Image-Pro Plus Version 6.

0 for Windows Start-Up Guide

Media Cybernetics, Inc. 8484 Georgia Avenue Silver Spring, MD 20910 (301) 495-3305, FAX (301) 495-5964

MAN SU 41N60000 20051031

COPYRIGHT NOTICE
Copyright 1993, 2005 Media Cybernetics, Inc. All Rights Reserved TRADEMARK ACKNOWLEDGMENTS HALO, HALO Desktop Imager, Image-Pro and Image-Pro Plus are registered trademarks of Media Cybernetics, Inc. All other trademarks in this document are trademarks or registered trademarks of their respective companies.

IMPORTANT: PLEASE READ CAREFULLY LICENSE AGREEMENT

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PROPRIETARY RIGHTS This Agreement gives you ownership only of the physical program media on which the PRODUCT is stored, but not of the PRODUCT itself. You acknowledge that Media owns all right, title and interest in the PRODUCT, and that you will acquire no rights in the PRODUCT through your use of it. You agree that you will take no action that interferes with Media's rights in the PRODUCT. TERM This Agreement is effective until terminated. You may terminate it at any time by destroying the PRODUCT together with all copies and documentation in any form. This Agreement will also terminate automatically and without notice from Media if you fail to comply with any term or condition of this Agreement. You agree upon such termination to destroy the PRODUCT and all copies of the PRODUCT. DISCLAIMER; LIMITED WARRANTY EXCEPT AS PROVIDED BELOW, THE PRODUCT IS PROVIDED "AS IS" WITHOUT WARRANTY OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING, BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE PRODUCT IS WITH YOU. SOME JURISDICTIONS DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES, SO THE ABOVE EXCLUSION MAY NOT APPLY TO YOU. NEITHER MEDIA NOR MANUFACTURER WARRANT THAT THE FUNCTIONS CONTAINED IN THE PRODUCT WILL MEET YOUR REQUIREMENTS OR THAT THE OPERATION OF THE PRODUCT WILL BE UNINTERRUPTED OR ERROR-FREE. However, where Media is the Manufacturer, Media warrants that the program media on which the software is furnished will be free from defects in materials and workmanship under normal use for a period of ninety (90) days from the date of delivery as evidenced by a copy of your receipt. LIMITATION OF REMEDIES Where Media is the Manufacturer, Manufacturers entire liability and your exclusive remedy shall be: 1. the replacement of the program media not meeting the Limited Warranty, which is returned to Manufacturer with a copy of your receipt; 2. if Manufacturer is unable to deliver replacement program media which is free of defects in materials or workmanship, you may terminate this Agreement by returning the PRODUCT and a copy of your receipt to the place of purchase, and your money will be refunded. Where Media is not the Manufacturer, Media shall have no liability to replace or refund, and you agree to look to Manufacturer to meet the obligations described above. LIMITATION OF LIABILITY IN NO EVENT WILL MEDIA OR MANUFACTURER BE LIABLE TO YOU FOR ANY DAMAGES, INCLUDING, BUT NOT LIMITED TO, ANY LOST PROFITS, LOST SAVINGS, OR OTHER INDIRECT, SPECIAL, EXEMPLARY, INCIDENTAL OR CONSEQUENTIAL DAMAGES, ARISING OUT OF THE USE OR INABILITY TO USE THIS PRODUCT, EVEN IF MEDIA OR MANUFACTURER HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. FURTHER, IN NO EVENT WILL MEDIA OR MANUFACTURER BE LIABLE FOR ANY CLAIM BY ANY OTHER PARTY ARISING OUT OF YOUR USE OF THE PRODUCT. SOME JURISDICTIONS DO NOT ALLOW THE LIMITATION OR EXCLUSION OF LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES, SO THE ABOVE LIMITATION OR EXCLUSION MAY NOT APPLY TO YOU. TRADEMARKS Image-Pro Plus is a trademark of Media Cybernetics, Inc. and Media Cybernetics is a registered trademark of Media Cybernetics, Inc. Microsoft, VisualBasic and the VisualBasic logo are registered trademarks of Microsoft Corporation. No right, license, or interest in such trademarks is granted hereunder. U.S. GOVERNMENT RESTRICTED RIGHTS IN DATA This computer software product and documentation are provided with Restricted Rights. Use, duplication, or disclosure by the U.S. Government is subject to restrictions as set forth in the Rights in Technical Data and Computer Software clause at DFARS 252.227-7013, or subparagraphs (c)(1) and (2) of the Commercial Computer Software - Restricted Rights at 48 CFR 52.227-19, as applicable. Contractor/Manufacturer is Media Cybernetics, Inc., 8484 Georgia Avenue, Silver Spring, Maryland 20910.

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You agree not to export or re-export the PRODUCT, directly or indirectly, to any countries, end-users or for any end uses that are restricted by U.S. export laws and regulations, without first obtaining permission to do so as required by the U.S. Department of Commerce's Bureau of Industry and Security, or other appropriate government agency. These restrictions change from time to time. If you have any questions regarding your obligations under U.S. export regulations, you should contact the Bureau of Industry and Security, U.S. Department of Commerce, Exporter Counseling Division, Washington D.C. (202) 482-4811, http://www.bis.doc.gov. GENERAL 1. You may not sublicense, assign or transfer the license or the PRODUCT, in whole or in part, except as expressly provided in this Agreement. Any attempt to do so is null and void. 2. This Agreement will be governed by the law of the State of Maryland applicable to agreements made and to be performed in the State of Maryland, but shall not be governed by the Uniform Computer Information Transactions Act as adopted in Maryland, or the United Nations Convention on Contracts for the International Sale of Goods. 3. Should any part of this agreement be declared void or unenforceable by a court of competent jurisdiction, the remaining terms shall remain in full effect. 4. Failure of Media to enforce any of its rights in this agreement shall not be considered a waiver of its rights, including its rights to respond to subsequent breaches. 5. This Agreement is the complete and exclusive statement of the agreement between you and Media and supersedes any proposal or prior agreement, oral or written, any other communication between you and Media relating to the subject matter of this Agreement. Should you have any questions concerning this Agreement, you may contact Media in writing at the address above. BY USING THIS SOFTWARE YOU ACKNOWLEDGE THAT YOU HAVE READ THIS AGREEMENT, UNDERSTAND IT, AND AGREE TO BE BOUND BY ITS TERMS AND CONDITIONS.

Contents
Introducing Image-Pro Plus Version 6.0........................................................ 1-1 Product Features ........................................................................................ 1-1 What's New in Version 6.0........................................................................ 1-3 Package Contents....................................................................................... 1-4 System Requirements ................................................................................ 1-5 Upgrading From A Previous Version Of Image-Pro Plus ....................... 1-6 Installing Image-Pro Plus.......................................................................... 1-7 Installing Image-Pro Plus with a multi-user (red) network key.............. 1-15 Installing Image Capture Support............................................................ 1-26 Installing IQbase Support........................................................................ 1-28 Starting the Image-Pro Plus Program ..................................................... 1-29 Technical Support On Line ..................................................................... 1-29 Contacting Technical Support ................................................................. 1-30 Solutions Zone......................................................................................... 1-31 Imaging Discussion ....................................................................................... 1-33 What is Image Processing?...................................................................... 1-33 Image Digitization ................................................................................... 1-33 Pixel Depth .............................................................................................. 1-34 Image Class ............................................................................................. 1-35 Color Models ........................................................................................... 1-39 Enhancing Your Image............................................................................ 1-41 Modifying the Intensity Index ................................................................. 1-41 Spatial Filtering ....................................................................................... 1-52 Frequency Filtering ................................................................................. 1-61 Intensity Analysis .................................................................................... 1-62 Measuring and Counting ......................................................................... 1-72 Customizing the Command Menus and Buttons ..................................... 1-97 Getting Started ................................................................................................. 2-1 The Image-Pro Plus Application Window................................................ 2-1 Image-Pro Plus Demonstration Macros .................................................. 2-11 Learning the Basics ......................................................................................... 3-1 Loading the Image..................................................................................... 3-2 Copying and Pasting.................................................................................. 3-4 Enhancing the Image ................................................................................. 3-5 Closing the Image Windows and Exiting Image-Pro Plus ...................... 3-17

vii

Filtering, Calibrating, and Measuring............................................................ 4-1 Loading an Image from the Database........................................................ 4-2 Sharpening the Image ................................................................................ 4-3 Calibrating the Spatial Scale...................................................................... 4-6 Measuring Length .................................................................................... 4-11 Using the Trace Measurement Tool ....................................................... 4-16 Saving the Measurements........................................................................ 4-24 Closing all the Windows and Exiting Image-Pro Plus............................ 4-26 Optical Density Analysis.................................................................................. 5-1 Loading the Image..................................................................................... 5-2 Specifying the Black and Incident Levels ................................................. 5-6 Plotting the Density of a Lane ................................................................... 5-9 Plotting the Density Against a Baseline .................................................. 5-13 Storing the Plot to an Image File ............................................................. 5-16 Closing all the Windows and Exiting Image-Pro Plus............................ 5-18 Filtering with Fast Fourier Transforms ......................................................... 6-1 Transforming the Image ............................................................................ 6-2 Editing the Spectrum ................................................................................. 6-4 Returning the Spectrum to Spatial Form ................................................... 6-7 Sending as an E-mail Attachment............................................................ 6-10 Closing all the Windows and Exiting Image-Pro Plus............................ 6-12 Counting, Measuring, and Classifying ........................................................... 7-1 Perform Gray Scale Thresholding ............................................................. 7-2 Select Measurements ................................................................................. 7-6 Counting the Objects ............................................................................... 7-10 Graphical Exclusion of Ranges ............................................................... 7-14 Splitting Objects ...................................................................................... 7-17 Obtaining Measurement Distributions..................................................... 7-20 Classifying the Measurement Data .......................................................... 7-22 Color-coding the Objects ......................................................................... 7-26 Saving the Measurement Data ................................................................. 7-29 Creating a Report ..................................................................................... 7-31 Using Templates ...................................................................................... 7-37 Closing All the Windows and Exiting Image-Pro Plus .......................... 7-45

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Color Segmentation ......................................................................................... 8-1 Contrast Enhancement............................................................................... 8-2 Performing Color Segmentation ................................................................ 8-4 Counting the Spots .................................................................................... 8-9 Closing all the Windows and Exiting Image-Pro Plus............................ 8-15 Calculating Population Density ...................................................................... 9-1 Copying the Image to a New Window ...................................................... 9-2 Identifying Cell Sites ................................................................................. 9-3 Removing Extraneous Grains.................................................................... 9-5 Outlining the Cell Sites.............................................................................. 9-6 Saving the Cell Outlines .......................................................................... 9-10 Preparing the Grain Image....................................................................... 9-13 Counting the Grains................................................................................. 9-16 Calculating Population Density ............................................................... 9-19 Closing all the Windows ......................................................................... 9-22 Advanced Exercises ....................................................................................... 10-1 Using the Calibration Wizard .................................................................. 10-1 Tracking Objects ................................................................................... 10-17 Creating a Workflow Toolbar ............................................................... 10-31 Closing all the Windows and Exiting Image-Pro Plus.......................... 10-40 Glossary........................................................................................................... G-1 Index..........................................................................................................Index-1

ix

Product Features

Introducing Image-Pro Plus Version 6.0

Welcome to Image-Pro Plus version 6.0, the most complete and powerful image analysis program available today. This version of Image-Pro Plus is designed to run under the Microsoft Windows 32-bit systems: Windows 2000 (service pack 4), and Windows XP Professional (service pack 2) . These advanced image processing features are provided through the Microsoft Windows graphical user interface (GUI), delivering powerful imaging capabilities in an easy-to-use tool. In corresponding package materials, Image-Pro Plus is also referred to as Image-Pro and IPP.

Product Features
Image-Pro Plus gives you state-of-the art imaging and analysis capability for acquiring, enhancing and analyzing your images. This includes the ability to: Acquire image data from a camera, microscope, VCR, or scanner Read and write image data in all the standard image file formats including, TIFF, JPEG, BMP, TGA, and many others. Work with gray scale data in 8, 12, 16, or 32-bit floating-point depths. Work with color data in palletized or 24-, 36-, or 48-bit format. Manipulate color data using RGB, HSI, HSV or YIQ models. Perform image enhancement using powerful color and contrast filters, including Fast Fourier Transforms (FFT), morphology, field flattening, background subtraction and other spatial and geometric operations. 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. View collected data numerically, statistically or in graphic form (histogram and scattergram). Save the measurement data to disk. Sort and classify your measurement data according to your predefined criteria. Color code your objects by class.

Page 1-1

Image-Pro Plus Start-Up Guide Collect intensity data for an entire image, an area of interest, a single line or a band of lines. Measure intensity using the standard intensity or Optical Density curves supplied by Image-Pro Plus, or create your own custom curve. View intensity data in graph or table form. Save the intensity data to disk. Extract features with spatial tools that isolate an Area of Interest (AOI) from the rest of the image, or with segmentation tools that extract features by color or intensity value. Create composite in-focus images from partially focused source images. Create composite images from multiple fluorescent probes. Generate reports that can include images, measurement data, text, and pictures. Manage your images using Media Cybernetics new IQbase product. Create organized collections of image thumbnails that can be easily loaded, located, and retrieved. Automate repetitive tasks and customize Image-Pro Plus to your particular needs using Auto-Pro, the powerful macro language that saves you real time. You can call Auto-Pro functions from high-level languages like Visual Basic and Visual C++. You can combine Auto-Pro calls with your own programs to create custom user-interface, processing and I/O routines.

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Product Features

What's New in Version 6.0

The following improvements have been made to Image-Pro Plus v. 6.0: IQbase 2.0, the newest version of the Media Cybernetics image information management tool, is included with IPP 6.0 to provide advanced storage, information management, and reporting ability. Image-Pro Plus 6.0 is compatible with IQBase at all levels. You can now import your legacy Image Database (IDB) files into IQbase. Due to size constraints, the Image-Pro Plus Reference Guide and Auto-Pro Reference are no longer included as part of this package. All the information from those manuals appears in the online help. These manuals may be ordered from Media Cybernetics Customer Service department. A new menu editor allows you to customize the Image-Pro Plus menus New demonstration macros have been added to IPP 6.0. DDE now supports exporting files to Origin 7.5 Image-Pro Plus v. 6.0 now supports Olympus FV-1000 file formats There are new charting windows for the Data Collector, including Histogram, Scatterplot, and Data Graph. You can now synchronize data among the data collector windows, graph windows, and count/size. The Data Collector includes a new equation editor. The Measure Distances feature is now included, on the Measure menu 3D filters are now a standard feature of IPP, on the Process menu IPP now includes Integrated color management and ICC profiles based on Microsoft CMM Many new Auto-Pro functions have been developed to support the advanced features of Image-Pro Plus.

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Image-Pro Plus Start-Up Guide

Package Contents
Before you get started, please check that you have received everything in your ImagePro Plus package: Image-Pro Plus CD Image-Pro Plus Capture CD 1 USB port copy-protection plug Image-Pro Plus Start-Up Guide (this book) Registration/warranty card Documentation Feedback Card Image-Pro Plus Brochure

If any piece is missing, please contact Customer Service at (301) 495-3305. Important: The Capture Drivers are supplied on a separate CD. To complete the Image-Pro Plus installation, the capture information from the CD must be installed on your system. From time to time, we provide new and updated capture drivers on our web site. To check for new drivers, please visit www.solutions-zone.com .

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System Requirements

System Requirements
To install and work with Image-Pro Plus 6.0, you will need the following equipment and software: Pentium III CPU, running at 1.0 Ghz or higher configured with Windows 2000 (service pack 4), or Windows XP Professional (service pack 2) A minimum of 512 MB of RAM ( 1 GB recommended) 3 GB disk drive with available disk space for installation with additional space to accommodate four times the image size with four bytes/pixel An SVGA (or better) high color, 16-bit resolution graphics card with Open GL graphics, a hardware accelerator, and a minimum of 32M memory that supports 2D texturing A Windows-compatible mouse, keyboard, and CD-ROM.

The following hardware is optional: Digital Camera Image Capture Board (frame-grabber) Scanner Printer.

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Image-Pro Plus Start-Up Guide

Upgrading From A Previous Version Of Image-Pro Plus

If you are upgrading from an earlier version of Image-Pro Plus, the Image-Pro Plus setup will guide you through the process of upgrading from version 5. 0/5.1 to version 6.0. If you are upgrading from version 4.X or earlier, we recommend that you consider the following before installing version 6.0. (You may have more than one version of Image-Pro Plus installed on your computer.) Image-Pro Plus version 6.0 contains many new and enhanced features. Some of these features may perform differently than similar features in previous versions of ImagePro. To insure a smooth migration to Image-Pro Plus version 6.0 we suggest that you leave your previous version of Image-Pro Plus installed on your system and install Image-Pro Plus version 5.0/5.1 in a separate directory. After installing Image-Pro Plus version 6.0, use both versions and when you are satisfied that your old macros, settings, etc., perform to your satisfaction in Image-Pro Plus version 6.0, remove the previous version of Image-Pro Plus. For additional information regarding possible migration issues, please see the Release Notes.

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Installing Image-Pro Plus

Installing Image-Pro Plus

To install Image-Pro Plus, follow the steps below. If you are installing Image-Pro Plus as an upgrade to your existing version, please see the section entitled Upgrading Your Image-Pro Plus Software later in this chapter. Note that Image-Pro Plus 6.0 will not install on versions of Windows older than Windows 2000. STEP 1. PROCEDURE Attach the copy protection plug to an operable parallel (LPT1, LPT2). or USB port. Without this plug, Image-Pro will only operate in demo mode. More information about the plug appears in the following section. Place the Image-Pro CD in your CD-ROM drive. Within a few seconds, the Setup program will start running automatically. The Image-Pro Selection screen will appear. Indicate that you are installing Image-Pro.

2.

Make your selection from the screen above to continue installing Image-Pro or its components. Note: Most installations of Image-Pro will be importing images from a TWAIN-compatible scanner, and therefore do not need to install any capture drivers. continued on next page

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Image-Pro Plus Start-Up Guide STEP 3. PROCEDURE If the Setup program does not start automatically, click the Start button on your Windows desktop, and choose Run. Use the Browse button to find the your Image-Pro CD. Select the Start program. The path is: CD Drive letter:\start.exe 4. After clicking Install Image-Pro Plus on the Autostart dialog, Image-Pro will ask you to wait for a few minutes to prepare the Install program. Note that this may take some time, depending on your operating system. 5. You will see the Image-Pro Welcome screen. After reading it, click Next to continue installing Image-Pro.

Note that you may have only one installation of IPP 6.0 on your computer at any one time. continued on next page

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Installing Image-Pro Plus STEP 6. PROCEDURE The next screen asks if you want to install, modify, or remove the selected program. This screen will not appear if you are installing IPP 6.0 for the first time.

Select one of the options, and click Next. 7. If you have choosen to install or modify IPP, you will see the Image-Pro license agreement. Click Yes to continue the installation process.

8.

Indicate if you want other users of this computer to have access to your Image-Pro installation, or if it is for your use alone:

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Image-Pro Plus Start-Up Guide STEP 9. PROCEDURE Select the type of installation you require from the following choices, as shown on the next screen:

9a.

Typical is the recommended installation for most computers. Typical gives you all the Image-Pro files, including the online help for Image-Pro and Auto-Pro, as well as the image database function, and sample images. If you are installing Image-Pro for the first time, this is the setup you should choose. If you select Typical, continue the installation process from step 10. If you are installing Image-Pro to be used with a red network key, choose the Typical installation and then follow the instructions on page 1-15 for installing the network key License Server and Clients. Compact allows you to install a reduced set of files to save space on your hard drive. Custom allows you to choose which Image-Pro components you want to install. If you choose a Custom installation, continue from Step 14. Click on the button for your selected installation. continued on next page

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Installing Image-Pro Plus STEP 10. PROCEDURE Select the location for Image-Pro. The default destination for Image-Pro is the C:\ drive in the directory IPWIN51. You can specify a different location by clicking the Browse button, and selecting or typing in a new name.Click Next to continue the installation process.

10a.

If you want to install Image-Pro in another folder, use the Browse button to select a folder. Highlight the name of the folder and click OK. Click Next to continue installing ImagePro.

continued on next page

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Image-Pro Plus Start-Up Guide STEP 11. PROCEDURE Specify the folder where you want the Image-Pro program icons installed. The default folder is called Image Pro Plus 6.0 . This folder name appears on the Start menu under Programs. Click Next to continue installing Image-Pro. Follow the instructions on the Image-Pro setup screen. The Progress Indicator shows that the Image-Pro files are being loaded onto your computer.

12.

13.

When the installation process is complete Image-Pro folder will appear on the Program List when you click the Start button. From this screen, you can view the Release Notes, start ImagePro, or exit and do something else.

The final installation screen indicates that installing Image-Pro has changed your system configuration. It may ask if you want to restart your computer right away, or wait until later. Click the appropriate radio button for your choice. continued on next page Page 1-12

Installing Image-Pro Plus STEP 14. PROCEDURE If you select Custom, you will see the Select Components dialog. This dialog allows the you to selectively install any components desired. This dialog only appears in a Custom installation. Check the components to install, then click Next.

To continue with a custom installation, click Next. (Please go back to step 10.

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Image-Pro Plus Start-Up Guide

Installing the Copy-Protection Plug

Image-Pro Plus is hardware-locked. A special security plug (dongle) is included with Image-Pro Plus to unlock the program. Only when this plug is connected to the parallel or USB port on your system will Image-Pro Plus remain active. If the plug is not installed, or if it is removed while Image-Pro Plus is operating, Image-Pro Plus will revert to the demo mode. To install the plug, simply connect it to any operable port of the correct type on your system. If a printer or scanner is currently attached to the port, it must be disconnected and reconnected after the plug has been installed (the device can simply be plugged right into the back of the copy-protection plug). Important: DO NOT connect any device other than a printer to this plug (e.g.,scanner, tape backup unit, or laptop data-transfer link) as this can permanently damage the plug. If your plug does not seem to work, please see the Diagnosing Protecting Key Problems section, below.

