset button 552, 570, 573 placing more than one ROI in an
image 285

657
Index

resizing 287 Saving an Image 74

rotating 289 Saving Annotations 320
saving a collection 284 Scale Bar 172
Setting the appearance of 293 Scatterplot 459
tools described 279 3D 459
Rotate 175 Search group 490
Rotate tools 175, 177 See-saw animation 625
Rotating Segmentation
an annotation 312 Example 407
Rotating an Image 175 recipe summary 408
Rotating an ROI 289 Segmentation bias 327
Rotation type 626 Segmentation Bias 395

S best-fit 396

Saturation 95 range maximum 395

Saturation Warning 119 standard deviation 396

Save 74 Segmentation objects

Save All 75 Defined 592

Save As 77 Segmentation Options 394

Save as Movie 76 Segmentation type 399

Save Calibration dialog box 136 Segmetation options 326

Save Data Table Measurements 352-353, Select All tool 305

602 Select and Count 400
Save to File 75 Select measurements 586
Save Tracks 429 Select mode 572
Saving Select tab 88, 277, 279, 303-304, 391
an ROI collection 284 Select tool 305, 312
calibrations 136 Select Tool 105

658
 Select/Pick Button – Sorting

Select/Pick Button 631 Show Shadows 562

Selected Measurements list 325 Show tab 480
Selection tool Show VOI axes 569
for ROIs 279 Show VOI handles 569
Guided Segmentation 594 Show/Hide mask 402
Selection Tools 329 Shrink object 397
Send Measurements to File 352, 354, Single Variable Classification 410
602, 613
Slicer
Send Measurements to Report 352, 603
Layout tools 619
Sequence Files 634
Slicer Position group 621
Sequence toolbar 239-240, 424
Slicer ribbon 619
Set
Slicer tab 619
Open from file 67, 266
Slicer Viewer 159
Set Builder 68
Slicer Viewer ribbon 160
Set measurement 456
Smart Segmentation 401
Set Navigator 274
Smart Segmentation button 596
Set Order 307
Smart Segmentation Mode
Shading Correction 213
Guided Segmentation 595
Shadow screen 563
Smart Segmentation Training 402
Shapes
Smart Segmentation. 399
Finding 417
Smoothing 327
Share tab 89, 256, 530-531, 537
Smoothness 562
Sharpen filter 226
Snap to Edge 211, 333
Show All Tracks 429
Software license
Show Data Overlay 243
returning 38
Show group 482
Software version 26
Show Image 560
Sorting 521

659
Index

Sorting objects 384 System Calibration 126

spatial calibration 129 System requirements 3, 5
Spatial calibration 125-126, 130 T
Spatial Calibration 132-133, 136 Table button 116
Spatial Calibration group 127-128, 130 Tabs
Split objects 382, 587 2D Count/Size xxi
Split polyline into lines 589 2D Measure xxi, 89
Statistics 353-354, 603 3D Measure 91, 550, 572
Image histogram 98 3D View 91, 550-551
Status bar 84 Adjust 88
Status bar, described 84 Animation 623
Step Size 340 App 505
Step Tablet Wizard 145 Apps 90, 494
Stereo Alignment 249 Automate 90
Stereo View 556 Capture xxi, 87
Stitching 211 Compare 92
Streampix 635 Context-sensitive 91
Style Count/Size 380, 399
Surface Plot 151 Custom 90
Sub-sampling 194 Extras 91
Sub-volume 570 Hide or show 480
Sub sampling 573 hiding 480
Subsample Sequence Dialog Box 194 Home xxi, 87
Sum 565 Image 89, 146
Support 30 Latest news 57
Surface Plot 146 Measure 424
Synch with Time 629 Optional 91

660
 Tabs and Ribbons – Tracking Objects

Process 88, 193, 232 Threshold 112-113, 559

Project Workbench 536 Threshold Type 573
Report Contents 534 Thresholding 391
Report Home 534 class 392
Report Insert 534 Thresholding Tools 393
Report Layout 534 Tiling 209
Select 88, 277, 303, 391 Tiling and Stitching 209
Share 89, 256, 531, 537 Tiling Display 215
Slicer 619 Tiling Options 217
Tiling View 209 Tiling Result 215
Training resources 56 Tiling View tab 209
View 47, 89, 475 Tools 260
Tabs and Ribbons 87 Layout 619
Tagged Image File Format 634 Redock 51, 475
Task Manager 524 Reset 51, 475
Task Scheduling 527-528 thresholding 393
Technical support xxii Tooltips 46, 177, 325, 468
Templates Viewing 46, 468
predefined 532 Topographic Map 206-207
Test Strips Tracking
EDF 205 Automatic 431
Text annotations 309 Intensity 433
Text tool 305, 309 Manual 424
Texture mode Tracking Data Table 425-428, 436
Surface Plot 148 Tracking histogram 433
The Image Tab 146 Tracking Histogram 438
Thickness group 621 Tracking Objects 424

661
Index

Tracking Options 426-427 View mode 572

Tracking Statistics 427 View Style group 556
Training Resources tab 56 View tab 47-48, 89, 465, 471, 475
Transparency 557 View Tab
Trim borders 199 Layouts 51, 475

U tools 51, 475

Undo split/merge 589 Viewing Image Document

Information 55
Update Available 24
Viewing Intensity Measurements 94
Updates 22, 30
VLI Tools 260
Software 26
VOI shape
Upgrade
sub-volume 571
Software 35
Volume
Upgrading your License 39
exporting 613
Upgrading your Product 23, 35
importing 613
User Interface
Volume Measurements 578, 605
Customizing 461
Volume Measurements group 576
User method 199
Volume of Interest group 568
Users Guide 30
Volumen Display group 551
Using Quick Save 75
Voroni map type
V
center-to-center 422
Validate button 595 edge-to-edge 422
Valleys 110 Voronoi Map 422
Very Large Images 259 Voxel 548, 554
View All 556 Voxel Size group 553
View Angle slide 566 Voxelize 557
View Button 553, 631 Voxels Size 163
View group 489, 602, 619

662
 Wand Mode – Zooming images

Wand Mode
Guided Segmentation 595
Welcome screen 56, 121
Windows Media Video 635
Windows Security Alert 21
Wire mode
Surface Plot 150
Workspace 549

XOR pixels 281, 287

Z
Illumination” 206
Z-Propagation sub-panel
Guided Segmentation 597
Zeiss LSM 635
Zeiss Widefield 635
Zoom and Pan controls 47, 469
Zoom Button 170, 173
Zooming images 170

663