Diagnosing Protection Key Problems

If you are having problems with your copy-protection plug, please consider the following: What is your parallel port mode setting? It should be set to bi-directional if that is an option, otherwise you should choose something other than EPP or Standard, usually ECP. To find out, check your BIOS when you start your computer. You will find information about accessing and changing your BIOS in your computers documentation. On many computers, you can access the BIOS by pressing the <F1>, <F2>, or <Del> keys during the startup process. Your computer should display a prompt telling you which key to press. If you do not see his prompt, it may be hidden by a startup screen; pressing <ESC> or <Tab> will often clear the screen and allow you to see the startup messages. Navigate through the BIOS using your arrow keys. Look for settings for Peripherals and Parallel Port mode. Download the HASP Diagnostic program ( HaspDg.exe) from the HASP section of the Software Updates page of the Media Cybernetics Technical Support web page (http:\\support.mediacy.com). Run this program to see if the key is recognized by the program.

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Installing Image-Pro Plus with a multi-user (red) network key If the HASP program does not recognize your key, please download and run the HDD32.zip program from Software Updates page of the Media Cybernetics Technical Support web page (http:\\support.mediacy.com). Using this program will reinstall the HASP drivers. Then you can re-run the HASP Diagnostic program ( HaspDg.exe). If the key still does not work, please call Media Cybernetics customer service at 301-495-3305, and request a replacement key.

Installing Image-Pro Plus with a multi-user (red) network key

Setting up a multi-user network license is a two-step process. First, the License Server must be installed and configured on the Server computer. Next, the License Client must be installed on every computer that will actually run Image-Pro (including the Server, if it will be used to run Image-Pro). NOTE: You MUST be logged on as an Administrator when installing the network key License Server or Client under Windows 2000 or Windows XP. Installing the License Server NOTE: It is strongly recommended that the License Server be configured with a Static IP Address. If the License Server is using a Dynamic IP Address (i.e. DHCP) you need to reconfigure each Image-Pro client machine every time the License Server IP Address changes (e.g. when it is rebooted). If you are not sure whether the License Server is using a Static or Dynamic IP Address, please contact your System Administrator.

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Image-Pro Plus Start-Up Guide STEP 1. PROCEDURE If you will also be running Image-Pro on the License Server, do a Typical installation of Image-Pro, according to the instructions earlier in this chapter. Otherwise, skip to Step 2. Click Install Network Support. You will see the Welcome screen.

2.

3.

Click Next to advance past the Welcome screen. Indicate where you want to install the NetHasp, and then click Next.

continued on next page

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Installing Image-Pro Plus with a multi-user (red) network key STEP 4. PROCEDURE Select a Server installation, and click Next.

5.

When you installing the License Server as a service on Windows 2000 and XP systems, this screen asks whether to install the License Server as a service. Doing so is the recommended option, since this allows the License Server to run, even if no one is logged into the system.

Click Next after making your selection. continued on next page

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Image-Pro Plus Start-Up Guide STEP 6. PROCEDURE Click Next to accept the default directory, and begin copying files, or click Browse to change the directory in which the License Server will be installed.

7.

Make a note of the License Servers IP Address; you will need this during the client installation. If you do not know the License Servers IP Address, start a command prompt (Start: Programs:Accessories:Command Prompt) and type ipconfig. This command will produce output like that shown here:

Microsoft Windows XP [Version 6.0.2600] (C) Copyright 1985-2001 Microsoft Corp. C:\>ipconfig Windows IP Configuration Ethernet adapter Local Area Connection: Connection-specific IP Address. . . . . Subnet Mask . . . . Default Gateway . . C:\> DNS . . . . . . Suffix . . . . . . . . . . . . . . . . : : 192.168.1.100 : 255.255.256.0 : 192.168.1.1

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Installing Image-Pro Plus with a multi-user (red) network key

Installing a License Client

STEP 1. 2. PROCEDURE Do a Typical installation of Image-Pro, according to the instructions earlier in this chapter. Select Install Network Support.

3.

Click Next to advance past the Welcome screen.

continued on next page

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Image-Pro Plus Start-Up Guide STEP 4. PROCEDURE Select a Client installation, and click Next.

5.

Select the application that will be using NetHASP licensing (Image-Pro should be selected by default), and click Next.

continued on next page

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Installing Image-Pro Plus with a multi-user (red) network key STEP 6. PROCEDURE Click Next to accept the default directory, or click Browse to change the directory in which the License Server will be installed.

7.

You will now be prompted for the IP Address of the License Server (copied down in Step 7 of the License Server installation).

Click Next to continue. 8. Click Finish to begin copying files.

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Image-Pro Plus Start-Up Guide

Upgrading Your Image-Pro Plus Software

If you are installing Image-Pro Plus as an upgrade from versions prior to IPP 4.0, you will be prompted to enter an Upgrade Authorization Code (UAC) before starting the program. The code is located on the back outside cover of the Start-Up Guide (this manual) and on the sleeve containing the Image-Pro Plus version 6.0 CD. Follow the steps below: Note that Image-Pro Plus 6.0 will not install on versions of Windows older than Windows 2000. STEP 1. PROCEDURE If you know your Upgrade Authorization Code (UAC), select the radio button for an upgrade.

Press Continue. continued on next page

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Installing Image-Pro Plus with a multi-user (red) network key STEP 2. PROCEDURE The next screen asks you to enter your UAC in the spaces provided:

Your serial number will appear automatically. Enter the 12-digit number that appears on the back cover of the Start-Up Guide or on the CD sleeve. The click the Upgrade button. 3. A message window asks you to verify your UAC:

Click Yes. Now you can use Image-Pro Plus version 6.0. continued on next page

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Image-Pro Plus Start-Up Guide STEP 4. PROCEDURE If you dont have an Upgrade Authorization Code, you can continue in Demo mode for 30 days. Click the radio button for Continue in Demo Mode.

Press Continue to work with Image-Pro Plus version 6.0. 5. To receive information about an Upgrade Authorization Code, click the radio button next to I would like to purchase an Upgrade Authorization Code.

Press Continue. continued on next page

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Installing Image-Pro Plus with a multi-user (red) network key STEP 6. PROCEDURE The next screen shows your serial number and copy protection key ID number.

Press Contact Information to see the Media Cybernetics Customer Support telephone number, fax number, and e-mail address. Contact us for your Upgrade Authorization Code (UAC).

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Image-Pro Plus Start-Up Guide

Installing Image Capture Support

To use a camera or frame-grabber with Image-Pro, you will need to install the appropriate capture drivers from the separate Image Capture CD-Rom. You must install the 32-bit version of the capture drivers to work with Image-Pro.

STEP 1.

PROCEDURE Insert the Image Capture Drivers CD in your CD-ROM drive.

Within a few seconds, the Setup program will start running automatically. 2. If the Setup program does not start automatically, click the Start button on your Windows taskbar, and use the Browse button to find the setup.exe file on your CD, and choose Run to start the Capture Drivers setup. Click Install Drivers to select the Media Cybernetics capture drivers. You will see the Install Drivers menu screen: Use your cursor to select the kind of drivers that you wish to install The Capture program will ask your for a destination location. You must choose the directory where you installed ImagePro.If you are adding capture drivers to a network installation, the drivers may be added to either the local directory or to the main network location. The Capture Drivers is the only component that Image-Pro uses. Checking the other components has no effect on your system.

3. 4. 5.

6.

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Installing Image Capture Support STEP 7. PROCEDURE The Progress Indicator shows that the Capture files are being loaded onto your computer. The program will give you a message when installation is completed, and any drivers that you installed will become available the next time that you start Image-Pro. Choosing Web Update allows you to download the most recently updated drivers from the Media Cybernetics web site:

8.

Follow the instructions on this web page to download the drivers.

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Image-Pro Plus Start-Up Guide

Installing IQbase Support

If you already have a copy of IQbase running on your computer, you can install IQbase support for Image-Pro Plus 6.0. This will enable you to manage your images using the advanced features of IQbase instead of the Image Database provided with Image-Pro Plus. STEP
1.

PROCEDURE Choose Install IQbase Support.

2.

The system will ask you to select a target for the database upgrade. Select Image-Pro Plus 6.0. Then click Next. Follow the prompt instructions on the screen to continue installing support for IQbase. Click Finish to complete the installation.

3. 4.

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Starting the Image-Pro Plus Program

Starting the Image-Pro Plus Program

The Image-Pro Plus setup program creates a program folder called Image-Pro Plus 6.0 under the Programs group in the Windows 2000/XP Start menu. A typical installation includes the following items: Image-Pro Plus Image-Pro Plus Reporter.

Image-Pro Plus starts the program. Select this item to open the Image-Pro Plus application window. Image-Pro Plus Reporter starts the Report Generator program, which can be used to create formatted reports, incorporating results from Image-Pro Plus. Note: Under, Windows 2000 or Windows XP Professional you should remove ImagePro Plus using the Add/Remove Programs option from the Control Panel group under Settings on the Start menu.

Technical Support On Line

Please refer to the Technical Support page, (accessible from the Media Cybernetics home page on the World Wide Web at http://support.mediacy.com )where valuable technical information is available to you. This information includes:

Answers to frequently asked questions A collection of technical notes, including How To... tips, sample macros, and more. Technical information on built-in and third-party video acquisition hardware and software. A Technical Support E-mail form

You may obtain additional useful information via e-mail by joining the Image-Pro Users Group. This service facilitates information exchange among Image-Pro users. You will be able to learn how others use Image-Pro, and possibly find solutions to your needs. To join the Image-Pro Plus Users Group, visit our web page at http://support.mediacy.com.

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Image-Pro Plus Start-Up Guide

Contacting Technical Support

Before you contact Media Cybernetics Technical Support for further assistance, please be prepared to provide the following information:

Your registration information: the product serial number (*), your name, phone number, organization name, mailing address, and e-mail address. The software version number (*) and system information. A description of the problem, and any relevant information regarding prior technical support assistance.

(*) These numbers are displayed on the About screen (open from the Help menu) You may contact Media Cybernetics Technical Support in either one of the following ways:

To speak with a technical support representative, please call 301-495-3305, and select the option for Technical Support. The Technical Support line operates on regular business days, between 9:00 a.m. and 5:00 p.m., EST. To use e-mail, you may fill out the e-mail form available from the Technical Support page on the World Wide Web (see above); or e-mail your message directly to: [email protected]. Please remember to include your IPP serial number in your message. To send a fax, address your fax to Image-Pro Plus Technical Support, and send it to 301-495-5964. Please remember to include your IPP serial number in your message.

Note: Technical support is available only to registered users, during the basic or extended warranty period. Please remember to return your registration card.

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Solutions Zone

Solutions Zone
The Solutions Zone is an information and resources web page which can be accessed freely by Media Cybernetics customers. This page contains downloadable solutions that range from device drivers, plug-ins, macros, hardware, and services. Solutions are created by Media Cyberneticsemployees, dealers, and users. Solutions created by Media Cybernetics employees can be downloaded at no cost. Some solutions created by dealers or other users may have a cost listed in their descriptions. Media Cybernetics does not set any of these prices. The Solutions Zone is searchable by keyword, product (i.e. Image-Pro Plus, Scope-Pro, etc.), by solution type (i.e. device driver, plug-in, etc.), or application (biological sciences, industrial, or forensics). Click this link in the online system to visit the Media Cybernetics' Solutions Zone web page: http://www.solutions-zone.com/

Device Drivers
Device drivers are software plug-ins that help a user control a specific piece of hardware that is connected to their computer. From time to time, Media Cybernetics will post new and updated drivers on the Solutions Zone. If your device is not working correctly, please check first to see if there is an updated driver available before contacting Technical Support. To search for a device driver, type the name of the device for which you are seeking a driver (e.g. PVCAM). After the search is complete, click on a driver so that you can read the description and updated information (if applicable). The next screen will allow you to download the driver directly to your computer.

Plug-ins
Plug-ins are software programs that work within Image-Pro to provide a new function or feature. Many times, users have asked how rather complicated functions can be accomplished using Image-Pro Plus. In some cases, our engineers and other Image-Pro Plus users have taken it upon themselves to write new plug-ins so that these complex or novel tasks can be accomplished. We feel that if one user asks a question, there are probably many more that will have to have the same question answered. That is why these plug-ins are placed on the Solutions Zone. A plug-in search is best accomplished by selecting Application Programs/Plug-Ins from the Solutions Type drop-down menu on the front page of the Solutions Zone. You should then limit your search with a keywordbased on what you would want the plug-in to do (e.g. trace object). Once you have some options to choose from, click on the plug-in title for Page 1-31

Image-Pro Plus Start-Up Guide more information. The page with information on the plug-in will have a link toallow you to download the plug-in directly to your computer.

Macros
One of the major advantages of using Image-Pro Plus is the ability to record and use macros. Macros are programming scripts that call the programming language of Image-Pro Plus to automatically execute tasks. Our engineers and other users have written macros to solve problems and add to the capabilities of Image-Pro Plus. A macro search is accomplished in a similar manner as a plug-in search. Select Macros from the drop-down menu on the front page of the Solutions Zone. You should then limit your search with a keyword based on what you would want the macro to do (e.g. sort objects). Once you have some options to choose from, click on the macro title for more information. The page with information on the macro will have a link to allow you to download the plug-in directly to your computer.

Hardware and Services

Dealers and other users have specialized hardware and macro/plug-in authoring services that are listed on the Solutions Zone. Hardware devices can best be found by selecting Other Hardware Driver/Component from the Solutions Type drop-down menu on the front page of the Solutions Zone. Macro/plug-in authoring services can best be found by selecting Consultants/Programmer Services from the Solutions Type drop-down menu on the front page of the Solutions Zone. After selecting a specific item for more information, a description of the hardware or service will appear as well as contact information. Do you have a solution that you think others could benefit from? Submit it to the Solutions Zone today! Just click http://www.solutions-zone.com/addedit.htm and fill out the form on the webpage.

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What is Image Processing?

Imaging Discussion
What is Image Processing?
An image is a visual representation of an object or group of objects. Image processing manipulates information within an image to make it more useful. Digital image processing is a specific type of image processing performed with a computer. You are probably most familiar with photographic images; however, photographic images do not lend themselves to computer analysis because computers work with numerical rather than pictorial information. In order to process an image with a computer, the image must be converted into numeric form. This process is known as image digitization.

Image Digitization
The digitization process divides an image into a horizontal grid, or array, of very small regions called picture elements, or pixels. In the computer the image is represented by this digital grid, or bitmap. Each pixel in the bitmap is identified by its position in the grid, as referenced by its row (x) and column (y) number. By convention, pixels are referenced from the upper-left position of the bitmap, which is considered position 0,0 (row 0, column 0).

pixel bitmap Note - for illustrative purposes, the pixels in the drawing above are shown much larger than their actual size. A pixel usually represents a very small region within an image, often 1/300th of an inch square, or less.

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Image-Pro Plus Start-Up Guide When a source image, such as a photograph, is digitized, it is examined in grid fashion. That is, each pixel in the image is individually sampled, and its brightness is measured and quantified. This measurement results in a value for the pixel, usually an integer, which represents the brightness or darkness of the image at that point. This value is stored in the corresponding pixel of the computer's image bitmap. When the image is digitized, the width and height of the array are chosen and fixed. Together, the bitmap's pixel width and height are known as its spatial resolution.

Pixel Depth
Depending upon the capability of the measuring hardware and the complexity of the image, anywhere from 1 to 32 bits might be used to store each pixel value. Pixel values for line art images, which contain only black and white information, can be easily represented by a single bit: 0=black, 1=white. However, a photographic-like image contains much more information...it takes 24 bits to represent all the possible colors that might occur in a true color image. Given 24 bits, over 16 million colors, far more than the human eye can differentiate, can be represented.

an example of a simple line drawing that can easily be represented with a single bit per pixel The number of bits used to represent the pixel values in an image is referred to as its pixel depth, or bits-per-pixel (BPP). The number of bits per pixel used to represent each pixel value determines the images class.

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Image Class

Image Class
While the bit depth (BPP) tells us how many unique colors an image can possess, it does not tell us what colors are actually contained within the image. Color interpretation is determined by bit depth and one of several conventions, which Image-Pro refers to as image class. The following classes are supported by Image-Pro: Gray Scale 8 Gray Scale 12 Gray Scale 16 Floating Point (Gray Scale 32) RGB 24 (True Color) RGB 36 RGB 48 Palette Each class is discussed below. Note - Although Image-Pro supports all the classes listed above, not every operation can be used with every class. For example, FFT( Fast Fourier Transform) operations cannot be performed on a True Color image. The Command Reference in Section 2 of this manual documents such class considerations for each Image-Pro command. Commands that are not supported for a given image class are ghosted (dim) in the menu.

Bilevel
A Bilevel image stores image data with 1 bit-per-pixel (1 BPP). Each pixel is interpreted as either completely black (0) or completely white (1). Bilevel images are commonly referred to as either Black and White images, Line Art or Halftones. Image-Pro Plus no longer actively supports the Bilevel image class. Bilevel images imported into Image-Pro Plus will be converted to Gray Scale 8 images.

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Image-Pro Plus Start-Up Guide

Gray Scale
Gray Scale pixel values represent a level of grayness or brightness, ranging from completely black to completely white. This class is sometimes referred to as monochrome. In an 8-bit Gray Scale image, a pixel with a value of 0 is completely black, and a pixel with a value of 255 is completely white. A value of 127 represents a gray color exactly halfway between black and white (medium-gray), and a pixel value of 64 has a gray color halfway between medium-gray and black.

256 shades of gray Although Gray Scale images with bit depths of 2, 4, 6, 12, 16 and 32 exist, 8 BPP Gray Scale images are the most common. This is for two reasons: 1) its 1-byte-per-pixel size makes it easy to manipulate with a computer, and 2) it can faithfully represent any gray scale image because it provides 256 distinct levels of gray (the human eye can distinguish less than 200 gray levels).

Image-Pro supports the following gray scale bit depths: NAME Gray Scale BPP 8 DESCRIPTION The most common gray scale format in use. Used by most popular monochrome image formats (e.g., TIFF, PCX). Intensity values are represented with 8-bit integers. Provides 256 (0 - 255) levels of gray. A gray scale format generated by many specialized imaging systems. Intensity values are represented with 12-bit integers. Provides 4096 (0 - 4095) levels of gray.

Gray Scale 12

12

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Image Class NAME Gray Scale 16 BPP 16 DESCRIPTION A gray scale format generated by many specialized imaging systems. Intensity values are represented with 16-bit integers. Provides 65,536 (0 - 65,535) levels of gray. A gray scale format that is not native to any device or image format, but is useful for certain arithmetic and filtering operations. Intensity values are represented with 32-bit floating point values. Provides a virtually unlimited number of gray levels.

Floating Point

32

The Gray Scale 12, Gray Scale 16, and Floating Point classes are used for specialized operations and applications. Gray Scale 12 and Gray Scale 16 images are often generated by specialized imaging equipment such as infrared cameras and medical imaging devices. A Floating Point image is not native to any application or device. Unlike all other image classes, a Floating Point image does not have a fixed intensity range. The lowest value in the image is displayed as black, and the highest value in the image as white. A Floating Point image is created within Image-Pro using its Convert To command. The advantage of a Floating Point image is that data are not lost to truncation or clipping when arithmetic and filtering operations are performed upon it (clipping refers to the loss of data that occurs when a pixel value falls outside a fixed intensity scale. An example is a negative value that is generated during a filtering or subtractive operation in a fixed-point image, these negative values get clipped to 0). This characteristic makes it especially valuable for image restoration applications. Although Image-Pro supports Gray Scale 12, Gray Scale 16, and Floating Point images for analysis purposes, most popular file formats do not support these image classes. The TIFF file format does support these image classes, but not all programs that support TIFF files support these variants. These image types can be saved in the Image-Pro Workspace (IPW) or flat file formats.

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Image-Pro Plus Start-Up Guide

RGB
The RGB image class uses the most straightforward way of representing color images. RGB stands for Red, Green, Blue, the three primary colors of light. From the development of color photography and color television we have learned that any color can be represented as a mixture of varying levels of pure red, green, and blue light. RGB 24 is referred to as True Color. In a True Color bitmap, each pixel contains a 24-bit value, called an RGB triplet or chunk. This RGB-triple is made up of three separate 8-bit samples. Each sample represents the level of brightness of its respective color channel: Red, Green, or Blue. These brightness values represent levels within a 256-level scale, just as they do in a Gray Scale image. The first sample is a level of Red, ranging from 0 (black) to 255 (brightest red). The second sample is interpreted as a level of green, and the third sample is the level of blue. Equal levels of Red, Green, and Blue always generate a level of gray. Due to the increasing popularity of digital cameras, Image-Pro Plus now supports 36- and 48-bit color images. The storage for the classes is similar to the method used to store the 24-bit images: triplets of 16-bit words (16-bit red, followed by 16-bit green, and 16-bit blue, followed by the triplet for the next pixel). The two classes are different only in the maximum range for intensity-related values (4095 for RGB-36 versus 65535 for RGB-48). Planar forms of the two new classes will not be supported. Although Image-Pro supports RGB-36, and RGB-48 images for analysis purposes, most popular file formats do not support these image classes. The TIFF file format does support these image classes, but not all programs that support TIFF files support these variants. These image types can be saved in the Image-Pro Workspace (IPW) or flat file formats.

Palette
The Palette image class uses 8 bits-per-pixel to store color information. Palette format is a convenient and efficient way to store images that have fewer than 256 colors. It requires far less storage space than encoding an image in True Color (RGB-triple) form. Unlike all other image classes, the pixel value in a Palette image does not represent a brightness value. Instead, the value within the pixel is an index (pointer) to an entry in the images palette. The palette, which is stored with the image, is a 256-entry table of RGB values. Each entry in the table contains the 24-bit RGB value for that particular index. In a Palette image, you could change the color of all pixels with a certain value, say 10, by simply redefining the contents of entry 10 in the palette. Page 1-38

Color Models It is important to understand that the values in a Palette image have no significance in terms of intensity; i.e., you cannot assume that a pixel value of 0 means black, a value of 255 means white, or that low values are dark and high values are bright. Because of this, Palette images should not be used for intensity-driven calculations, such as filtering, or intensity analysis operations such as histogram and line analysis. If you want to perform such operations on a Palette image, you should first convert it to a continuous-tone image class such as True Color or Gray Scale.

Color Models
Although Image-Pro internally maintains a color image in RGB-chunky form, it can express a color value in terms of other color models. A color model is simply a standard way to represent color in mathematic terms. Most color models use a 3Dcoordinate system. Each point within the system's subspace represents a unique color. The RGB color model, for example, can be visualized as a cube where Red is the X-axis, Blue is the Y-axis and Green is the Z-axis. Each one of the 16.8 million colors is described by a unique point within this cube. There are many color models in use today: the RGB (Red, Green, Blue), HSI (Hue, Saturation, Intensity) and HSV (Hue, Saturation, Value) models are most frequently used in digital image processing. The CMY (Cyan, Magenta, Yellow) model is a standard used to describe color in the color printing industry, and YIQ (Y-axis, Inphase, Quadrature) is used in broadcast television. Image-Pro lets you analyze and modify your image using any of these models. The RGB (Red, Green, Blue) color model is an especially important one in digital image processing because it is used by most digital imaging devices (e.g., monitors and color cameras). In the RGB model, a color is expressed in terms that define the amounts of Red, Green, and Blue light it contains. In a 24-bit, color image, pure red would be represented as 255/000/000, where 255 represents the highest level of red light possible, untainted by any green (000) or blue (000) light. Various combinations of the Red, Green and Blue values allow us to define 224 (over 16 million) colors. The HSI (Hue, Saturation, Intensity) color model describes a color in terms of how it is perceived by the human eye. Hue is what an artist refers to as pigment; it is what we think of as color -- yellow, orange, cyan and magenta are examples of different hues. An artist usually starts with a highly saturated (i.e., pure), and intense (i.e., bright) pigment, and then adds some white to reduce its saturation and some black to reduce its intensity. Red and Pink are two different saturations of the same hue, Red.

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Image-Pro Plus Start-Up Guide Green: 120 Cyan: 180 Yellow: 60 Red: 0 In the HSI model, a hue is specified by its position on a color hexagon as measured by its distance, in degrees, from the red axis (e.g., a Hue value of 120 would indicate Green, which is 120o from Red). A color's Saturation and Intensity components are identified using additional coordinates in 3D space (see the HSI glossary entry for details).

Blue: 240 Magenta: 300 Hue Hexagon

The HSI model is useful when processing images to compare two colors, or for changing a color from one to another. For example, changing a value from Cyan to Magenta is more easily accomplished in an HSI model; only the H value needs to be changed (from 180 to 300). Making the same change in an RGB view is less intuitive; since you must know the correct amounts of Red, Green and Blue needed to create Magenta. The HSI model is also a more useful model for evaluating or measuring an object's color characteristics, such as the redness of a berry or the yellowness of an autumn leaf. The HSV model is very similar to the HSI model. The main difference between the two is the calculation used to produce the brightness value. In the HSI model, a pixels brightness (I) is derived from the mean of its three (R, G and B) color values. In the HSV model, a pixels brightness (V) is determined from the mean of the minimum and maximum value of its three color values. The YIQ model is used in commercial color TV broadcasting. It was designed to take advantage of the human visual systems greater sensitivity to changes in luminance rather than changes in hue or saturation. YIQ standards call for more bits to be used to represent the Y channel (luminance) and few representing the I and Q channels (hue and saturation).1 Within Image-Pro, a color model can be specified when performing channel analysis operations with the Histogram and Line Profile Analysis commands. You can also specify a color model when editing or adding a color in the Palette window, or when performing a channel extraction process using the Extract Channel tools.

Digital Image Processing, Gonzales & Woods, 1992, Addison-Wesley Publishing Co., pg. 228. Page 1-40

Enhancing Your Image

Enhancing Your Image

More often than not, an image will require some form of enhancement to improve its appearance for aesthetic reasons, or to improve your ability to extract data from it. Enhancement techniques range from simple operations such as brightness and contrast adjustment, to the sophisticated and complex spatial and morphological filtering operations designed to tease out and refine visual information. There are three basic ways to enhance an image: modify the intensity index apply a spatial filter, or manipulate the image frequencies. Image-Pro Plus gives you a variety of tools in each category, allowing image enhancement for a variety of purposes and applications. Depending upon your application, you may use some techniques and not others, or you may find that you need to combine several techniques to achieve the results you need. The following section contains material to help you select the appropriate tool for the task at hand. It describes Image-Pro Plus's enhancement features, and gives you some insight into the uses for each.

Modifying the Intensity Index

One way to enhance an image is to change the way intensity values are interpreted. For example, if your image was very dark overall, you could boost all the values by a certain amount. You might boost all values by 20 points, or flatten a range of intensities to a single value (e.g., set all intensities from 75 to 150 to 127). Image-Pro Plus gives you many tools and controls to manipulate the intensities within your image. Although you will see the effect of an index modification immediately upon your image, pixel changes are not actually written to your image. They are written to an intermediate table called a Lookup Table (LUT). This is done so that intensity changes can be executed quickly, and so that they can easily be undone. When you are satisfied with the intensity changes you have made, you must make the changes permanent by either storing your image or explicitly writing the changes to the image bitmap using the Contrast Enhancement command. The following intensity manipulation tools are provided by Image-Pro Plus.

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Image-Pro Plus Start-Up Guide

Brightness
Brightness is a term used to describe the overall amount of light in an image. In Image-Pro Plus, brightness is modified using the Brightness ( ) slider control on the BCG Controls. This control affects the overall image. In a color image, the Brightness slider control adjusts luminance, which is the combined intensity of the RGB channels. Brightness for an individual color channel can be modified using the Brightness control in the Contrast Enhancement dialog box. When brightness is increased, you increase the value of every pixel in the image, moving each pixel closer to 255, or white. When brightness is decreased, you reduce the value in each pixel, moving it closer to 0, or black.

Contrast
Contrast is a term used to denote the degree of difference between the brightest and darkest components in an image. An image has poor contrast if it contains only harsh black and white transitions, or contains pixel values within a narrow range (an image whose values ranged from 100 to 140 would have poor contrast). An image has good contrast if it is composed of a wide range of brightness values from black to white. The amount of the intensity scale used by an image is called its dynamic range. An image with good contrast will have good dynamic range. In Image-Pro Plus, contrast is modified using the Contrast ( ) slider control on the BCG Controls. This control affects the overall image. In a color image, the Contrast slider control operates upon the luminance channel, which is the combined intensity of the RGB channels. Contrast for an individual color channel can be modified using the Contrast control in the Contrast Enhancement dialog box. During a contrast operation, each pixel value is scaled by a contrast value, which serves to redistribute the intensities over a wider or narrower range. Increasing the contrast spreads the pixel values across a wider range, while decreasing contrast squeezes the values into a narrower range.

Gamma Correction
Gamma correction is a specialized form of contrast enhancement that is designed to enhance contrast in the very dark or very light areas of an image. It does this by Page 1-42

Modifying the Intensity Index changing the midtone values, particularly those at the low end, without affecting the highlight (255) and shadow (0) points. Gamma correction can be used to improve the appearance of an image, or to compensate for differences in the way different input and output devices respond to an image. In Image-Pro Plus, Gamma adjustments are made using the Gamma ( ) slider control on the BCG Controls. In a color image, the Gamma slider control operates upon the luminance channel, which is the combined intensity of the RGB channels. The Gamma value for an individual color channel can be modified using the Gamma control in the Contrast Enhancement dialog box. The Gamma control modifies an image by applying standard, nonlinear gamma curves to the intensity scale. A gamma value of 1 is equivalent to the identity curve, which has no effect on the image. An increase in the gamma value (setting it to a value greater than 1) will generally lighten an image and increase the contrast in its darker areas. A decrease in the gamma value (setting it to a value less than 1) will generally darken the image and emphasize contrast in the lighter areas. The figure below illustrates the effect of the gamma curves on pixel values 0 through 255.

Gamma Correction Curve

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.

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Image-Pro Plus Start-Up Guide Thresholding is performed using the Threshold command on the Process menu. When this 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 thresholded.

Histograms
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 (discussed in more detail in the Intensity Analysis section), or can be manipulated for image enhancement purposes. A histogram is created using the Histogram command on the Measure menu. As you can see in the example below, the X-axis in a histogram represents the intensity scale (0 to 255 in this example), and the Y-axis measures the number of pixels in the image possessing that value. When you are working with Gray Scale 8 images, the x-axis represents gray values 0 through 255. For other Gray Scale image types, the x-axis will represent the intensity range ( 0 to 4095 for Gray Scale 12). When working with True Color images, you can choose to measure either the images combined luminosity or its separate color channels (e.g., Red or Green or Blue, Hue or Saturation or Intensity...).

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Modifying the Intensity Index A histogram will show you what kind of brightness/contrast deficiencies exist in an image. Images with low contrast will have histograms that are clustered around a very narrow portion of the color range. The position of the cluster will indicate whether the image is too dark, too light, or simply too gray, as shown in the three examples below.

Poor contrast histograms Brightness, contrast, and gamma adjustments modify the shape of a histogram. The series of images on the pages that follow illustrate the effect of a Brightness, Contrast, or Gamma adjustment upon the histogram. This first set of images demonstrates the effect of a brightness change on the histogram.

Original image

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Image-Pro Plus Start-Up Guide

Image with decreased brightness

Image with increased brightness As you can see from the series above, a brightness change affects the position of the histogram on the scale, sliding the entire histogram towards black when it is decreased, and towards white when it is increased.

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Modifying the Intensity Index The following series illustrates the effect of a contrast operation upon an image and its histogram.

Original image

Image with decreased contrast

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Image-Pro Plus Start-Up Guide

Image with increased contrast In the image series above, the contrast has been decreased and increased. As you can see, contrast operations affect the width of a histogram; compressing it when it is decreased, and stretching it when it is increased. The following series illustrates the effect of a gamma operation upon an image and its histogram.

Original image

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Modifying the Intensity Index

Image with decreased gamma

Image with increased gamma In the image series above, the gamma has been decreased and increased. As you can see, a decrease in gamma brings out features in the lighter area of the image, by stretching the histogram in the upper region. An increase in gamma stretches the lower values, providing increased contrast in the darker areas.

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Image-Pro Plus Start-Up Guide

Accumulated Histograms
A cumulative, or accumulated, histogram can also be used to assess the brightness and contrast characteristics of an image. An accumulated histogram measures the number of pixels that have a given pixel value or a lesser value. The result is an integral of the regular histogram distribution function. The accumulated histogram indicates the evenness of the intensity distribution. An even distribution will produce a histogram that resembles a linear progression, like the example shown below.

Image with good contrast and dynamic range As you can see in the very dark and very light examples below, the accumulated histogram is skewed to the dark and light ends of the scale.

Bright image with poor contrast Page 1-50

Modifying the Intensity Index

Dark image with poor contrast Accumulated histograms are created using the Accumulated command on the Report menu in the Histogram command window.

Histogram Equalization
Image-Pro Plus provides several ways to automatically reshape a histogram. Using the Contrast Enhancement command on the Enhance menu, you can optimize the brightness and contrast characteristics by directing Image-Pro Plus to stretch the histogram to achieve the best possible contrast distribution for the given image. You can also use one of the specialized distribution functions provided by the Equalize command on the Enhance menu. These histogram redistribution functions enhance contrast and dynamic range in a nonlinear manner. When you use the Equalize command, Image-Pro Plus analyzes your images accumulated histogram and redistributes it to fit the shape you specify: Linear: Distributes the histogram equally across the intensity scale. This function produces a high contrast image with the highest possible dynamic range. Bell: Distributes the histogram evenly around the center of the intensity scale. This function produces a high contrast image with less dynamic range than the Linear distribution. Logarithmic: Concentrates the histogram at the low end of the scale. This function produces a high contrast image with little dynamic range. It will tend to darken the image overall. Useful for stretching the contrast in a very light image. Page 1-51

Image-Pro Plus Start-Up Guide Exponential: Concentrates the histogram at the high end of the scale. This function produces a high contrast image with little dynamic range. It will tend to lighten the image overall. Useful for stretching the contrast in a very dark image. Best Fit: Use the Best Fit command to instruct Image-Pro Plus to optimize the values for your particular image. The results are achieved by stretching the histogram to achieve the best possible contrast distribution of pixel values in your image in this way: Best fit assigns the bottom 3% of the values to the shadow point, (0) and the top 3% of the values to the highlight point (255). The rest of the values are distributed evenly across the scale).

Spatial Filtering
Filtering operations reduce or increase the rate of change that occurs in the intensity transitions within an image. Areas in which there are sudden or rapid changes in intensity appear as hard edges in an image. Areas where there are gradual changes produce soft edges. Filtering acts to detect and modify the rate of change at these edges. It can increase the intensity differences in a soft edge to make it appear sharper, or reduce the intensity differences in a hard edge to smooth and soften it. Filtering operations produce their effect by modifying a pixels value based upon the values of the pixels that surround it. For example, blurring is accomplished by averaging all of the pixel values in a specified region, and replacing the center pixel with the averaged value. This produces reduced variation among neighboring pixels, which visually softens the image. A sharp black/white edge would be softened with intervening levels of gray. Filtering techniques are divided into two categories: convolution filters (linear filters) and nonconvolution (nonlinear) filters. Both techniques accomplish their results by examining and processing an image in small regions, called pixel neighborhoods. A neighborhood is a square region of image pixels, typically 3x3, 5x5, or 7x7 in size. In Image-Pro Plus, spatial filtering is performed using the Filters command on the Process menu.

Convolution/Enhancement Filters
Enhancement or convolution filters process image neighborhoods by multiplying the values within a neighborhood by a matrix of filtering coefficients (integer values). This matrix is called a kernel. It is the same size as the neighborhood that it is being applied to. The results of this multiplication are summed and divided by the Page 1-52

Spatial Filtering sum of the filter kernel. The result replaces the center pixel in the image neighborhood. This process is further illustrated and described on the following page.

The Convolution Filtering Process

THE CONVOLUTION FILTERING PROCESS STAGE DESCRIPTION 1. 2. 3. Each pixel in the image neighborhood is multiplied by the contents of the corresponding element in the filtering kernel. The results from the multiplication are summed and divided by the sum of the kernel. The result is scaled and boosted, and used to replace the center pixel in the image neighborhood.

Note - The convolution process always uses a neighborhood's original (unfiltered) pixel values as input. When, in the example above, the kernel's focus is moved to pixel 6, the filtering process will use pixel 5's original value, not the one it was just assigned by the convolution. The following Enhancement filters are provided by Image-Pro Plus: Lo-Pass: This filter blurs an image by modifying a pixel value to be more like its neighbors. This eliminates harsh edges by reducing the intensity differences between adjacent pixels. The Lo-Pass filter can be used to blur an image for aesthetic reasons, to eliminate detail in preparation for object segmentation, or to eliminate random image noise (see also Median filter in the Nonconvolution Filters section). Page 1-53

Image-Pro Plus Start-Up Guide

Use of Low-Pass filter to eliminate contouring in a 32 level (5 BPP) Gray Scale image Hi-Pass: This filter accentuates intensity changes in an image by modifying a pixel's value to exaggerate its intensity difference from its neighbors. This filter produces an image with harsh intensity transitions, and generally results in an image with only edges of high contrast visible. Fine detail with low contrast is usually lost to the background. This filter can be used when you need to pull out just the elements having high contrast to the image background. Sharpen: This filter accentuates all edges within an image by significantly enhancing all intensity transitions in the image. This is accomplished using a technique called unsharp masking, which essentially sharpens an image by subtracting its low-pass results from the original image (although this is the result, it is accomplished using the convolution process, not by actually subtracting a blurred image from the original). Sharpening is used to bring out fine detail in an image, or to re-focus an image that has been blurred.

Use of Sharpen Filter to accentuate image elements and enhance fine detail Gauss: Use this filter to soften an image by eliminating high-frequency information using a Gauss function. This has the effect of blurring sharp edges. Page 1-54

Spatial Filtering The operation of the Gauss filter is similar to the LoPass filter, but it degrades the image less than the LoPass filter. Higauss: Use this filter when you want to enhance fine details. Its operation is similar to the unsharp masking technique (see the Sharpen filter), but it introduces less noise in the process. It uses a Gaussian curve type of kernel. Available in 7x7 and 9x9 kernel sizes. Local Equalize: This filter is used to enhance pixel contrast based on the histogram of the local neighborhood. Flatten: Use the Flatten filter to even out background variations. This is often done to prepare an image for a count/size operation if its objects are difficult to isolate because the background contains pixels of the same intensity as the objects of interest. Flatten reduces the intensity variations in the background pixels. Median: This filter smoothes an image by modifying pixels that vary significantly from their surroundings. This is accomplished by replacing the center pixel in a neighborhood with the median value of the neighborhood. Although median filtering will soften an image, it generally preserves its edges. This filter is particularly effective at removing random, high-impulse noise from an image (e.g., spots or points that vary significantly from the background), as shown in the example below.

Use of Median filter to eliminate impulse noise Rank: Select this filter if you want to remove impulse noise from an image. The Rank filter replaces the center pixel with a ranked pixel value from the kernel when the gray value difference is larger than the threshold value. Convolution filters can be applied in kernels of 3 x 3, 5 x 5, or 7 x 7. Generally, the smaller the kernel, the more subtle the result. Appendix B, File Format Specifications, identifies the coefficients contained in each of the kernels used by the convolution filters described above. Page 1-55

Image-Pro Plus Start-Up Guide

Edge Filters
Roberts: This filter extracts and enhances fine edges in an image by expressing the differences between neighboring pixels (cross pairs) as an intensity value. In neighborhoods where there is no difference among values in the neighborhood the pixels intensity is set to 0 (black); where there is the greatest possible difference, the pixel is set to 255 (white). Intermediate levels of gray reflect varying amounts of difference. The result is an image in which edges and contours are highlighted against a dark background. Unlike most other filters, which use an odd-sized square neighborhood, the Roberts filter operates upon a 2 x 2 neighborhood. Since this neighborhood has no center, the pixel in the upper left-corner is the one replaced with a new value. Its filtered value is calculated using the following formula:

[(A - D)2 + (C - B)2]

where the neighborhood is arranged as:

A C

B D

The Roberts filter enhances all edges within an image, even those introduced by noise. Note - if your image appears black following a Roberts filter, use your brightness, contrast and gamma controls to lighten the image and bring out the edge detail. Sobel: This filter extracts and enhances edges and contours in an image by expressing intensity differences (gradients) between neighboring pixels as an intensity value. This is done by combining the difference between the top and bottom rows in a neighborhood, with the difference between the left and right columns, using the following formula:

(X2 + Y2)
where: X = (C + 2F + I) - (A + 2D +G) Y = (A + 2B + C) - (G + 2H +I)

A
and the neighborhood is arranged as:

B E

C F I

G H
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Spatial Filtering

In neighborhoods where there is no difference among values in the neighborhood the pixel's intensity is set to 0 (black); where there is the greatest possible difference, the pixel is set to 255 (white). The results are similar to the Roberts filter: highlighted edges against a dark background, but the Sobel filter is less sensitive to image noise. This generally results in an image with smoother, more pronounced outlines of only the principal edges. Laplacian: The Laplacian filter is an edge filter that accentuates intensity changes in an image by modifying a pixel's value to exaggerate its intensity difference from its neighbors. Its results are very similar to those of Hi-Pass. It produces an image with harsh intensity transitions, and results in an image with only edges of high contrast visible. Horizontal Edge: This edge filter accentuates the horizontal edges in an image by highlighting pixels with significant intensity differences from those above and below it. It produces an image with just its horizontal edges visible against a flat background. Horizontal Edge filtering is used when horizontal features need to be extracted from an image. Vertical Edge: This edge filter accentuates the vertical edges in an image by highlighting pixels with significant intensity differences from those to the left and right of it. It produces an image with just its vertical edges visible against a flat background. Vertical Edge filtering is used when vertical features need to be extracted from an image. Phase: This filter produces an image that expresses the direction of intensity change (the gradient) in a neighborhood as an intensity value (this filter is a complement of the Sobel filter). The phase filter gives a 3-dimensional relief look to the image -- areas that have no intensity differences are flat, and those with any variation are coded to indicate whether they are brighter or darker than those above it. Generally, the lightest intensities depict vertical transitions to brighter intensities.

. Page 1-57

Image-Pro Plus Start-Up Guide Use of the Phase filter to locate edges and indicate direction of intensity changes. Note how minor imperfections also became visible in the filtered image. Variance: Select this filter if you want to detect and emphasize edges and textures. The Variance filter replaces the standard deviation for its neighborhood.

Nonconvolution/Morphological Filters
Nonconvolution filters also work with pixel neighborhoods; however, unlike convolution filters, they do not multiply the neighborhood values by a kernel of filtering coefficients. Instead, a nonconvolution filter works only with the data in the neighborhood itself, and uses either a statistical method or a mathematic formula to modify the pixel upon which it is focused. The following nonconvolution filters are provided by Image-Pro Plus: Erode: The Erosion filter is a morphological filter that changes the shape of objects in an image by eroding (reducing) the boundaries of bright objects, and enlarging the boundaries of dark ones. It is often used to reduce, or eliminate, small bright objects.

Use of Erosion filter to reduce bright object size Dilate: The Dilation filter is a morphological filter that changes the shape of objects in an image by dilating (enlarging) the boundaries of bright objects, and reducing the boundaries of dark ones. The dilation filter can be used to increase the size of small bright objects. Open: The Open filter is a morphological filter that performs an erosion, then a dilation. In images containing bright objects on a dark background, the opening filter smoothes object contours, breaks (opens) narrow connections, eliminates minor protrusions and removes small dark spots. In images with dark objects on a bright background, the opening filter fills narrow gaps between objects. Page 1-58

Spatial Filtering

Use of 5 x 5 Octagonal Opening filter to eliminate connections between bright objects Close: The Close filter is a morphological filter that performs a dilation followed by an erosion. In images containing dark objects on a bright background, the opening filter smoothes object contours, breaks narrow connections, eliminates minor protrusions and removes small bright spots. In images with bright objects on a dark background, the closing filter fills narrow gaps between objects.

Use of 11 x 11 Octagonal Closing filter to fuse breaks between bright objects Note - The morphological filters: Erosion, Dilation, Open and Close, are named for their effect on bright objects on a dark field. For example, the erosion filter erodes bright objects, the closing filter closes gaps between bright objects, and so forth. To obtain a morphological effect upon dark objects on a bright field, use the opposite morphological filter -- e.g., use the dilation filter to do an erosion, use the opening filter to close gaps. etc. Tophat: Use this filter to detect and emphasize points, or grains, that are brighter than the background. Available in 3 kernel sizes; click the radio button indicating the kernel size that most closely matches the size of the grains you want to detect. Well: Use this filter to detect and emphasize points, or grains, that are darker than the background. Available in 3 kernel sizes; click the radio button indicating the kernel size that most closely matches the size of the grains you want to detect. Page 1-59

Image-Pro Plus Start-Up Guide Watershed: Select this filter to separate objects that are touching. The Watershed filter erodes objects until they disappear, then dilates them again, but will not allow them to touch. The Watershed filter will not operate upon True Color images. If you want to separate objects in a True Color image, you must first convert it to Gray Scale. Thinning: This filter reduces an image to its skeleton. It operates on a binary basis; pixels are part of an object if their intensity is greater than 127, otherwise, they belong to the background. To identify what objects the thinning filter will operate upon, it is best to threshold the image before applying the thinning filter. Pruning: Select this filter to eliminate projecting arms from an object. Reduce: Select this filter to reduce the objects in an image to a single point or group of points. Branch/Endpoints: Use this filter to identify morphological branch and endpoints in an image.

Special Filters
Sculpt: Use this filter to apply a sculpted effect to the image Background: Select this filter to extract the background from an image. The Background filter works by filtering out objects using a very large filtering kernel. The background image, with objects removed, is placed into a new, untitled image window. Distance: The distance filter is used for showing the distances of pixels within blobs to the outer boundaries of those blobs. After applying the distance filter, the background will be black (i.e., pixels with value 0). Only the area within the blobs will have non-zero values (will be white). The values of each pixel within the blob will be a count of the shortest distance from that pixel to the edge of the blob. Thus, all pixels along the blob's border will have a value of 1 (since they are one pixel away from the edge of the blob), pixels that are a distance of 2 from the border will have the value 2, and so on. This creates a distance map of the image. Phase: Select this filter if you want to enhance edges in a manner that also indicates the direction of the intensity change.

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Frequency Filtering

Frequency Filtering
Image interference that presents itself as a regular pattern across an image can be especially difficult to remove using a spatial approach. The best way to eliminate such periodic or coherent noise is by converting the image to a set of frequencies, and editing out the frequencies causing the problem. Converting an image to its frequency domain is called a Fourier Transform (named for its inventor, Joseph Fourier), and is performed in Image-Pro Plus using the FFT (Fast Fourier Transform) command on the Process menu. Using the Forward button within the FFT command window, you can express the images frequency domain as a symmetrically centered cloud of points, where brightness represents amplitude of the waveform, and the position represents the frequency of the waveform. In a normal image, the spectrum will appear as a roughly circular cloud that is brighter and denser near its center. Images containing a regular pattern will reflect pattern-like effects in their spectra. It is this characteristic that can be manipulated to remove coherent noise. Coherent noise usually manifests itself as bright points outside of the central cloud. Removing these points eliminates the frequency causing the noise.

Image with coherent noise with its Fourier spectrum

Image after removing frequency spikes Page 1-61

Image-Pro Plus Start-Up Guide

In Image-Pro Plus, frequency spikes are removed by defining an AOI around the spike, then selecting the Spike Cut option on the FFT command window. The frequencies within the AOI are either removed (set to zero), or attenuated depending upon the Transition value you have specified in the Inverse/Filter Options dialog box. Just as frequencies can be removed or reduced, they can also be boosted. You might want to do this to accentuate a periodic pattern in an image. Fourier transformations can also be used to perform image sharpening and blurring. In fact, the Lo-Pass, Hi-Pass and Sharpen convolution filters, and their Lo-Pass and Hi-Pass counterparts on the FFT command window, serve exactly the same purpose -- they both allow only the specified frequencies (low or high) to remain in an image. When you use these filtering options from the FFT command window, you define an AOI in the spectrum, within which, or outside of which, frequencies will be eliminated or attenuated according to the Transition value you have specified in the Inverse/Filter Options dialog box.

Intensity Analysis
Intensity analysis operations let you collect data from your image based upon the intensity values it contains. Image-Pro Plus provides the following intensity analysis tools on the Measure menu: Histogram Analysis Line Profile Analysis Bitmap Analysis. Histogram Analysis lets you create a histogram of an image or AOI. Line Profile Analysis lets you plot the intensity values along a given line. Bitmap Analysis allows you to display the values of individual pixels in a bitmapped image. Using the Save commands on the Histogram, Line Profile , and Bitmap Analysis: File menus, you can store the results of your analysis to a file for use with other applications. Although the analysis tools actually measure the intensity levels within your image, you can calibrate Image-Pro Plus to express these values in units relevant to your application. For example, by setting Image-Pro Plus's Standard Optical Density option on the Intensity Calibration dialog box, the intensity scale can be calibrated to standard optical density values used by most transmitted light experiments. You could also calibrate the intensity scale to indicate temperature, or protein content, for example. Page 1-62

Intensity Analysis Intensity calibration is performed using the Calibration command on the Measure menu.

Optical Density
Optical Density (OD) analysis is a common image processing application. Optical Density analysis determines the amount of matter in a material by measuring the amount of light it transmits (lets pass through it). Because OD analysis measures the amount of light passing through a material, OD measurements are meaningful only in the analysis of images that have been captured with the light source radiating from behind them. Some examples are: images captured from a microscopic slide (where light passes through the sample from underneath the slide) or gel samples captured in a light box (where light passes through the gel from the back of the box). Optical density measurement is not useful in the analysis of images captured under reflected light. The Standard Optical Density option in the Intensity Calibration dialog box, uses the following standard light transmission formula for calculating OD from intensity. This formula assumes an exponential decay of light inside the transmitting material:

OPTICAL DENSITY(x,y) = -log[(INTENSITY(x,y) - BLACK) / (INCIDENT BLACK)]

where: INTENSITY(x,y) is the intensity at pixel (x,y) BLACK is the intensity generated when no light goes through the material, INCIDENT is the intensity of the incident light. A material through which no light is transmitted has an infinite optical density, and one through which all light is transmitted, has zero optical density. As you will see by viewing the OD curve, the OD scale is inversely related to the intensity scale that is, on an optical density scale, dark pixels produce high values, and light pixels produce low values.

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Standard Optical Density Curve X-axis depicts intensity, Y-axis depicts density When working with optical density, it is important to establish the intensity values representing the black and incident levels in your image. By default, Image-Pro Plus assumes these values are 0 and 255, respectively; however, due to camera noise and other interference, these pure values are rarely appropriate for real-life images. The black and incident values should always be calibrated to your camera or image. For accuracy, this calibration should be performed for each experiment or image. To calibrate the black level to your camera you must acquire an image while your cameras lens is shut. Then, use the Black Level Image button in the Optical Density Calibration dialog box to select a pixel from this image (you should select a pixel located near the center of the image). The value of this pixel will indicate the black level for your camera. It represents the value that your camera registers when no light at all is transmitted to its lens. You must similarly set the incident level to your camera. To calculate incident light, you must acquire an image with nothing between the camera's lens and the light source. Then, use the Incident Light Image button to measure the intensity of a pixel near the center of the empty image. Hint - With the proper preparation, you can also obtain black and incident levels from within an image itself. If a solid material (such as a penny) can be affixed to your sample, your image will contain an area through which absolutely no light is passed. You can then select a pixel in this area to calibrate the black level. Similarly, you can set the incident level if your image contains an area where there is no absorbing material present.

Custom Calibration
If Image-Pro Plus's intensity or Optical Density scales do not fit your application, you can define a scale that meets your needs. This is done using the Free Form Page 1-64

Intensity Analysis option in the Intensity Calibration dialog box. Within this dialog box, you can define the name of your unit (e.g., degrees, luminance, disintegration/minute) and calibrate the scale. Custom calibration is accomplished by specifying reference points that define how your scale relates to the intensity scale associated with the image. Reference points are specified in the Free Form Intensity Calibration dialog box. If your scale is linear, only two points on the scale need to be identified. In the example below, intensity has been linearly mapped to temperature. This curve was established by setting: 0 = 0 and 255 = 100.

Linear Intensity Scale X-axis depicts actual intensity, Y-axis depicts calibrated intensity If your scale is nonlinear, more than two reference points must be used to define your scale (many medical imaging devices have nonlinear scales). The scale below is an example of a nonlinear intensity relationship.

Nonlinear Scale Intensity scale on bottom; your scale on left Generally, test strips, with documented reference points, can be purchased for calibrating a non-linear curve. By capturing an image containing this strip, you can Page 1-65

Image-Pro Plus Start-Up Guide calibrate Image-Pro Plus to the values it represents (for optical density analysis, this strip is referred to as a density wedge). If the reference values are not readily available, a test image, made up of various index levels and known intensity values, should be created.

Intensity Analysis Tools

The intensity analysis commands, Histogram, Line Profile, and Bitmap Analysis, are located on the Measure menu. If you have calibrated an intensity and/or spatial scale of your own, these tools will express their results in your units of measure. When one of the intensity analysis commands is selected, a command window is presented. This window contains a graph of the intensity data and, optionally, a list of statistics. This command window is specifically linked to the image under analysis. The name of the image to which an analysis window is linked is displayed in the title bar.

The title bar will indicate the name of the image to hi h Statistics Graph

The graph and statistics are obtained from the intensity values contained in the images bitmap. They do not reflect intensity adjustments contained in the Lookup Table (LUT). If you want your analysis to measure the pixel values as modified by the Lookup Table, you must first apply the table values using the Apply Contrast command on the Enhance menu.

Histogram Analysis
As discussed earlier in this manual, histograms measure and illustrate in graph form, the intensity characteristics of an image. They can be used to manipulate the image for enhancement purposes (as discussed under Enhancing Your Image), or can be Page 1-66

Intensity Analysis used for data gathering purposes to measure the area associated with a specific intensity value. In a histogram, the X-axis represents the intensity scale (as defined by the current intensity calibration), and the Y-axis measures the spatial value (as defined by the current spatial calibration). A satellite image that had been calibrated to express intensity as temperature, would show temperature along the X-axis, and area (perhaps in square miles) along the Y-axis. The axes may be scaled using the Scaling menu in the command window. You can produce a cumulative histogram by selecting the Accumulated option on the Histogram windows Report menu. An accumulated histogram measures the number of pixels (in terms of the current spatial unit) that have the measured value, or a lesser value.

Accumulated Histogram Histogram data can be viewed in graphic or numerical form using one of the graphing options on the Report menu in the Histogram command window. When an accumulated histogram is selected, the numerical data will reflect the accumulated index values at each point along the X-axis. Histogram data (the numerical data) can be saved into a file using the Data to File command on the Histogram window's File menu. Data and statistics are stored in an ASCII file (assigned the extension .HST), which contains descriptive information followed by the data points. An excerpt from a *.HST file is shown below. You can learn more about .HST files in Appendix B, File Format Specifications.
# Image-Pro Plus Histogram Data # # # # # # Time: Mon Jun 12 17:16:55 2003 Image: newmoon.tif Spatial Unit: Pixels Intensity Unit: Pixels Number of Data Sets: 1 Set Size: 256

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Image-Pro Plus Start-Up Guide

# Mean 175.3897 # Gray Level 0.0 1.0 2.0 3.0 4.0 6.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 . . . Min 4.0 Pixels 0.0 0.0 0.0 0.0 874.0 38.0 19.0 22.0 0.0 24.0 0.0 1404.0 0.0 560.0 Max 254.0 Std.Dev 69.91221

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Intensity Analysis

Line Profile Analysis

Line profile analysis allows you to collect the actual index values along a line that you define. A profile plot shows the pixel positions of the line along the X-axis, and, on the Y-axis, measures the index value for each position along the line. In a line profile, the X-axis represents the spatial scale (as defined by the spatial calibration), and the Y-axis measures the intensity value (as defined by the current intensity calibration). A DNA gel that had been calibrated to express intensity as density, would show density along the Y-axis. The profile below illustrates the intensity changes along a line drawn horizontally across the image.

Line profile of DNA image Image-Pro Plus lets you obtain data for a single line (as shown above), or for a band of lines. A profile for a band of lines is called a thick profile.
Vertical Band
Average taken this direction

Horizontal Band
Average taken this direction

A thick profile plots the average (or standard deviation) for each row in a vertical band, or each column in a horizontal band.

Both single-line and thick-line profiles are created using the Line Profile command on the Measure menu. By default, a single line profile is created when this command is initially invoked. You can create a band profile by selecting the Thick Horz or Thick Vert options Page 1-69

Image-Pro Plus Start-Up Guide on the Report menu. A line profile can be of any length and position. While the line for a Normal line profile can be of any orientation, a thick profile can only be obtained for a horizontal or vertical band of pixels. If the pixels you want to measure are at an angle, you will need to rotate your image until they are aligned vertically or horizontally, before defining your band. Profile data can be written to a file using the Data to File command on the File menu in the command window. Like histogram data, line profile data is stored into an ASCII file, which is assigned a *.HST extension. An excerpt from a line profile .HST file is shown below. You can learn more about .HST files in Appendix B, File Format Specifications.

# Image-Pro Plus Line Profile Data # # # # # # # Time: Mon Jun 12 17:53:35 1998 Image: stripes.tif Spatial Unit: Pixels Intensity Unit: Pixels Number of Data Sets: 1 Set Size: 148 Mean 153.7973 1 2 3 4 5 6 7 8 9 10 11 Min 33.0 231.0 231.0 231.0 231.0 231.0 231.0 231.0 231.0 231.0 231.0 231.0 Max 231.0 Std.Dev 80.95961

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Bitmap Analysis
Bitmap Analysis command on the Measure menu is used to view the pixel values of the active window (or AOI) 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. When the Bitmap Analysis command is selected, the Bitmap Analysis window is opened and the pixel values associated with the active image or AOI are displayed.

PixelsY Coordinate

Pixels X Coordinate

Pixel Values

Bitmap analysis of a Gray Scale image Bitmap data can be written to a file using the Data to File command on the File menu in the command window. Like histogram data, bitmap data is stored into an ASCII file, which is assigned a *.BIT extension. The bitmap data are saved in a spreadsheet format, as shown above. You can learn more about .BIT files in Appendix B, File Format Specifications.

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Measuring and Counting

One of Image-Pro Plus's most powerful capabilities is its ability to let you perform spatial measurements upon your image, manually or automatically. There are two basic ways to perform measurements within Image-Pro Plus: Manually measuring single objects. Using the Measurements command in the Measure menu, you can measure the length of lines or polylines that you define, the area of polygons that you define, and the angles of arcs that you define. The Measurements command also lets you automatically trace and measure the edge of an object or feature in your image. You can measure distances between any two features, and perform tolerance testing using the new features of Image-Pro Plus. Automatically counting and measuring multiple objects. Using the tools on the Measure menu, you can collect multiple measurements of multiple objects within a single image. If your objective is to count the number of cells in a sample, and measure the area, roundness or perimeter of these cells, this is the tool to use. Once the objects have been counted and measured, you can use the Count/Size windows Measure menu options to automatically sort and classify the objects by any of the measured characteristics. You can also visualize the classified data by plotting it on a scattergram, or pseudo-coloring the counted objects by class. Manually counting and measuring multiple objects. Using the Manual Tag selection on the Measure menu, you can collect multiple measurements of multiple objects within a single image. You may select the number of objects in a class (up to 256) and the number of classes in a single image (up to 16), as well as the color, symbol, and name to identify each class. When a measurement is made, Image-Pro Plus highlights the measuring outline and assigns a reference number to it as shown in the object trace example below.

By default, Image-Pro Plus displays its measurement outlines in yellow, and reference numbers in blue, but if you prefer other colors, you can assign them using the Options button in the Count/Size or Measurements window. Measurements remain on the screen Page 1-72

Measuring and Counting until you close the image or explicitly delete the measurements using the Delete Measurements button. When you have taken automatic measurements using the Count/Size command, the measurement values are recorded on a data sheet. You can view measurement information by simply double-clicking the measured object within the image. You can view all current measurements by selecting the Measurement Data command on the Count/Size windows View menu. This will open the Measurement Data window, into which the data sheet for the active AOI or image will be displayed. Measurement data can be saved using the Data to File command on the Measurement Data window's File menu. Measurements are stored in ASCII form (*.cnt). The format for a measurement file is defined in Appendix B, File Format Specifications.

Before Measuring, Calibrate Your Image

Measurement operations are performed in terms of image pixel positions, e.g., the length of a measurement is determined by the number of pixels along the line, the area of an outlined object is determined by the number of pixels within the outline, and so forth. Note - When Image-Pro Plus performs a measurement, the pixels included in the outline are included in the measurement. For example, when you are measuring the area of an object you have outlined, the pixels making up the outline are included in the area calculation. Image-Pro Plus's pixel-level measurements can be scaled to fit any coordinate system. This allows you to obtain measurements which are reported in terms meaningful to your application. For example, you can calibrate the measurement scale so that one pixel is equal to one foot, or so that five pixels are equal to one foot (or inch, or mile, or micron ). Fractional values are allowed. Image-Pro Plus will express your measurements in terms of that unit. Additionally, if your image contains a measurable object of the unit length, you can calibrate your scale directly from that object, using the Image button in the Spatial Calibration dialog box. Important - Calibrate your scale before taking your measurements. Once measurements are recorded, their values are not affected by a subsequent change in spatial calibration. For example, if after taking a line measurement of 50 pixels where 1 unit equals 1 pixel, you changed calibration to 1 unit equal to 10 pixels, the first measurement you recorded would remain at 50; Image-Pro Plus will not automatically re-scale it to 5 to after the calibration is changed. You must take the measurement again in order for the new calibration to be reflected.

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Aspect Ratio
With respect to spatial calibration, the aspect ratio reflects the relationship between the length of the vertical and horizontal axes in your image, and is expressed as X/Y. Aspect Ratio is also calibrated using the Calibration command. If your camera's aspect ratio is not equal to your video card's aspect ratio, an image of a square object will not appear square when viewed on your display. To correct for this inconsistency, you can calibrate the aspect ratio from a reference square within an image. Note - Camera/video card inconsistencies are not the only reason a square might not appear square on your display. It might simply be a misadjustment of your monitor's Vertical Size control.

Taking Manual Measurements

The manual measuring tools are accessed using the Measurements command on the Measure menu. The manual tools are useful for obtaining individual measurements, and they are the only way to obtain straight-line length and thickness measurements. You can define features that have measurements of their own (the length of a line, for example) or take measurements between two existing features (i.e. distance, angle, or thickness). The following tools are used to create the primary features: Selection: This button is used to select existing features and/or measurements, i.e. to move or delete them. You may select more than one feature by holding down the <Ctrl> key while you use the mouse to click on the feature. Remove: This button is used to remove all of the currently selected features or measurements from the image. All related measurements in the Measurement tab and the corresponding row(s) in the Feature tab will be removed. Point: This button is used to create a point feature on the current image. Straight Line: This button is used to create a straight line feature on the current image. (This is the equivalent of the Length tool in previous versions of Image-Pro Plus.) Best Fit Line: This button is used to create a straight line that best fits several points on the current image. Click on the image to add up to 1000 points, and double-click to end the list of points. A setting on the Options page determines a user-defined limit to the number of points and the feature will be added when this limit is reached. For instance, to draw a two-point line (with two clicks) this option Page 1-74

Measuring and Counting could be set to a user limit of 2. The end points of the line will be the point where the line is perpendicular to the first and last points. Circle: This button is used to create a circle feature on the current image. Best Fit Circle: This button is used to create a circle that best fits several points on the current image. Click on the image to add up to 20 points, and double-click to end the list of points, creating the best-fit circle. A setting on the Options page determines a user-defined limit to the number of points and the feature will be added when this limit is reached. Best Fit Arc: This button is used to create an arc that best fits several points on the current image. Click on the image to add up to 20 points, and double-click to end the list of points. The points of the arc will need to be ordered, i.e. clicked in order from the beginning to the end of the arc. A setting on the Options page determines a userdefined limit to the number of points and the feature will be added when this limit is reached. Rectangle: This button is used to create a rectangle feature on the current image. Polygon: This button is used to create a polygon feature on the current image. This will be created using the current trace/wand tool, and will always yield a closed figure. (This is the equivalent of the Area tool in previous versions of Image-Pro Plus.) Trace: This button is used to create a trace on the current image. This will be created using the current trace tool, and will always yield a open figure. (This is the equivalent of the Trace tool in previous versions of Image-Pro Plus.) The automatic tracing tool works by following an edge of significant contrast. When you start a trace, select the point at which you want the trace to begin by clicking your mouse button. Then, click a second point along the edge to indicate the direction that Image-Pro Plus should begin following the edge. If Image-Pro Plus gets lost during its trace (loses the edge), it will stop. If it has wandered beyond the edge of your object, you can press Backspace to back up the trace to the edge, then manually lead the trace by moving the cursor to the next significant point along the boundary and clicking the left mouse button. Successful tracing is dependent primarily on how well differentiated your object is from the background. If there is good contrast between it and the pixels that surround it, your automatic traces will perform very well. Edges that have low contrast from the background, or have lots of competing edges intersecting them, will be interrupted more frequently. These controls let you fine-tune Image-Pro Pluss edge detection mechanism, used by the Trace and Freeform tools. Magic Wand: The magic wand tool makes it easier to outline an irregular AOI. Simply place the wand cursor inside the area that you want to trace, and click the left Page 1-75

Image-Pro Plus Start-Up Guide 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. Threshold: this value (from 1 - 10) describes the level of contrast distinguishing the edge you want to track from its surrounding elements. If there is low contrast, this value should be set to a low number. Smooth: this value (0-9) specifies the amount of post-filtering of the outline you want. 0 = no smoothing, 9 = high degree of smoothing. . Noise: this value (1 - 6) specifies the number of pixels Image-Pro Plus advances when moving to the next edge position. A larger value here results in a smoother outline (it won't follow every nook and cranny) and faster trace, but may cause it to get lost more frequently. Specifying values over 3 or 4 generally causes the Trace to overshoot curves along your edge. Speed: this value (0 - 5) specifies the rate at which the outline will be drawn. A small value here will slow down the trace function. You might do this to make it easier for you to watch the trace as it progresses The following measurement tools are available to take measurements between features created with the feature tools: Distance Measurement: This button creates a measurement of the distance between two user-selected features. Angle Measurement: This button is used to create two lines and do an angle measurement between these two lines. Note that this tool is different from the five other measurement tools in that rather than selecting two features for the measurement, two features are created and then the measurement derived from them. Angle Measurement: This button creates a measurement of the angle between two user-selected line features. (This is the equivalent of the Angle tool in previous versions of Image-Pro Plus.) Horizontal Thickness: This button creates a measurement of the horizontal minimum or maximum distance between two user-selected traces. Vertical Thickness: This button creates a measurement of the vertical minimum or maximum distance between two user-selected traces. Curve Thickness: This button creates a measurement of the minimum or maximum distance between two user-selected traces in any direction

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Measuring and Counting

Automatic Counting and Sizing

Using the Count/Size command on the Measure menu you can count and measure multiple objects automatically. Once the objects have been counted and measured, you can use the Count/Size windows Measure menu to automatically classify them by any of the measured characteristics. You can also visualize the data by plotting it using the Count/Size windows View menu options, or color the counted objects by class using the Auto Classification command on the Count/Size: Measure menu. A new feature of Image-Pro Plus version 6.0 allows you to sort your counted objects according to area or other measurements. Use the Sort Objects selection on the Measure menu to generate a new image of your objects in sorted order.

Identifying Objects to be Counted

For counting purposes, objects are identified by their intensity (monochrome images) or color (True Color-class images). Therefore, it is best to begin with an image that contains objects that are clearly distinguishable from the background, and have an intensity range/color different from other elements in the image. Because many images do not initially fit such ideal conditions, there are a number of pre-processing steps you can take to correct such deficiencies (pre-processing is discussed a little later in this section). The first step in performing a count is to identify the objects you want to include in your count. This is done by specifying the range of intensities or colors that uniquely identifies your objects. The way in which you do this first step depends upon whether your image is Gray Scale or True Color. Note - If your image is a Palette-class image, it must first be converted before the Count/Size command can be used with it. If you are working with a gray scale image, selecting your objects intensity range in a Gray Scale image can be done manually, or automatically. To manually specify object intensity, use the Manual option and the Select Range button. With your image on the screen, move the range markers until all your objects are highlighted. If you highlight a few extraneous objects in addition to the ones you want, dont worry they can be manually removed from the object group after the count using the Hide option in the Object Window command. However, if you find that your objects cant be highlighted without including numerous unwanted elements, you will need to use one of the pre-processing tools to increase object differentiation or eliminate unwanted noise. See Pre-Processing below.

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Image-Pro Plus Start-Up Guide To have Image-Pro Plus automatically set the intensity range that defines your objects, you can use its Automatic option. When a count is performed with the Automatic option, Image-Pro Plus will analyze your image, calculate the intensities distinguishing objects from background, and establish the intensity range based upon the objects that it finds. When running a count with the Automatic option, you must be sure to set the Bright Objects or Dark Objects option to describe your objects. If you are working with a True Color Image, selecting your object's color range is done by identifying an AOI that encompasses the colors of your objects. Selecting a color for counting purposes is essentially performed in the same way as extracting a set of colors using the Segmentation tool -- that is, you select a set of colors that separates certain features (in this case, objects to be counted) from the rest of your image. To confirm that your color selection is extracting just the objects you want, use the Create Image button to view your image with all but the selected colors grayed out. Refine your selection by adjusting the color ranges, until just the objects you want to count are visible.

Pre-Processing
If you are having difficulty separating your objects from other elements within your image, a number of pre-processing techniques are offered to help you enhance object definition. Contrast Enhancement: Often, simply increasing the contrast in your image will improve your ability to extract objects more successfully. Background Flattening: The Flatten Background command on the Count/Size window's Image menu is used to even out intensity variations in the background of a Gray Scale image (flattening is not applicable to True Color images). When flattening the background, you will be required to describe your object size and color in the Flatten Background dialog box. If your image has lots of objects, or your objects are very large, leaving only small patches of background showing, you may need to set the Background option to specify the color of your objects, rather than the background. You can also perform background flattening using Image-Pro Plus's Filter command. Background Subtraction: The Background Correction command on the Process menu can be used to produce a flat background, and compensate for nonuniform lighting, nonuniform camera response or minor optic artifacts (such as dust specks that mar the background of images captured from a microscope). To use the Background Correction tools, you must have, or be able to capture, an image of the background light (to produce a background image, capture an image with the slide Page 1-78

Measuring and Counting removed from the stage, or with the optics completely defocused). When the background image is subtracted from your image, areas that are similar to the background will be replaced with values close to the mean background intensity. Note - within the Background Correction dialog box, are two correction options: Background Correction and Background Subtraction. If the purpose of the correction is to facilitate object segmentation, use Background Subtraction. If the purpose of the correction is to obtain accurate density measurements, use Background Correction instead.

Selecting Your Measurements

Once you have defined your objects, you must choose the measurements you want to take. This is done in the Select Measurements command on the Count/Size window's Measure menu. Image-Pro Plus gives you the following measurement options, and you can select as many as you need. All spatial measurements are reported in the current spatial unit; all intensity measurements are reported in terms of the current intensity calibration. Angle: Reports the angle between the vertical axis and the major axis of the ellipse equivalent to the object (i.e., an ellipse with the same area, first and second degree moments), where 0 Angle 180. The vertical angle is 0, unless an offset has been set with the Calibration command. Area: Reports the area of each object (minus any holes). The area comprised of pixels having intensity values within the selected range is reported unless the Fill Holes option has been enabled. If Fill Holes is enabled, all pixels within the object perimeter are included in the area measurement. Area/Box: Reports the ratio between the area of each object, and the area of its imaginary bounding box, as determined by Area of Object / Area of Box. Area (Polygon): Reports the area of the polygon that defines the objects outline. Aspect: Reports the ratio between the major axis and the minor axis of the ellipse equivalent to the object (i.e., an ellipse with the same area, first and second degree moments), as determined by Major Axis/Minor Axis. Aspect is always 1. Average Diameter: Reports the average length of diameters measured at 5 intervals around the centroid of each object. Box Height: Reports the height of the bounding box along the major axis (i.e., the bounding box is the smallest rectangle that completely encompasses the whole object). Page 1-79

Image-Pro Plus Start-Up Guide Box Length: Reports the length of the bounding box along the major axis (i.e., the bounding box is the smallest rectangle that completely encompasses the whole object). Box Width: Reports the width of the bounding box along the major axis (i.e., the bounding box is the smallest rectangle that completely encompasses the whole object). Box X/Y: Reports the ratio between the width (X) and height (Y) of each object's imaginary bounding box, as determined by Box Width / Box Height. Centroid X: Reports the X-coordinate position of the centroid of the object from the left side of the image. Centroid Y: Reports the Y coordinate position of the centroid pixel of the object from the top of the image. Center Mass-X: Reports the X-coordinate position of the centroid of the object based on intensity measurements. Center Mass-Y: Reports the Y coordinate position of the centroid pixel based on intensity measurements. Class: Reports the class number to which the object belongs. This value will only be reported if the objects have been previously classified using the Single Variable Class command. If they have not, a Class measurement will not appear on the Measurements data sheet. Cluster: A cluster is a group of objects defined by an AOI. Cluster reports the number of individual objects contained within the outline. Clumpiness: Derived from Heterogeneity measurement; The fractions of heterogeneous pixels remaining in an object after an erosion process. It reflects the object texture. Count (adjusted): Reports the size-weighted object count. Only works when clean border flag is turned on. Dendrites/Dendritic Length: Reports the number of dendrites (1-pixel thick open branches) and the total length of all the dendrites. Measurements made of dendrite number and dendrite length should be performed on objects which have been skeletonized, or thinned using the Thinning filter in Filters: Morphological. Dendrite measurements performed on non-thinned objects may return unacceptable results. Density Blue: Reports the mean blue value for the measured object in a true color image. Density Green: Reports the mean Green value for the measured object in a true color image. Density Red: Reports the mean red value for the measured object in a true color image. Page 1-80

Measuring and Counting Density Sum: Reports the sum of the intensity values of all the pixels of a counted object. Diameter (max): Reports the length of the longest line joining two outline points and passing through the centroid. Diameter (mean): Reports the average length of the diameters measured at two degree intervals joining two outline points and passing through the centroid. Diameter (min): Reports the length of the shortest line joining two outline points and passing through the centroid. Feret (max): Reports the longest caliper (feret) length. Feret (mean): Reports the shortest caliper (feret) length. Feret (min): Reports the average caliper (feret) length. Fractal Dimension: Reports the fractal dimension of the objects outline.2 Heterogenity: Reports the fraction of pixels that vary more than 10% from the average intensity of the object. Holes: Reports the number of holes inside an object. A hole is defined as any contiguous set of pixels within an object that have intensity values outside the selected range for objects. If the Fill Holes option is set, this value will be 0. Hole Area: Reports the area of holes within an object. A hole is defined as any contiguous set of pixels within an object that have intensity values outside the selected range for objects. If the Fill Holes option is set, this value will be 0. Hole Ratio: Reports the ratio of the object area excluding holes, to the total area of the object, as determined by Area / (Area + Holes Area). Remember, IPP implements a variation on the hand and divider method (first introduced by Richardson). IPP accounts for variations in the perimeter measurement as a result of different starting points along the outline (i.e. depending on the where you first put the ruler to start measuring the outline, the perimeter is going to vary slightly even if the ruler stays the same). The original method doesn't compensate, which is one of its main drawbacks. The fractal dimension is defined as the slope of the linear part of the function that relates the log of the outline length to the log of the stride length, where the stride length is how long a ruler we attempt to lay along the perimeter of the object. Perimeters change with different size rulers, as the rulers bridge various small wriggles in the outline. We calculate the fractal dimension as 1 minus the slope of the regression line obtained when plotting the log of the perimeter (using a particular stride) against the log of the stride length, as calculated with multiple starting points in the outline for the strides. The stride length limits can be set using BLBCMD_SETFRACTDIM (see the SDK for details on how to use this), although we stop when the stride length is close to the minimum diameter of the object in any case. Page 1-81
2

Image-Pro Plus Start-Up Guide when a Hole measurement is selected, Area is the area of the object less the area of the holes. If the Fill Holes option is set, this value will be 1. Integrated Optical Density (IOD): Reports the average intensity/density of each object. This value will be expressed in terms of the current intensity/density mode and calibration. Major Axis: Reports the length of the main axis of the ellipse equivalent to the object (i.e., an ellipse with the same area, first and second degree moments).3 Minor Axis: Reports the length of the minor axis of the ellipse equivalent to the object (i.e., an ellipse with the same area, first and second degree moments)3 Max Diameter: Reports the length of the longest line that can be drawn to pass through the centroid position and join two points on each object's perimeter. Min Diameter: Reports the length of the shortest line that can be drawn to pass through the centroid position and join two points on each object's perimeter. Max Radius: Reports the maximum distance between each object's centroid pixel position and its perimeter. Min Radius: Reports the minimum distance between each object's centroid pixel position and its perimeter. Max Density: Reports the maximum intensity or density inside the object. Min Density: Reports the minimum intensity or density inside the object. Margination: Reports the distribution of intensity between the center of an object and the edge of the object.4 Perimeter: Measurement to report the length of the outline of each object using a polygonal outline. The perimeter of interior holes are not included in this measurement. Perimeter2: Old measurement from version 3.0. Faster but less accurate than current perimeter measurement. Reports the length of the outline of each object. When holes are outlined, the perimeters of the holes are added to the perimeter of the object.

3 The Equivalent Ellipse is an ellipse with the same moments of inertia (firstand second moments) of the object. Note that this will in general NOT have thesame area (the '0' moment) of an object, as two objects with similar areas but different shapes can have different moments of inertia. As a conceptual example, consider the moment of inertia of a barbell around its center of gravity with the weights at the ends of the bar, compared to the same barbell with the weights slid to the center of the bar. Both have the same weight, but the second example is much easier to rotate, since its weight is concentrated at the center of gravity. In two dimensional terms, a long capital letter "I" will have the same equivalent ellipse as a much larger filled circle, even though the filled circle will have a larger area. 4 Margination algorithm courtesy of Professor Ian T. Young, Pattern Recognition Group Delft University of Technology Department of Applied Physics Lorentzweg 1 NL-2628 CJ Delft The Netherlands

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Measuring and Counting Perimeter3: New measurement in Image-Pro Plus 6.0. Reports a corrected chain code length of the object perimeter, not including holes. Much less biased than Perimeter2.5 Perimeter (Convex): Reports the perimeter of the convex outline of each object. Perimeter (Ellipse): Reports the perimeter of the ellipse surrounding the outline of each object. Perimeter (Ratio): Reports the ratio of the convex perimeter to the perimeter of the outline of each object. PerArea: Reports the ratio between the area of the counted object to that of the entire area. The ratio is determined by (Object Area/Total Area) where Total Area is the area of the active image or AOI (whichever was in effect when the measurement was taken). Radius Ratio: Reports the ratio between Max Radius and Min Radius for each object, as determined by Max Radius / Min Radius. Roundness: Reports the roundness of each object, as determined by the following formula: (perimeter2) / (4 * pi * area). Circular objects will have a roundness = 1; other shapes will have a roundness > 1. Size Count: Reports the count of multiple objects of varying sizes in multiple frames. Size (length): Reports the feret diameter (caliper length) along a major axis of the object. Size (width): Reports the feret diameter (caliper length) along a minor axis of the object. Std. Dev. Density: Reports the standard deviation of density or intensity inside the object.

Selecting Your Ranges

Once you have identified the measurements you want to take, you can set measurement criteria that will determine which objects Image-Pro Plus includes in the count. For example, you can instruct Image-Pro Plus to include only objects larger than a certain size. This is done by establishing a valid range (i.e., specifying minimum and maximum values) for each measurement using the Set Ranges command in the Measure menu. If
5 Kulpa, Z., Area and perimeter measurement of blobs in discrete binary pictures. Computer Vision, Graphics and Image Processing, 1977. 6: p. 434-454.

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Image-Pro Plus Start-Up Guide the object's measurement is within this range (min and max, inclusive), it will be included in the count. Objects with values outside the range are ignored.

Selecting Other Measurement Options

Image-Pro Plus offers several additional options that let you tailor your count to your needs, including how partial objects will be treated, and how objects will be labeled. These miscellaneous options are selected using the Options button in the Count/Size window: Smoothing: This option lets you set a factor that determines whether or not the perimeters of the counted objects will be smoothed. A factor of 0 indicates that no smoothing should be done. Values greater than 0 turn smoothing on, and indicate the degree of smoothing that should take place. If perimeter and roundness are critical measurements to your analysis, you may want to use this option. Bear in mind that smoothing will lengthen the time it takes to perform your count, so you will want to use this option only when it is truly necessary. Clean Borders: This option determines whether partial objects that lie at the border on an image should be included in the count (partial objects can skew your results). If Clean Borders is selected, these partial objects will be ignored. Outlines: This option determines whether outlines of the counted objects will be shown or not. If objects are to be outlined you can select one of several outline formats: perimeter, elliptical, filled or class. When Outline is selected, the object's perimeter is outlined. When Ellipse is selected, the object's equivalent ellipse (an ellipse with the same area, first and second degree moments as the object) is displayed. The Ellipse option is particularly meaningful in graincounting experiments, because it identifies an object's ideal domain, from which binding sites can be identified. The Filled option shows counted objects in solid color, and the Class option colors the objects by category (see Visualizing Measurement Data below). This option also allows you to select the color in which you want the outlines displayed. Dot marks each counted object with a small cross hair positioned in the center of each object's bounding box. Label: This option determines whether an object's unique numeric label should be displayed following the counting process, and what color should be used to display it. The displayed labels allow you to visually match objects with your results, and should rarely be turned off. The only times you may want to turn labeling off are to eliminate screen clutter or inspect the details of a particular outline.

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Measuring and Counting Object Limits: Up to 16,000 objects can be included in a single count. If your image contains more than 16,000 objects, consider using AOIs to process the count in a series of small batches. Image-Pro Plus also has an internal limit of 250,000 object segments. A counted object is built of segments, which are horizontal, single-pixel slices of the object. Each segment is represented internally by its beginning and endpoint coordinates.
a counted object is composed of horizontal "segments"
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

a counted object with holes and/or fissures

will be composed of many, many segments

3 6 9 12 15 18 21 24 26 28 30 32 29 31 1 4 7 10 13 16 19 22 25 27 2 5 8 11 14 17 20 23

Objects containing lots of fissures or holes will require many segments to describe their shape. If you have many such objects, the internal limit of 250,000 segments could be exceeded. Consider using AOIs to process the image in a series of small batches if this limit is encountered. The Outline option in the Count/Size Options dialog box can outline object perimeters comprised of up to 4096 vertices. If your object has a very, very large, irregular perimeter, you may exceed this limit and its outline will not be displayed correctly (all measurements taken for the object are correctly reported, however). To display an outline of a very, very large object, select either the Filled or With Holes outline styles.

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Image-Pro Plus Start-Up Guide

Performing the Count

Once you have identified your objects, selected your measurements and tailored the count with the options you need, you are ready to initiate the count itself. To do this, click the Count button. Image-Pro Plus will analyze your image, and will outline and measure the objects it finds meeting your selection criteria. When it is done measuring, it will display the object outlines, and write all the requested measurements to the measurement data sheet. You can view the measurements for an individual object by double-clicking the object in the image. This produces a pop-up window that contains the measurements for the selected object. The measurement data sheet, which is a table of all the measurements, can be viewed by selecting the Measurement Data command in the View menu.

Editing the Count

Before working with the data sheet, you should take a moment to review the display to make sure you are satisfied with the objects that have been extracted you may want to eliminate extraneous objects, separate clusters that have been counted as a single object, or combine separate outlines into one. On the Edit menu, Image-Pro Plus gives you a number of tools that let you edit and finalize your count. Draw/Merge Objects: This option lets you manually outline an object that was not included in the count, or combine two objects so that they are counted as one. To manually include an outline, select Draw/Merge then draw a polygon that traces the object, using any of the standard AOI tools: rectangular, elliptical or freeform (you can even use the Freeform tools Auto Trace feature to automatically trace an edge: See AOI Tools.) When you have finished drawing the polygon, the specified measurements for that shape will also be added to the data sheet. Note - You can draw a polygon that defines an object that doesn't visibly exist in your image. This is usually done to define a region from which population density can be calculated. To combine two closely positioned objects into a single object, simply draw a narrow polygon that connects (not outlines) the two existing objects (see example below).

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Measuring and Counting

polygon drawn to connect objects 6 & 7 single object produced by merging objects 6&7

Once connected, the measurements of the two objects will be outlined and considered as a single object. In the data sheet, the combined measurement will replace the measurements of one of the original objects and the other's measurements will be deleted. You can also merge more than two objects into a single object. The objects needn't be close together, either. As shown in the drawing below, you can merge multiple objects into a group by drawing a polygon around them, or by drawing a polygon with its vertices inside the individual objects.
polygon drawn around 3 objects single object it produced

or
polygon drawn inside the 3 objects single object it produced

Merging many objects in such a manner might be done to develop area outlines for population density analysis (see Population Density Analysis later in this section). Split Objects: This option lets you manually divide clusters into single objects. Dividing is done drawing a division line between the objects you want to separate (see example below).
clustered objects line drawn to separate the cluster

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Image-Pro Plus Start-Up Guide After separation, the measurement data sheet will be updated to reflect the additional object, and the measurements of the original object will be adjusted to reflect the remaining object. Auto Split: You can also use the Auto Split command to instruct Image-Pro Plus to analyze all existing outlines and automatically split any clustered objects it finds. Of course, not all clustered objects can be separated with Auto Split; in general, circular objects with minimal overlap work best. Use the following criteria for determining whether your objects are clustered in a way that can be easily separated by the Auto Split operation: Individual objects within the cluster should be convex-shaped. An object is considered convex if all lines joining two points of the outline fall within the object. Objects should not overlap each other by more than 30%. The intersection of two overlapping objects should be identifiable by two concave points. Object outlines should be smooth enough to readily distinguish a concavity created by an overlap from a concavity that is simply part of the object. Note - Image-Pro Plus splits objects with a straight line. It does not attempt to reconstruct the missing or hidden portions of an object. If the Auto Split command does not yield satisfactory results, try using the Watershed Split. This method does not rely on object concavities to determine lines of separation. Instead it erodes an object until it disappears, then dilates it back to its initial size, but does not allow it to touch its neighbors. Watershed Split: Use the Watershed Split to automatically analyze every counted object and split clusters using the Watershed separation technique. The Watershed method erodes objects until they disappear, then dilates them again such that they do not touch. The Watershed Split command may be more effective than the Auto-Split method if your objects are not convex. You may remove an extraneous object from your count by using the Toggle Objects On/Off feature from the Count/Size:Edit menu. When an object is hidden, its outline and label are removed from the display and its measurements are eliminated from the data sheet. Deactivated measurements are also excluded from global statistic, classification and visualization operations. To permanently remove hidden objects, choose Delete Hidden Objects from the Count/Size:Edit menu.

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Measuring and Counting

Manual Tagging
Another option to identify objects in your image is to use the Manual Tag option from the Measure menu. Manual Tag allows you to select points in an image, and assign them to a specific class. You can define up to 16 classes of objects for a single image, and assign each class a name, color, and identifying symbol. Image-Pro Plus automatically keeps track of the points as you select them. You can display information about each class including minimum, maximum, mean, standard deviation, and total by using the Manual Tag : View: Class Stats menu. Manual Tag : Options allows you to see the x and y position, intensity, and class of each tagged object in the image. Manual Tag measurement information can be stored using the Data to File command on the Manual Tag : File menu. Data and statistics are stored in an ASCII file (assigned the extension .CNT), which contains location information about the different markers. A portion of a .CNT file appears below. Marker 1 2 3 4 Class Class 3 Class 3 Class 3 Class 3 X 78 178 181 79 Y 88 88 179 177 Intensity 169 180 185 168

A Manual Tag file is created when you store your class and/or tag marker information in the Manual Tag selection from the Measure menu. A *.TAG file is created using the Save Points command on the File menu in the Manual Tag dialog box. A *.TAG file is an Image-Pro Plus file that contains the class and marker information that is to be applied to the objects in an image. This data is not in ASCII format. Users should not attempt to modify a *.TAG file

Using the Outlines

There may be situations where you will need to generate your outlines from one image, then superimpose the outlines onto another image to identify object position, or to obtain an average intensity measurement. Such is necessary in Auto-Radiography applications, where cell position and shape is captured in visible light, but cell contents are captured with a different apparatus which does not produce a cell outline. To measure the Page 1-89

Image-Pro Plus Start-Up Guide intensity within a cell, the outlines are generated by the first image, then superimposed upon the second image. Then, an intensity measurement is performed using the Population Density measurement in the Count/Size commands Measure menu. Object outlines can also be stored using the Save Outlines command on the Count/Size windows File menu. Outlines are stored to Image-Pro Pluss outline files (.OUT). An outline file contains a list of all the polygonal shapes comprising your outlines. The format of this file is described in Appendix B, File Format Specifications. Stored outlines can be used as input to an external program, or can be reloaded into Image-Pro Plus. A stored outline can be subsequently loaded into Image-Pro Plus for positioning purposes or for additional measuring. Outline files are also an essential component to the population density process (see Population Density Analysis later in this section).

Viewing Measurement Data

When a count operation is performed, the requested measurements (those selected with the Select Measurements command from the Count/Size:Measure menu) for all counted objects are written to the measurement data sheet. This data sheet can be viewed by selecting the Measurement Data command from the Count/Size:View menu. The data sheet contains only values for the requested measurements; if you want to incorporate additional measurements in your data sheet you will need to select them, then re-measure the outlines by clicking the Measure button (keep the original measurements selected too, if you want them to appear in the data sheet). Statistics, such as minimum, maximum, mean and standard deviation can be viewed using the Statistics command on the View menu. A set of these statistics is produced for each measurement you have selected. Measurement data can be stored to a file using the Data to File command on the Count/Size window's File menu, or on the Measurement window's File menu. Data can be stored in ASCII or LOTUS 123 WK1 format.

Visualizing Your Measurement Data

Two plotting devices, scattergram and histogram, are provided to let you visualize your data in chart form. You can also render your objects using color to represent the measurement values they possess.

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Measuring and Counting Scattergrams A scattergram plots two measurements against each other. Scattergrams can be used to illustrate a correlation between two measurements. For example, a scattergram can graphically show you whether or not there is any relationship between two measurements, or whether there is any clustering of the data points. A scattergram is created by selecting the Scattergram command from the Count/Size:View menu. Image-Pro Plus displays the scattergram along with its correlation factor and a best fit line drawn through the points.

To select the measurements you want to plot on each axis, use the list boxes provided in the Scattergram window. Measurement histograms (not to be confused with intensity histograms) provide another way to plot your measurement data. Measurement histograms illustrate the distribution of your object population over a measurement range. The histograms X-scale identifies the measurements range (from its minimum value to its maximum value), and the Yscale measures the number of objects within each measured interval.

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Image-Pro Plus Start-Up Guide Measurement histograms are created using the Histogram command on the Count/Size windows View menu. Within this menu, you must select both the measurement you want to plot, and the number of intervals (from 2 - 100, inclusive), into which you want your X-axis segmented. Scattergrams and histograms can be captured and saved into an image file using ImagePro Plus's Screen Capture facility. The captured image file can be printed in Image-Pro Plus, or imported into your word processing program, or included in a report (see The Report Generator). You can also visualize your measurement data with color, using the classification commands on the Count/Size windows Measure menu and coloring the objects by class. This lets you distinguish the objects with a color that represent their measurement values. Objects may be classified using one of two methods. The Single Variable Class. command lets you divide the results of a single measurement type into the number of categories that you define. If you then select the Class outline options in the Count/Size options dialog box, your objects will be colored according to the class to which they belong. In the example below, the spores have been classified and colored according their Area measurement.

You may also classify your measurement results using the Auto Classification command. This lets you categorize data into groups of objects with similar measurement characteristics. In the Auto-Classification dialog box, you must define the number of groups you want your data sorted into and the measurements you want used as classifiers. Image-Pro Plus will look for clusters of data points that are created when the selected measurements are plotted against each other (see figure below). Data clusters (classes) are isolated using a cluster-seeking technique.

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Measuring and Counting

Clusters (classe

Clusters identified during automatic classification by Area and Roundness Although Image-Pro Plus does not display a plot like the one above during its automatic classification process (it would be impossible to produce for more than 3 classifiers), you can check for clusters yourself by generating a Scattergram for each measurement pair in your classifier selection list (see Scattergram discussion earlier in this section). Creating a scattergram is a good way to determine whether or not you have selected meaningful classifiers - if your scattergram yields a correlation close to 1, you only need to use one of the plotted measurements as a classifier because the values of the two are so similar. After your measurements have been classified, you can select the Class outline options in the Count/Size options dialog box, and your objects will be colored according to the class in which they belong.

Saving Your Counting Environment

You may spend a considerable amount of time tailoring the counting options, intensity ranges and classifiers to a particular image. Instead of manually resetting these parameters next time you work with a similar image, you can save all these settings, then reload them when they are needed. This is done using the Save Settings command on the Count/Size window's File menu. Environment parameters are stored in .ENV files. A counting environment can be restored by loading the appropriate .ENV file using the Load Settings option.

Population Density Analysis

Population Density is the measurement of the number of objects per unit of area. It is often used to determine the density of a cell sample. Page 1-93

Image-Pro Plus Start-Up Guide

Calculating population density is performed using the Population Density command on the Count/Size windows Measure menu. However, there are two very important steps that must be performed before the Population Density command is selected. The three steps required to produce a population density analysis are: Step 1 - The outlines of the sites to be measured must be created and saved to a file. Step 2 - The individual objects within the areas to be measured must be counted. Step 3 - The Population Density command must be called. Each step is discussed below. You may also want to refer to the Population Density tutorial in the Image-Pro Plus Installation Guide for additional background. Step 1 - Defining the Areas to be Measured Defining the sites to be measured can be done in two basic ways: If your image contains small objects (cells or nuclei, for example) inside of a visibly defined larger object (site), you can use the normal object counting process to measure and outline the large objects (sites). If your small objects are not contained within any visible entity, you can use Count/Size window's Draw/Merge command or the Measurements window's Area tool to manually draw outlines of the sites to be measured. Regardless of which method you use to define the outlines of the sites, once you are satisfied with the outlines on the screen, they must be saved to an outline file using the Save Outlines command on the windows File menu. This file will be requested when you perform the Population Density command in Step 3. Step 2 - Counting the Individual Objects Next, you must count the individual objects (grains, nuclei, or whatever the case may be). If the site outlines and measurements from the previous step are still active, be sure to clear them before you begin. Then, count (and measure, if needed) all the small objects using any the normal counting procedure. It is important that you have an accurate count of the number of individual small objects in your image. If some of your small objects are overlapping and clumped into groups, the objects within the groups will need to be measured. This is done by selecting the Clusters measurement when you perform the count (select Cluster in the Select Measurements dialog box). A cluster measurement calculates and records the number Page 1-94

Measuring and Counting of objects contained in each counted outline. This value is written to the measurement data sheet. Step 3 - Calculating Density Once you have the small-objects outlined and count on the screen, you are ready to begin the density analysis. To do this, select the Population Density command from the Count/Size window's Measure menu and select the file that contains the site outlines created in Step 1. The site outlines will be superimposed over the small objects you've just counted, and the object density within the outlines will be calculated. The results of the density calculations are displayed in the Population Density window. The following data is reported: Site Number: This number identifies a site outline within the image. Each site is given a unique numeric label, which is displayed in brackets, [ ], to distinguish it from the small object labels. Object Count: This number identifies the number of objects within the site. An object is considered within a site if its center of gravity lies within the site outline. Site Area: This number reports the area of the site. It will be reported in terms of the current spatial unit. Density (raw): This number reports the gross density for the site as calculated by dividing Object Count by Site Area. Density (corrected): This number reports an adjusted density for the site as calculated by subtracting the AOI's background density from the Site's raw density. This gives a value that represents the density of the site independent from the background population. Sites (mean): This row of number reports the average object count, area density statistics for all sites within the AOI. Sites (sum): This row reports the total number of objects and the total area for all sites within the AOI. Background: This row reports the number of objects that fell outside of the site outlines, the area of the AOI that fell outside the site outlines (the background area), and the density of the background calculated by dividing the Background Site Area value by the Background Object Count value. The background density value is used to create a Corrected Density value for each site.

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Image-Pro Plus Start-Up Guide Total: This row reports the total number of objects within the AOI (those within the sites and those within the background), the total area of the AOI and the population density of the entire AOI.

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Customizing the Command Menus and Buttons

Customizing the Command Menus and Buttons

Image-Pro Plus adds new capabilities for customizing the users interaction with the application. While previous versions maintained a single *.MNU file that defined the applications menu and toolbar, Image-Pro Plus supports multiple menu files and user selection of the menu file to be used. Image-Pro Plus ships with 4 menu files, which you can modify to customize the application for your purposes, but you can also use one of these menus as the source for creating a new menu file of your own that is added to the list shipped with Image-Pro. This is a very powerful addition to the customization capabilities of Image-Pro. The rest of this section will discuss the contents of an MNU file, and while the examples will refer to the Image-Pro Plus reference MNU file, the discussion is relevant to the other standard MNU files as well as any new ones that you create. Note - You can completely customize Image-Pro using Image-Pro's Software Development Kit (SDK). The SDK allows you to incorporate your own, customdesigned DLL's and toolbar icons, letting you create powerful, vertical-market applications based upon Image-Pro. For more information about the Image-Pro SDK, contact Media Cybernetics or your Image-Pro dealer. The selected MNU file in the applications resources folder defines the main menu and tool bar. By editing this file (using any plain text editor) you can add your plugin to a menu or attach it to a button on the tool bar. When you add it to the tool bar, you can use one of the icons provided with Image-Pro or design one of your own. Along with the ability to select a different menu and toolbar arrangement, Image-Pro Plus 6.0 also adds the ability for the MNU file to give the application hints about the intended purpose of the specialized menu. Individual features of Image-Pro may customize their appearance or tool set based on the selected menus hints. Note - You may need to close any open dialogs before switching menus to see changes in the feature set.

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Image-Pro Plus Start-Up Guide You may therefore wish to define new Image-Pro menus for a number of different reasons: To change the set of features available to your users to customize the product for their experience and workflow, and/or To change the menu hints An MNU file is made up of a series of definition statements. The first set of statements defines any application hints. The second set of statements defines the menu structure. The third set of statements is a portion of .MNU which defines the tool bar buttons.

While editing a .MNU file, the following rules must be followed: Keywords must appear entirely in lower case (i.e., progitem, not PROGITEM or ProgItem) The first parameter must be separated from its keyword with a space. Subsequent parameters must be separated from one another with a comma. You may use spaces or tabs to indent statements (usually done to improve readability).

The # character is the comment character. It can be used to designate a statement that should not be processed. This is usually done to include lines of descriptive information, or to comment out a statement that is to be excluded from the file. The # character must appear in the first position of the line. Note: Although you may edit the MNU file while your application is active, your changes will not take effect until the program is restarted, or until you re-select the menu from the Select Menu item in Image-Pros Window menu. Also, be sure to close the MNU file in your editor before starting the application, otherwise it may not load properly. The presentation of the menus and buttons is defined by the current *.MNU file. This file is located in your Image-Pro resources directory. By editing the contents of this file using any ASCII editor (the Window's Write could be used, for example), you can modify the appearance of your menus and command buttons. Note - although you may edit the *.MNU file while Image-Pro is active, your changes will not take effect until Image-Pro is restarted, or the menu is reloaded using the Select Menu item on the Windows menu.. Also, be sure to close the *.MNU file in your editor before starting Image-Pro; otherwise Image-Pro may not load properly. Important - always make a copy of your *.MNU file before making any changes to it. Mistakes in this file can easily affect the operation of Image-Pro, and render Page 1-98

Customizing the Command Menus and Buttons commands inoperable. A back-up file will ensure that you can revert to a previous, good, version of this file in the event of problems with your edits.

Customizing the Menus

The first section of the *.MNU file defines the contents of the command bar. You can edit this section to remove, rename or re-organize the Image-Pro main menu and commands therein.

Menu statements
The example below defines a File menu (though not the Image-Pro Plus standard File menu). The menu begins with a columnbegin statement and ends with an endcolumn statement.
columnbegin &File, 0 Menu items progitem &New...,Make document.,0,101 (commands) progitem &Open..., Open image file.,0,102 progitem &Reload, Reload image file.,0,112 separator Separating line group &LUT item &Load LUT. . ., Load LUT file.,iplut32.DLL,14,1 item &Save LUT. . ., Save LUT to file.,iplut32.DLL,14,2 endgroup item &Convert..., Convert image on disk.,ipconv32.dll,32,0 endcolumn

The Menu

A sub-menu

Each statement in the MNU file is made up of a keyword followed by several parameters. A complete, syntactical description of the allowed statements is documented at the end of this chapter. Important Note: When adding columns to the menu, (which adds a new menu to the applications command bar) new menus must be added after the File column and before the Window column. Note - *.MNU provides the controls for modifying the main Image-Pro menu. Menus that are presented within command windows, such as Count/Size and Histogram, are not defined by the *.MNU file and are not modifiable. Before editing this file, you should familiarize yourself with the elements of its structure. The following table introduces you to the basic statements that are used to Page 1-99

Image-Pro Plus Start-Up Guide create a menu in Image-Pro. A complete, syntactical description of each of these statements is documented at the end of this section under *.MNU Syntax.

STATEMENT columnbegin endcolumn progitem

DESCRIPTION Marks the beginning of a menu Marks the end of a menu Defines a menu item i.e., a command listed in the menu. Differs from item, below, in that it invokes a procedure, or macro, that is resident in the main Image-Pro program. Defines a menu item i.e., a command listed in the menu. Differs from progitem, above, in that it invokes a procedure that resides in an Image-Pro DLL. Marks the beginning of a sub-menu within the current menu (i.e., a pop-out menu). Marks the end of a sub-menu within a menu. Draws a line between items in a menu.

item

group endgroup separator

The example below shows a portion of the Image-Pro File menu description, and points out the menu components within it.
File Menu Title Menu Items columnbegin &File progitem &New...\tCtrl+N,Make new document.,0x0000,101 progitem &Open...\tCtrl+O,Open document from disk.,0x0000,102 item Image &Database...,Open document from image database.,ipgali32, 0x0480, 0 %item Database Stub,Start database.,ipgali32,0x0480,20 progitem &Reload,Reload document from disk.,0x6B0F,112 progitem &Close...,Close current document.,0x6B0F,103 progitem &Save...\tCtrl+S,Save current document to disk.,0x6B0F,104 progitem Save &As...,Save current document to disk with new name.,0x6B0F,105 item Arc&hive...,Store image in the image database.,ipgali32.dll,0x6B0F,10 item Con&vert...,Convert file format.,ipfile32,0x0000,0 separator Separating Line item Send &Mail...,Send Mail.,ipmail32,0x0480,0 group Remote File &Transfer Submenus item &Open Remote File...,Open file on remote FTP server.,ipftp32,0x0480,0 item &Save Remote File...,Save file on remote FTP server.,ipftp32,0x6B0F,1 item &Personal File Server...,Configure personal FTP file server.,ipftp32,0,2 endgroup

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Customizing the Command Menus and Buttons

Removing a Menu Item

You can remove an item from a menu by placing the # character in the first position of the item or progitem line. The # character tells Image-Pro to ignore that particular statement. In the following example, the three color-processing commands have been removed from the Process menu.
columnbegin &Process item &Background Correction...,Perform Background Ops.,ipflt32,0x6BAF,101 item Restricted &Dilation...,Perform masked image dilation.,ipflt32,0x6BAF,103 item &Threshold..., Binarize Image.,ipseg32,0x0B82,100 item &FFT...,Fast Fourier Transform operations.,ipfft32,0x0080, 100 item &Stretch..., Slide/Stretch.,iplut32,0x6B02,111 item Fi&lters...,Perform Spatial or Morphological Filters.,ipflt32,0x6B8F, 100 item &Operations...,Perform arithmetic & Logical Operations.,ipflt32,0x6BAF,102 # item Color C&hannel...,Color channel processing.,ipcolr32,0x6BAF,100 # item &Segmentation...,Perform Segmentation.,ipseg32,0x6BAE,100 # item &Pseudo-Color..., Colorize Image.,iplut32,0x0B02,201 item &Registration...,Register two images.,ipreg32,0x698A,100 progitem Image Overla&y...,Overlay an image with another.,0x6B2E,3008 item &Mosaic Image...,Combine several images into a new mosaic image., IPMos32, 0x6BAE, 1 item Trace Ob&jects...,Draw overlay tracing objects.,iptrce32,0x698A,100 item &Grid Mask..., Creates grid mask, ipgrid32, 0x0080, 1 endcolumn

Moving a Menu Item

You can move a command from one menu to another by simply moving its item (or progitem) statement to another menu. Be sure you move the entire statement, including the carriage-return character that terminates it. Note - be sure to consider the documentation impact when modifying your menus. Commands in the Image-Pro Reference Manual are documented in the order in which they appear on the default menus. Rearranging commands will reduce your user's ability to thumb-through the manual to locate a command.

Renaming a Menu Item

You can rename a command on the menu by changing the contents of the Title parameter in its item or progitem statement (see *.MNU Syntax for the position and content of the Title parameter). Within the Title parameter, the & character must precede the character designated as the access key (sometimes called accelerator key); the access character Page 1-101

Image-Pro Plus Start-Up Guide specifies the key that can be used to select the command once the menu is opened it appears as an underscored character when the menu is displayed. Try to choose a character that is not in use as an access key by any other command in the menu. If you want an ellipsis (i.e., ...) to appear after your command name, be sure to include it as part of your command name in the Title parameter (by convention, an ellipsis is used to denote commands that produce a dialog box when selected e.g., Print...). Note - keep in mind that if you rename a command, your users may have difficulty locating information about it in the Image-Pro Reference Manual and on-line help system.

Adding a Macro to a Menu

Macros can be added to the tool bar by creating a macrobutton statement for it in the MNU file. The macrobutton statement identifies the icon that is to be displayed on the tool bar, and specifies the macro that is to be executed when it is clicked (including the script file that contains the macro). See the MNU statements at the end of this chapter for a complete description of the macrobutton statement. The following example shows a macrobutton statement defined for a macro called Histogram in the C:\IPWIN5\DEFAULT.IPM script file. In this example, the icon called USER1 is assigned to the tool bar.
macrobutton USER1,Macro: Histogram,C:\IPWIN5\DEFAULT.IPM ,0,Histogram

You may use any of the predefined icons for the macro button, but the application will set it back to USERx if the buttons are recreated. . An ID of... 5000 identifies the first macro in the selected script file, 5001 identifies the second macro in the selected script file, 5002 identifies the third macro in the selected script file, and so forth. The following example shows a progitem statement defined for the 4th macro (#5003) in a script file.
progitem &Sub-Sample Sequence...,Create sub-sampled sequence.,0x6B0F,5003

Important - a macro is always invoked from the currently selected script file. If you change script files mid-session, a menu item will invoke the specified macro number from Page 1-102

Customizing the Command Menus and Buttons the new script file. To ensure that the macro you intended is always the one invoked by a menu command, do not change script files during your Image-Pro session.

Advanced Menu Editing

By following the syntax outlined under *.MNU Syntax at the end of this section, you can structure your *.MNU file such that it creates, deletes or renames entire menus. You can also regroup the existing menu commands so that they appear in any order you'd like.

Customizing Command Buttons

The second section in the *.MNU file contains the statements that determine which command buttons are shown on the tool bar. You can even assign a macro that you have developed to a command button on the toolbar. This allows you to customize your tool bar with just the tools you want to have close at hand. A command button is defined by one of the following statements in the *.MNU file: STATEMENT progbutton DESCRIPTION Defines a command button that is to be displayed on the Toolbar. Differs from button, below, in that it invokes a command that is resident in the main ImagePro program. The progbutton statement is also used to assign a user-defined macro to a command button. Defines a command button that is to be displayed on the Toolbar. Differs from progbutton, above, in that it invokes a command that is resident in an Image-Pro DLL.

button

The *.MNU file contains definitions for all of the Image-Pro commands that can be assigned to the Toolbar. By default, only a few of these statements are active. You can selectively activate/deactivate buttons by including or removing the # character (the comment character) at the beginning of each statement. Button statements that begin with the # character are ignored by Image-Pro, and the buttons for such statements are not shown on the Toolbar. The example below shows an excerpt from the *.MNU file. In this example, the first 3 statements are active the command buttons for these will be displayed. The last 3 statements are commented out these buttons will not be displayed.
button FFT,Fast Fourier Transform,ipfft32,0x0080, 100 button FILTER, Spatial filtering,ipflt32,0x6B8F, 100

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Image-Pro Plus Start-Up Guide

button OPER,Arithmetic and logical perations,ipflt32,0x6BAF,102 #button TRANSFORM,Color channel processing,ipcolr32,0x608A,100 #button SEGMENT,Perform segmentation,ipseg32,0x6B8E,100 #button PSEUDO, Colorize gray scale images,iplut32,0x6B02,201

See the next section (*.MNU Syntax) for syntactical descriptions of the button and progbutton statements. Note - approximately 20 command buttons can be shown simultaneously, however, the exact number will depend upon the resolution of your monitor. You will need to experiment with the button statements in the *.MNU file to determine just how many buttons will fit on your Toolbar

Assigning an Image-Pro Command to a Button

You may assign any of the menu commands to a command button by following the procedure below: 1. Copy the item or progitem statement from the menu definition section at the top of the *.MNU file to the button description section (do not delete the statement from the menu definition section unless you deliberately intend to remove it from the menu). 2. Change the statement's keyword as follows: IF THE KEYWORD IS... progitem item CHANGE IT TO... progbutton button

3. Change the contents of the Bitmap parameter to the name of one of the predefined button bitmaps. See the progbutton or button definition under *.MNU Syntax for a description of the Bitmap parameter. The following example shows how a copy of the Video Average item statement would be modified to assign it to the Camera button. The underscoring denotes the parts of the statement that would be changed.

This item statement...

progitem &Next Image \tF2,Activate next document.,0x6B0F,657

would be changed to... progbutton NEXT,Activate next document.,0x6B0F,657

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Customizing the Command Menus and Buttons

Assigning a Macro to a Command Button

Macros can be added to the tool bar by creating a macrobutton statement for it in the MNU file. The macrobutton statement identifies the icon that is to be displayed on the tool bar, and specifies the macro that is to be executed when it is clicked (including the script file that contains the macro). See the MNU statements at the end of this chapter for a complete description of the macrobutton statement. The following example shows a macrobutton statement defined for a macro called Histogram in the C:\IPWIN5\DEFAULT.IPM script file. In this example, the icon called USER1 is assigned to the tool bar.
macrobutton USER1,Macro: Histogram,C:\IPWIN4\DEFAULT.IPM ,0,Histogram

You may use any of the predefined icons for the macro button, but the application will set it back to USERx if the buttons are recreated. You can assign a macro to a command button by creating a progbutton statement for it in the MNU file (the progbutton statement, not the button statement, must be used when assigning a macro to a button). Like the progbutton statement for a built-in feature, the progbutton statement for a macro consists of the Bitmap, Descrip, BitFlags and ID parameters (see *.MNU Syntax for more about the position and content of these fields). For a macro, however, the ID parameter, which is the last parameter in the statement, must contain a value in the 5000 to 5999 range. This value notifies Image-Pro that a macro is to be invoked, and specifies the macro's position in the current script file, as follows. An ID of... 5000 5001 5002 and so forth. identifies the first macro in the selected script file, identifies the second macro in the selected script file, identifies the third macro in the selected script file,

The following example shows a progbutton statement for the 4th macro (#5003) in a script file. progbutton REPORT,Launch Report Generator, 0x0000, 5003 Important - a macro is always invoked from the currently selected script file. If you change script files mid-session, a button will invoke the specified macro number from the Page 1-105

Image-Pro Plus Start-Up Guide new script file. To ensure that the macro you intended is always the one invoked by a button, do not change script files during your Image-Pro session The Bitmap field specifies the name of one of the predefined bitmaps that can be used as button icons. Any of the icons listed under Pre-defined Button Bitmaps at the end of this manual can be used. Note: You can also assign a macro to a button on the toolbar through the Toolbar tab in the Edit:Preferences menu. This is described in the Preferences section under the Edit menu later in this manual.

*.MNU Syntax
The *.MNU file contains the description of Image-Pro's main menu and sub-menus, as well as descriptions for the command buttons that appear on the Toolbar. Each statement in *.MNU is made up of a keyword followed by several parameters. Within the *.MNU file, the following rules must be followed: Keywords must appear entirely in lower case (i.e., progitem, not PROGITEM or ProgItem) The first parameter must be separated from its keyword with a space. Parameters must be separated from each other by commas. You may use spaces to indent statements (usually done to improve readability), but you must not use tabs. Note - the # character is the comment character. It can be used to designate a statement that should not be processed. This is usually done to include lines of descriptive information, or to "comment-out" a statement that is to be excluded from the file. The "#" character must appear in the first position of the line.

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Customizing the Command Menus and Buttons The following describes the syntax for each allowed *.MNU statement:
Statement: Description: Parameters:

columnbegin Title, Position

Marks the beginning of a menu description. Title The name of the menu as it will appear on the Image-Pro command bar. The & character precedes the accelerator key (the character that can be used with the Alt key to open the menu). Position A number denoting the position of the menu on the command bar. The first position on the bar is position 0, (not 1). By convention, the File menu occupies position 0, and the Help menu occupies the last position. columnbegin &File

Example:

progitem &Reload,Reload document from disk.,0x6B0F,112 progitem &Close...,Close current document.,0x6B0F,103 progitem &Save...\tCtrl+S,Save current document to disk.,0x6B0F,104 endcolumn

The columnbegin statement in this example marks the beginning of a File menu description.
Statement: Description: Example:

endcolumn
Marks the end of a menu description.
columnbegin &File progitem &Reload,Reload document from disk.,0x6B0F,112 progitem &Close...,Close current document.,0x6B0F,103 progitem &Save...\tCtrl+S,Save current document to disk.,0x6B0F,104 endcolumn

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Image-Pro Plus Start-Up Guide

Statement: Description: Parameters:

progitem Title, Descrip, BitFlags, ID

Defines a menu item (i.e., a command) that resides in Image-Pro's main program. It is also used to assign a user-developed macro to a menu. Title The name of the command as it will appear in the menu. The & character must precede the accelerator key (the character that can be used to select this command once the menu is open). The \t character string can be used to right-align a string of text with the right margin of a menu. Note - if the progitem statement is moved outside of a columnbegin/endcolumn structure, the command itself will be listed on the command bar. Do not include any tab-aligned text in such a statement (i.e., "\t"). Descrip BitFlags The string of text to be displayed in the status bar when this command is highlighted. A number that determines the image class to which the command can be applied, and, when selected, the dialogbox mode that will result. For built-in Image-Pro commands, this value must not be changed! For user-defined macros, refer to the Bit Flag Options at the end of this section. ID A number that identifies the command procedure or macro. For built-in Image-Pro commands, this value must not be changed! For user-defined macros, this parameter must specify a value between 5000 and 5999, which indicates the macro's position in the current script file. Where: 5000 specifies the first macro in the script file, 5001 specifies the second macro, 5002 specifies the third macro and so forth.

Example:

progitem &Save...\tCtrl+S,Save current document to disk.,0x6B0F,104

The example above defines the Save menu item. "&S" designates "S" as the accelerator key. Save current document will be displayed in the status bar when this command is highlighted.
REPORT,Launch Report Generator, 0x0000, 5003

The example above assigns the second macro in the script file to a menu item called REPORT. Launch Report Generator will be displayed in the status bar when this command is highlighted.

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Customizing the Command Menus and Buttons

Statement: Description: Parameters:

item Title, Descrip, DLL, BitFlags, ID

Defines a menu item (i.e., a command) that resides in a DLL. Title The name of the command as it will appear in the menu. The & character must precede the accelerator key (the character key that can be used to select this command when its menu is open). The "\t" character string can be used to right-align a string of text with the right margin of the menu. Note - if the item statement is moved outside of a columnbegin/endcolumn structure, the command itself will be listed on the command bar. Do not include any tab-aligned text in such a statement (i.e., "\t"). Descrip DLL BitFlags The string of text to be displayed in the status bar when this command is highlighted. The name of the DLL in which the command procedure resides. This parameter must not be changed! A number that determines the image class to which the command can be applied, and, when selected, the dialogbox mode that will result. This value must not be changed! A number that identifies the command procedure. This value must not be changed!

ID
Example:

item &Scan...,Scan to new document.,ipscan32,0x1020, 101

This example defines the Scan menu item. Scan will be displayed in the status bar when this command is highlighted.

continued on next page

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Image-Pro Plus Start-Up Guide

Statement: Description: Parameters: Example:

group Title
Defines a menu item that displays a pop-out menu when selected. This statement marks the beginning of a sub-menu definition. Title The name of the menu item (sub-menu) as it will appear in the menu.

group Sequence &Tools item &Toolbar...,Show sequence Toolbar.,ipmotn32, 0x0480, 0 item &Merge Files...,Merge images into current workspace.,ipmotn32,0x6B0F,100 endgroup

The group statement in this example creates a menu item called Rotate, and marks the beginning of the definition for the pop-out submenu, which is presented when the Rotate item is selected. &o designates O as the accelerator key.
Statement: Description: Example:

endgroup
Marks the end of a sub-menu description.
group Sequence &Tools item &Toolbar...,Show sequence Toolbar.,ipmotn32, 0x0480, 0 item &Merge Files...,Merge images into current workspace.,ipmotn32,0x6B0F,100

endgroup
Statement: Description: Example:

separator
Draws a line between menu items.
item Set&up Acquire,Choose acquisition driver.,mccapt32,0x0080,105 separator item &Scan...,Scan to new document.,ipscan32,0x1020,101

The separator statement in this example creates a line between the Save As and Print commands listed in the menu.
Statement: Description:

progbutton Bitmap, Descrip, BitFlags, ID

Creates a button on the Toolbar for a command that resides in ImagePro's main program. It is also used to assign a user-developed macro to a button on the Toolbar. Bitmap The name of one of the pre-defined icons (button bitmaps) supplied with Image-Pro. The icons and their names are listed at the end of this section.

Parameters:

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Customizing the Command Menus and Buttons progbutton (continued)

Descrip BitFlags The string of text to be displayed in the status bar when the cursor touches this button. A number that determines the image class to which this button can be applied, and, when selected, the dialog-box mode that will result. For built-in Image-Pro commands, this value must not be changed! For user-defined macros, refer to the Bit Flag Options at the end of this section. ID A number that identifies the command procedure or macro. For built-in Image-Pro commands, this value must not be changed! For user-defined macros, this parameter must specify a value between 5000 and 5999, which indicates the macro's position in the current script file. Where: 5000 specifies the first macro in the script file, 5001 specifies the second macro, 5002 specifies the third macro and so forth.
Example: progbutton OPEN,Open document from disk.,0x0000, 102

The above example displays the Open icon on the Toolbar. When the cursor touches this icon, the description Open document from disk will be displayed in the status bar. When selected, this button will execute procedure #102, which is the Open command.
progbutton SAVE,Save current document to disk.,0x6B0F, 104

The above example displays the SAVE icon on the Toolbar. When the cursor touches this icon, the description Save Document will be displayed in the status bar. When selected, this button will execute procedure #104, the Save command.
Statement: Description: Parameters:

button Bitmap, Descrip, DLL, BitFlags, ID

Creates a button on the Toolbar for a command that resides in an ImagePro DLL. Bitmap The name of one of the pre-defined icons (button bitmaps) supplied with Image-Pro. The icon names are listed at the end of this section. The string of text to be displayed in the status bar when the cursor touches this button.

Descrip

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Image-Pro Plus Start-Up Guide button (continued)

DLL BitFlags The name of the DLL in which the command procedure resides. This parameter must not be changed! A number that determines the image class to which this button can be applied, and, when selected, the dialog-box mode that will result. This value must not be changed! A number that identifies the command procedure. This value must not be changed!

ID
Example:

button CAMERA,Video/digital capture., mccapt32, 0x0080,100

This example displays the CAMERA icon on the Toolbar. When the cursor touches this icon, the description Video Capture will be displayed in the status bar. When selected, this button will load and execute procedure 100 from CAPT.DLL, which is the Acquire command.
Statement: Description:

% <normal menu item>

The % prefix can be applied to any menu item statement, including any using the comitem, item, macroitem, and progitem keywords. The item will not appear on the Image-Pro menu structure. The % prefix is only useful with items that have the STARTUP flag defined (see the Bit Flag Options topic later in this chapter). In this case, the item will be hidden from the menu, but will be executed exactly once, when the application starts up. This is especially useful for any plug-ins that need to be aware of any images that are loaded (i.e. in case those images contain plug-in specific data). It is also used with comitem to allow IPCOM plug-ins to register new Auto-Pro functions.
%item XYZ Startup,startup,xyz32.dll,0x480,100

Comments:

Example:

This example defines a Startup menu item for the fictional XYZ32 module. The XYZ32 module will be invoked at Image-Pro startup with an ID of -1, and remain loaded for the duration of that Image-Pro session. Note that the ID parameter of the item line is ignored when used with the STARTUP flag.

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Customizing the Command Menus and Buttons

Statement: Description Parameters

combutton Bitmap, Descrip, DLL, BitFlags, Server.Object[.Method] Creates a tool bar button for a command that resides in a COM server. Bitmap Descrip DLL The name of the bitmap to be shown on the tool bar. The tooltip to be displayed when the cursor is over this button. The name of the DLL in which the IPCOM architecture resides. Note: this parameter must always be ipcom32.dll unless a different COM support module is provided. A number that determines the image class to which this button can be applied, and, when selected, the dialog-box mode that will result. See more under Bit Flag Options at the end of this chapter. The name of the COM server that is registered as providing the specified object. The name of the COM object that is providing the plug-in method. An optional specification of the method to use when invoking the particular function. This parameter can be used to support multiple commands in a single COM object.

BitFlags

Server Object [.Method]

Example

combutton HISTO,Histogram.,ipcom32.dll,32,VBPlugin.Task1 This example displays the HISTO icon on the tool bar. When the cursor is over this icon, the description Histogram will be displayed near the cursor. When selected, this button will load and execute the Task1 COM object in the VBPlugin COM server.

See Also:

Comitem

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Image-Pro Plus Start-Up Guide

Statement: Description: Parameters:

comitem Title, Descrip, DLL, BitFlags, Server.Object[.Method] This statement defines a menu item (i.e., a command) that is supported by an object that resides in a COM server. Title The name of the command as it will appear in the menu. The & character indicates an accelerator key . The \t character string can be used to right-align a text string with the right margin of a column. Note - if the comitem statement is moved outside of a columnbegin/endcolumn structure, the command itself will be listed on the main menu. Do not include any tab-aligned text in such a statement (i.e., \t ) Descrip DLL The string of text to be displayed in Image-Pros status bar when this command is highlighted. The name of the DLL in which the IPCOM architecture resides. Note: this parameter must always be ipcom32.dll unless a different COM support module is provided. A number that determines the image class to which this command can be applied, and, when selected, the dialog-box mode that will result. See more under Bit Flag Options at the end of this chapter. The name of the COM server that is registered as providing the specified object. The name of the COM object that is providing the plug-in method. An optional specification of the method to use when invoking the particular function. This parameter can be used to support multiple commands in a single COM object.

BitFlags

Server Object [.Method]

Comments:

The methods of the default interface (like Histo in the example below), are automatically available in IPBasic as Object.Method thus allowing control of them from a macro.

continued on next page

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Customizing the Command Menus and Buttons

Example:

comitem &VB Plugin,VB Plugin.,ipcom32.dll,32,VBPlugin.Task1 This example defines a command VB plugin with an accelerator of V, which when selected will invoke the VB Plug-in COM server and Task1 object. The selection of this menu item by the user will result in the IPCOM architecture calling the Initialize and Invoke methods of the Task1 object. %comitem &Java Plugin,Java Plugin., ipcom32.dll, 0x480, JavaPlugin.Class1 comitem &Histogram, Histogram., ipcom32.dll, 0x80, JavaPlugin.Class1.Histo This example defines a hidden item that will invoke and initialize the COM object at application startup, as well as the command Histogram with an accelerator of H, which when selected will invoke the JavaPlugin COM server and Class1 object. The selection of this menu item by the user will result in the IPCOM architecture calling the Histo method of the Class1 object.

See Also:

% prefix, combutton, helpitem, item, macroitem, progitem

Statement: Description:

endhints The endhints statement marks the end of section defining hints for the MNU files menu and toolbar configuration. It must follow a hintsbegin statement. See the section on Hints Statements for a discussion of menu hints.. hintsbegin type = 2 level = 1 endhints This example defines this menu configuration as a biological application using a Basic (simplified) interface.

Comments:

Example:

See Also:

Hintsbegin, level, type

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Image-Pro Plus Start-Up Guide

Statement: Description: Parameters:

helpitem Title, Descrip, Helpfile, BitFlags, ID This statement defines a help menu item (i.e., a command) accessing a Windows help file that resides in a separate file. Title The name of the command as it will appear in the help menu. The & character indicates an accelerator key. Note: the helpitem statement must not be moved outside of the Help columnbegin/endcolumn structure, and should not include any tab-aligned text in the statement (i.e., \t). Descrip Helpfile BitFlags ID The string of text to be displayed in Image-Pros status bar when this help item is highlighted. The name of a Windows online help file. Ignored: set to 0. 0 = help is a Winhelp file (.hlp) 1 = help is an Acrobat file (.pdf) 2 = displays the Winhelp table of contents (.cnt)

Example:

helpitem &Macro Language,Auto-Pro help., autopro.hlp,0,0 This example defines the Macro Language menu item, which uses the AutoPro.hlp Windows online Auto-Pro help file. hintsbegin The hintsbegin statement marks the beginning of section defining hints for the MNU files menu and toolbar configuration. It can be followed by a type statement and/or a level statement, and must be followed by an endhints statement. See the section on Hints Statements for a discussion of menu hints.. hintsbegin type = 2 level = 1 endhints This example defines this menu configuration as a Biological application using a Basic (simplified) interface.

Statement: Description:

Comments: Example:

See Also:

endhints, level, type

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Customizing the Command Menus and Buttons

Statement: Description: Parameters:

htmlhelpitem Title, Descrip, Helpfile, BitFlags, ID This statement defines a help menu item (i.e., a command) accessing a Windows help file that resides in a separate file. Title The name of the command as it will appear in the help menu. The & character indicates an accelerator key. Note: the htmlhelpitem statement must not be moved outside of the Help columnbegin/endcolumn structure, and should not include any tab-aligned text in the statement (i.e., \t). Descrip Helpfile The string of text to be displayed in Image-Pros status bar when this help item is highlighted. The name of a Windows HTML help file (typically a .CHM file). The file name can be qualified with the desired page of the help file see the Comments section for details Ignored: set to 0. 0 = displays the help index 1 = displays the help table of contents

BitFlags ID
Example:

htmlhelpitem &Macro Language,Auto-Pro help., AutoPro5.chm,0,0 This example defines the Macro Language menu item, which uses the AutoPro5.chm Auto-Pro HTML help file. htmlhelpitem &Solutions Zone, Display Solutions Zone, help\Support5.chm::Solutions_Zone_.htm, 0, 0 This example defines the Solutions Zone menu item, which goes directly to the specified page of the technical support help file.

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Image-Pro Plus Start-Up Guide

Statement: Description: Comments:

level The level statement indicates the level of complexity desired from the application features. See the section on Hints Statements for a discussion of menu hints. The levels defined at this time are 0 (zero) for advanced and 1 for basic. See the hintsbegin statement for an example. endhints, hintsbegin, type macrobutton Bitmap, Descrip, Script, BitFlags, Macro Creates a button on the tool bar for a macro. Bitmap Descrip Script BitFlags The name of the bitmap to be shown on the tool bar The tooltip to be displayed when the cursor is over this button. The name of the script file in which the macro resides. A number that determines the image class to which this button can be applied, and, when selected, the dialog-box mode that will result. See more under Bit Flag Options at the end of this chapter. The name of the macro to execute.

See Also:

Statement Description: Parameters:

Macro
Example:

macrobutton USER1, Macro: Histogram, C:\IPWIN5\DEFAULT.IPM , 0, Histogram The example above displays the USER1 icon on the tool bar. When the cursor is over this icon, the description Macro: Histogram will be displayed near the cursor. When selected, this button will execute the Histogram macro from the script file c:\ipwin5\default.ipm

See Also:

button, combutton, macroitem, progbutton

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Customizing the Command Menus and Buttons

Statement: Description: Comments:

type The type statement indicates the type of the application configuration. See the section on Hints Statements for a discussion of menu hints. The types defined at this time are 0 (zero) for general-purpose applications, 1 for industrial applications, and 2 for biological applications. See the hintsbegin statement for an example. endhints, hintsbegin, type

See Also:

Statement: Description: Parameters:

macroitem Title, Descrip, Script, BitFlags, Macro Creates an item in the menu for a macro. Title The name of the command as it will appear in the menu. The & character indicates an accelerator key. The \t character string can be used to right-align a string of text with the right margin of a column. Descrip Script BitFlags The text string to be displayed in the status bar when this command is highlighted. The name of the script file in which the macro resides. A number that determines the image class to which this button can be applied, and, when selected, the dialog-box mode that will result. See more under Bit Flag Options at the end of this chapter. The name of the macro to execute.

Macro
Example:

macroitem &Histogram, Macro:Histogram The example above defines the Histogram menu item. &H designates H as the accelerator key. Macro:Histogram will appear in the status bar when this command is selected. When selected, this command will execute the Histogram macro from the script file c:\ipwin5\default.ipm

See Also:

comitem, macrobutton, progitem, item

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Image-Pro Plus Start-Up Guide

Bit Flag Options

The Bit Flag parameter is used by the item, comitem, progitem, macroitem button, combutton, macrobutton, and progbutton statements. It determines the class of image that must be active for the menu item to be enabled, and the mode in which a plug-ins dialog box (if there is one) will be opened. If the bit flag for a particular class is disabled (i.e., not on), the command will be disabled when an image of that class is active. The table below describes the basic Bit Flag parameter values and their meanings. The flags can be combined by simply adding their values (e.g., a value of 6 would denote Gray Scale (4) and Palette (2) images 4 + 2 = 6). The Bit Flag parameter can be specified in binary, decimal, or hexadecimal. It must use the appropriate prefix for the method selected. If you were to set all possible image class flags, the representation would be as follows: Binary format (0b prefix): 0b0110101100001111 Hexadecimal format(0x prefix): 0x06B0F Decimal format (no prefix): 27407 VALUE
2

HEX
0x0002

DESCRIPTION
GRAY SCALE specifies that the command can be invoked when a Gray Scale image is active. This value turns on the GRAY flag in bit position 1. PALETTE specifies that the command can be invoked when a Palette image is active. This value turns on the PALETTE flag in bit position 2. RGB specifies that the command can be invoked when a True Color (RGB 24) image is active. This value turns on the RGB flag in bit position 3. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use.

0x0004

0x0008

16 32 64

0x0010 0x0020 0x0040

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Customizing the Command Menus and Buttons VALUE

128

HEX
0x0080

DESCRIPTION
MODELESS specifies that a DLL procedure invokes a modeless dialog. This value turns on the MODELESS flag in bit 7. If you set the MODELESS flag, hVriInst in the Interface Function will be Null. This flag is used only by the button and item statements.

256

0x0100

GRAY12 specifies that the command can be invoked when a Gray Scale 12 image is active. This value turns on the GRAY12 flag in bit position 8. FLOAT specifies that the command can be invoked when a Floating Point image is active. This value turns on the FLOAT flag in bit position 9. STARTUP specifies that the DLL is to be invoked automatically when the application is loaded. This value turns on the STARTUP flag in bit 10. At start-up, the application calls the InterfaceFunction of the plug-in with Id = -1 Note: This flag is used only by the button, combutton, comitem, and item statements.

512

0x0200

1024

0x0400

2048

0x0800

GRAY16 specifies that the command can be invoked when a Gray Scale 16 image is active. This value turns on the GRAY16 flag in bit position 11. Reserved. Do not use. RGB36 specifies that the command can be invoked when a 36-bit True Color image is active. This value turns on the RGB36 flag in bit position 13. RGB48 specifies that the command can be invoked when a 48-bit True Color image is active. This value turns on the RGB48 flag in bit position 14. Reserved. Do not use.

4096 8192

0x1000 0x2000

16,384

0x4000

32,768

0x8000

65,536

0x00010000

Multi-frame specifies that the command can only be invoked when the active workspace contains multiple image frames. continued on the next page

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Image-Pro Plus Start-Up Guide VALUE (Use Hex values!) HEX 0x00020000 0x00040000 DESCRIPTION Multi-workspace specifies that the command can only be invoked when there are multiple image workspaces open. Multi-frame or workspace specifies that the command can only be invoked when there are multiple frames available, either from multiple frames in the active workspace, or from multiple image workspaces. Disallow on FFT specifies that the command cannot be invoked when the active workspace is an FFT workspace. Only when IQbase installed specifies that the command can only be invoked if the Media Cybernetics integration with IQbase is installed to the application. Hide the menu entry if the plug-in is not installed. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use. Reserved. Do not use.

0x00080000 0x00100000

0x00200000 0x00400000 0x00800000 0x01000000 0x02000000 0x04000000 0x08000000 0x10000000 0x20000000 0x40000000 0x80000000

If none of the image class flags (GRAY, PALETTE, RGB, GRAY12, GRAY16, FLOAT, RGB 36 or RGB 48) are set, your macro will never be dimmed, even no image is active. Therefore, to make a macro available to all image classes, set its Bit Flags parameter to 0. If any of the image class flags (GRAY, PALETTE, RGB, GRAY12, GRAY16, FLOAT, RGB 36 or RGB 48) are set (i.e., turned on), your macro will be available only if the active image is of one of the enabled types. Otherwise it will be dimmed. For example, if a macro were valid for Gray Scale and True Color images, its Bit Flags would be set to 10 (i.e., 2 + 8). Page 1-122

Customizing the Command Menus and Buttons

Pre-defined Button Bitmaps

The following table describes Image-Pro's pre-defined buttons and their bitmap names (required in the Bitmap parameter of the button and progbutton statements). It also shows the command to which each button is assigned in the *.MNU file delivered with Image-Pro (unused icons are denoted with a dash). These button assignments can be changed by editing the *.MNU file. See Customizing Command Buttons for procedures.
ICON BITMAP NAME ANNOTATE BACKSUB BESTLUT CAMERA CLOCK CLUB DEFAULT COMMAND ASSIGNMENT

The Show Annotation Toolbar command The Background Operations command The Best Fit Equalization command The Video/Digital Capture command The Contrast Enhancement command The Count and Measure Objects command The 3D Viewer command

CONTRAST
COUNT

3D VIEWER

DARK MODE DATABASE

DATA DIAM DRAW

The Dark Mode command The Image Database command The Annotate command Page 1-123

Image-Pro Plus Start-Up Guide continued on next page

ICON BITMAP NAME FFT FILTER HAND HAPPY HEART HISTO INFO IRREG MACRO MACREC ASSIGNED COMMAND

The FFT command The Spatial Filtering command The Pan Image tool The Histogram command The Info command The Freeform AOI command The Macro command The Record Macro command The Edit Macro command The Send Mail command The Measurements command The Open command The Arithmetic and Logical Operations command The Print command The Line Profile command continued on next page

MACEDIT MAIL
MEASURE OPEN OPER

PRINT PROFILE

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Customizing the Command Menus and Buttons

ICON BITMAP NAME PSEUDO ASSIGNED COMMAND

The Colorize Gray Scale Image command The Report Generator command The Resize command The Reset Contrast Table command The Save command The Perform Segmentation command The Spatial Calibration command The Scan command Sync Scroll, Pan, and Zoom commands The Snap command The Undo command First user-defined macro Second user-defined macro Third user-defined macro Page 1-125

REPORT
RESIZE RESET SAD SAVE

SEGMENT
SCAL SCAN SPAD SUM

SYNC SNAP
TRASH

UNDO
USER1 USER2 USER3

Image-Pro Plus Start-Up Guide

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The Image-Pro Plus Application Window

Getting Started
The Image-Pro Plus Application Window
When Image-Pro Plus is loaded, its application window becomes active. The application window consists of the Menu Bar, the Tool Bar, the Image Window(s), the Application Area, and the Status Bar. The following diagram introduces you to these and other features of the Image-Pro Plus application window.
Menu Bar

Tool Bar Image Window

Application Area

Menu Bar: The menu bar contains the Image-Pro Plus commands selected from within the menus listed on this bar. As with most applications, you must identify the object of your command before selecting the command itself (i.e., make sure to select the image data the command is to operate upon before selecting the command). Page 2-1

Status Bar

Image-Pro Plus Start-Up Guide Tool Bar: The tool bar is the button bar along the upper edge of the screen. This portion of the application window contains the basic intensity controls and imaging tools for daily Image-Pro Plus operations. The tool bar includes the following commonly used tools: BCG controls AOI tools Zoom & Pan tools.

As the cursor is moved over the buttons, yellow tooltips appear providing a brief description of the button. If no tooltip appears, the button tool/function does not apply for that image. This Tutorial addresses 34 buttons in the tool bar. A description of these buttons and their uses appears below: The following tools are located on the toolbar immediately below the menu bar. If no toolbar is being displayed below the menu bar, you may show it by invoking the Hide/Show Tools command on the Window menu or pressing <F5>. If the menu bar and title bar are not being displayed, you may invoke them by pressing <F4>.

When you position the mouse cursor over one of the icons, a brief description of the command appears.
ICON BITMAP NAME COMMAND

OPEN SAVE DATABASE

CAMERA SCAN PRINT

The File : Open command The File : Save command The Image Database command The Acquire command The File : Scan command The File : Print command The Report Generator command

REPORT

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The Image-Pro Plus Application Window

ICON BITMAP NAME COMMAND

MAIL UNDO NEW AOI RECTANGULAR AOI TOOL ELLIPTICAL AOI TOOL FREEFORM AOI TOOL MULTIPLE AOI TOOL
DRAW

The Electronic Mail command The Edit: Undo command The create New Area of Interest (AOI) Command The create Rectangular Area of Interest (AOI) Command The create Round (or Oval) Area of Interest (AOI) Command The create Irregular Area of Interest (AOI) Command The create Multiple Area of Interest (AOI) Command The Edit: Annotate command The Zoom (Enlarge/Reduce) command The Pan (Move Image) command The Synchronize Zoom/Pan/Scroll command The Enhance: Contrast Enhancement command The Enhance: Best Fit Equalization command The Enhance: Reset Contrast Table command The Process: Background Correction command

ZOOM PAN SYNC

CONTRAST

BESTLUT

RESET

BACKSUB

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Image-Pro Plus Start-Up Guide

ICON BITMAP NAME FFT FILTER OPER SEGMENT PSEUDO COMMAND

The Process: FFT command The Process: Filtering command The Process: Operations command The Process: Segmentation command The Process: Pseudo-Color command The Measure : Spatial Calibration command The Measure: Count/Size command The Measure: Measurements command The Measure:Snap command The Measure: Histogram command The Measure: Line Profile command Open the 3D Viewer command

SPATIAL

COUNT MEASURE

SNAP
HISTO PROFILE

3D VIEWER

DARK MODE
MACRO MACREC

Change to dark mode The Macro command The Record Macro command The Edit Macro command

MACEDIT

Page 2-4

The Image-Pro Plus Application Window

The Contrast Enhancement Tool: The Contrast Enhancement tool shows or hides the Contrast Enhancement control panel. This panel lets you easily adjust the Brightness, Contrast, and Gamma (BCG) attributes of the active image. There is an independent slider control panel for each characteristic. The slider button changes color (from red to blue) when the specific attribute is selected. Use the Brightness control to modify the overall amount of light in your image. Use the Contrast control to change the degree of difference between the brightest and darkest components in your image. Use the Gamma control to enhance the contrast in the very dark or very light areas in your image without significantly affecting the contrast in the midtone regions. Moving the Slider ( ) button up the scale increases the attribute. Moving it down the scale reduces the attribute. For single incremental changes, press the up () or down () arrow keys on the keypad.

Page 2-5

Image-Pro Plus Start-Up Guide

These four buttons are the Color Channel buttons. Select the Luminance, Red, Blue, or Green channel button corresponding to the color in your image. If your image is not in True Color, the Luminance channel will be your only option here. The Luminance channel is the intensity produced by the combined RGB channels. Click on the color button, and move the slider controls until the new color values are reflected in your image. Use this button to permanently apply the current contrast enhancement adjustments to the active image. Use this button to reset the BCG sliders to the default values. AOI Tools: The Area of Interest (AOI) tools define a portion of your image. Many of Image-Pro Plus commands can be constrained by defining an AOI. When such commands are executed, their effects are applied to just the pixels within the AOI, leaving the pixels outside the AOI unaffected. There are several AOI tools which can determine the size or shape of the AOI as follows:
ICON TOOL New AOI Rectangular AOI Tool Elliptical AOI Tool Freeform AOI Tool FUNCTION

Use this tool to create a new AOI. Use this tool to create and/or activate a square or rectangular AOI in the image. Use this tool to create and/or activate a circular or elliptical AOI in the image. Use this tool to create and/or activate any irregular AOI in the image. You can use the AutoTrace feature of the Freeform AOI tool to outline the edge of an object. Use this tool to create and work with multiple AOIs.

Multiple AOI Tool

Page 2-6

The Image-Pro Plus Application Window Zoom & Pan Tools: Uses for the Zoom and Pan tools are described in the following table:
ICON TOOL Zoom FUNCTION

Pan

Sync

Use this tool to magnify or reduce the appearance of the image in the window. Pressing the right mouse button accesses the Zoom sub-menu containing other zoom options. The Pan tool is used to position an image that does not entirely fit within the image window. It is an alternative to using the arrows on the scroll bars for accessing image data that falls outside the image window. The Sync Zoom/Pan/Scroll tool is used to move several image workspaces at once.

Image Windows: Images are maintained in separate image windows within the application area. An image window contains a single image. You Active image may work with several open images at window the same time. When you do so, Image-Pro Plus will perform a selected operation upon the active window. The active window is the frontmost image in the application area, and its title bar is highlighted, as shown in the diagram on the left.

Page 2-7

Image-Pro Plus Start-Up Guide Application Area: The application area is the workspace in the desktop where Image-Pro Plus performs its functions. This is where the image windows are displayed, where the dialog boxes appear, and where you find the graphs and tables produced by Image-Pro Plus. The Image-Pro Plus workspace is enclosed by the menubar and toolbar at the top edge, and by the status bar at the bottom. (For more information about the menu bar and toolbar components, please refer to the Image-Pro Plus Reference Guide.) Status Bar: The status bar is the bar along the bottom edge of the screen. It displays information about the image open on the screen, such as calibration and spatial resolution. It also conveys messages about the current Image-Pro Plus operation and indicates the memory available on your system. Use the Set Preferences dialog to control what information appears in the status bar (see the Edit menu section in the Image-Pro Plus Reference Guide).
Current Position Apply to Entire Sequence 202, 391 Apply to Active Frame Message Box Pixel Value AOI Dimension 151 251,193,328,27 Image Extent (w/h) Calib. Spatial Units Available Memory

Calib. Intensity Units

The Status Bar provides general-purpose information about the open image: Message Box describes a selected command or menu and acts as a progress indicator for lengthy operations. Current Position gives the location of the cursor in pixels. Pixel Value displays the intensity value of the pixel underneath the cursor. AOI Extent displays the location of the AOI in terms of four pixel locations. AOI Dimension gives the width and height of the AOI in pixels. Intensity Units indicates the intensity calibration units. Spatial Units indicates the spatial calibratio units. Available Memory indicates how much memory is available for Image-Pro operations.

Page 2-8

The Image-Pro Plus Application Window The Apply to Active Frame and Apply to Sequence buttons appear at the extreme right of the status bar. These buttons can be used to apply a specific operation to the frame currently in the application area, or to the active portion of a sequence of images. When an operation is in progress, the message bar will display a tracking bar, and the sequence buttons will be replaced with a Cancel button. To cancel the operation, click the Cancel button or press <Esc>. (Please refer to the description of the Sequence Toolbar later in this manual.) Image-Pro Plus v. 6.0 allows you to enable or disable the status bar fields by right clicking on the status bar. You will see a pop-up menu of the indicator fields:

Click the cursor next to the name of the field you wish to enable. Enabled fields are checked. To resize any of the fields in the status bar, place your cursor over one of the dividers between the fields. The cursor will change to a double-headed arrow. Click and drag the cursor left to make the field smaller, or to the right to enlarge the field.

Page 2-9

Image-Pro Plus Start-Up Guide Dialog Boxes: Image-Pro Plus provides many of its image processing features using dialog boxes which display data for, or apply commands to, any open image window. The FFT and Filter windows on the left are examples of common dialog boxes. They reflect the data or choices available, and/or apply their effects to whatever image is active. Many boxes of this type have additional options available with tabs (as shown in the Filter tabbed dialog box).

Page 2-10

The Image-Pro Plus Application Window

Image-Pro Plus Demonstration Macros

Image-Pro Plus version 6.0 contains a large number of macro scripts that include both demonstration and utility macros. The demonstration macros can be used to view highlighted features of Image-Pro, and the utility macros can be used to help automate your tasks. Many of the demonstration macros are contained in the main Demo script (the Demo.ipm script in the Scripts folder of Image-Pro Plus) that is loaded by the Load Demo Macros command on the Macro menu of Image-Pro Plus. However, Image-Pro Plus version 6.0 includes a new feature to help you explore these and other macros provided with Image-Pro, the Macro Player. The Macro Player will be displayed the first time you run Image-Pro Plus version 6.0, illustrated below:

The Macro Player lists all of the demonstration macros and most of the utility macros that are provided as a part of Image-Pro Plus 6.0. You can choose whether to display the Player on startup using the Show on Startup check box on the player. As you select each macro in the list, you will see a description of the macro in the Description box. You can select one or several macros and run them interactively or in free-running mode. We suggest that you explore the demonstration macros, which you can use to get a overview of many of the interesting features of Image-Pro Plus version 6.0. You can also view a web page illustrating and describing all of the macros by clicking the Show Overview button. A full description of the Macro Player can be found in the online help and the printed Reference Guide.

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Image-Pro Plus Start-Up Guide If you are running the demo macros for the first time, you will need to select the Load Demo Macros command from the Macro menu on the Image-Pro Plus menu bar. The macros will then be listed at the bottom of the Macro menu in the following order: Separate_Fluorochromes: This demo macro demonstrates separating a 24bit color image into its Red, Green, and Blue channels. After enhancing the blue channel, the individual channels will be re-combined to a 24-bit color image. CountSmall_Cells: This is a demo macro showing how to count the number of cells in a sample. MeasureStain: Using an image of a tissue section, this macro demonstrates using the Count/Size tools to select ovjects of interest based on color. MeasureAngles: Using an x-ray film image, this demo macro calculates the angle of the leg by first processing the image, defining areas of interest (AOIs), and then measuring angle using Count/Size. CountAndClassify: This macro demonstrates one possible way to count the number of holes (pores) in an object (or objects), and then determines the percent area of the holes compared to the objects of interest. MeasureIronPhases: This demo macro first locates and counts individual phases of a multiple phase material, then demonstrates how to segment individual phases and then measure them using the Count/Size tool. ThroughfocusSequence: This demo macro opens a sequence of images using the Sequencer tool and applies a pseudocolor to the sequence. For additional details, the macro opens the histogram of the image sequence to view the histogram from frame to frame. MeasureAutoradiograph: This macro demonstrates creating an intensity calibration curve and then deriving measurements from selected areas of an autoradiograph.

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The Image-Pro Plus Application Window SprocketCountDemo: This demo macro creates a mask image, and then uses such filters as Thinning and Branch/End-Point to locate endpoints and uses Count/Size to count the number of endpoints. Barcode: This demo macro shows how to locate edges and derive useful measurements based upon the location of the edges and the distances between them. Watermark: This macro applies a watermark to your images. Additional demonstration macros may appear in the list provided with your copy of Image-Pro Plus 6.0.

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Image-Pro Plus Start-Up Guide

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Learning the Basics

Learning the Basics

The Tutorial in this section is designed to introduce you to the basic image manipulation tools within Image-Pro Plus. In this first exercise you will: Load an image and create a working copy. Adjust the intensity with the Brightness, Contrast, and Gamma (BCG) controls. Enhance the working image with various filters. Cut and resize the working image for comparison with the original image. Print and save the final image. This exercise will take about 25 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now by

double-clicking on the Image-Pro Plus icon within the Image-Pro Plus folder. Once Image-Pro Plus is running, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

Loading the Image

In this step, you will open the first image, which is a captured image from a surveillance camera in a bank.. 1. Select the Open... command from the File menu. The Open File dialog box appears, displaying the Images folder.

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Loading the Image 2. Open the Bankvid.tif file in the Images folder. TIFF in the Files of type field indicates that this image is in Tagged Image File Format. Image-Pro Plus opens the Bankvid.tif image in its own image window.

This is an image from a bank surveillance camera, with an unclear image of a crime suspect. In this exercise, you will enhance this image to produce a clearer image of the suspect in the foreground.

Continue to the next set of steps in this exercise: Copying and Pasting.

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Image-Pro Plus Start-Up Guide

Copying and Pasting

In this step you will make a working copy of the original image. Any further changes you make will only apply to this working copy of the image, leaving the original image unaltered. 1. Select the Copy command from the Edit menu. The image is copied to the Clipboard. Select the Paste New command from the Edit menu. The Clipboard data appears in its own window Untitled1. Use this new image as the working copy.

2.

Continue to the next set of steps in this exercise: Enhancing the Image.

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Enhancing the Image

Enhancing the Image

In this step you will enhance the working image Untitled1 using the Brightness, Contrast, and Gamma (BCG) controls on the Contrast Enhancement panel to improve the visual effect and extract unique features currently hidden from view. In this case, you will clean up the image to distinguish the suspect in the foreground. Before beginning this exercise, display the Contrast Enhancement panel on the screen by clicking the Contrast Enhancement button on the tool bar at the top of the screen.

Contrast Enhancement button Move this panel to any convenient position on the screen by dragging its title bar. In the following steps, use the slider controls on this panel to adjust the visual characteristics of this image.

1.

Increase the Brightness value. Because the overall image is somewhat dark, lighten it using the Brightness control. Place the cursor on the Slider ( control:

) of the Brightness

Brightness control Drag the Brightness slider up, until the indicator beneath it reads 53, or press the up arrow key () on the keypad to increase the value by single increments. This increases the brightness of the image, by lightening it.

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Image-Pro Plus Start-Up Guide

2.

Increase the Contrast value. To better distinguish the suspect in the foreground from the background, heighten the contrast in the image. Drag the Slider ( ) of the Contrast control:

Contrast control until the indicator beneath it reads 69. This increases the intensity differences between the suspect and the background in the image.

3.

Increase the Gamma value. To bring out the variations in the dark foreground, increase the Gamma value. High gamma values increase contrast in dark areas of an image. Low gamma values increase contrast in the lighter areas of an image. Drag the Slider ( ) of the Gamma control:

Gamma control until the indicator beneath it reads 1.2. All the image elements, the suspect in the foreground and the background, should now be distinguishable in the image.

Note: Since this image is not true color, the Luminance channel (selected left button) is the only option for the color channel, which is represented by this four-button panel. The Luminance channel is the intensity produced by the combined RGB channels. Page 3-6

Enhancing the Image

4.

Apply the Brightness, Contrast, and Gamma Control changes. The BCG intensity adjustments performed in the preceding steps were not permanently applied to the image. Instead, they were recorded into a Lookup Table (LUT) through which the original image is interpreted. To permanently modify the image, the LUT must be applied to the image. Click Apply and the Apply Contrast dialog box appears.

5.

Click OK. Clicking OK uses the BCG settings to permanently modify your image bitmap. Notice that after clicking OK, all three sliders return to their initial, unaltered positions.

Continue to the next set of steps in this exercise: Working with Filters.

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Image-Pro Plus Start-Up Guide

Working with Filters

In this step you will further enhance the working image by using the Sharpen filter to clarify the suspects face. 1. Select the Filters option from the Process menu. Image-Pro Plus opens the Filters tabbed dialog box.

2.

Click on the Sharpen filter radio button from the Enhancement tab label. Increase the number of passes to 2 in the Passes edit box. This demonstrates how multiple applications of filters can be combined. Click Apply. Image-Pro Plus applies the selected filter to the working image.

3.

4.

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Enhancing the Image

Defining an AOI
In this step you will define an Area of Interest (AOI) around the suspect and cut this portion from the working image. The cut portion will be used for later comparison to the original image. 1. Select the Rectangular AOI tool. Click the Rectangular tool located on the Tool Bar along the top of the screen. Notice that the Rectangular button illuminates, indicating that it is the activated tool. Move the cursor to the Untitled1 image window. The cursor appears as a right-angle.

2.

Place this cursor above the left side of the suspects head in the image, as shown in the following example.

Position the cursor as shown here

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Image-Pro Plus Start-Up Guide

3.

Define the Area of Interest (AOI). Then, press the mouse button and drag the cursor past the lowerright corner of the suspects head.

Drag and stretch the AOI box down and to the right

When the cursor is below the right-hand corner of suspects head, release the mouse button. The AOI rectangle you have just defined will remain in the image.

Definition...

Area of Interest (AOI) An AOI is an area that is isolated from the rest of the image. It can be any shape you choose: a rectangle, an ellipse, or a freeform polygon. Most, but not all, of Image-Pro Plus commands can be constrained by an AOI. The command applies to only the pixels within the AOI, when one is defined. Examples of commands that are constrained by an AOI are: Save As, Print, Filter, and Rotate. These commands will perform the requested operation using just the data within the AOI. A few operations affect the entire image regardless of whether an AOI is active or not. Examples of functions not constrained by an AOI are: Color Segmentation and Save.

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Enhancing the Image 4. Enlarge the AOI by selecting the Resize command from the Edit menu. The Resize Image dialog box opens.

5.

Increase the Width percentage value to 300%. Notice as the Width value changes, the Height value changes too. This occurs because the Allow Distortion checkbox is turned off, which forces the image to scale proportionally. Click OK. An enlarged image of the AOI appears as Untitled2.

6.

Move the Untitled2 window over to the right side of the application area to view the original image Bankvid.tif.

Continue to the next set of steps in this exercise: Zooming in on an Image.

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Image-Pro Plus Start-Up Guide

Zooming in on the Image

In this step you will magnify the original image (Bankvid.tif) of the suspect to compare against the enhanced image of the suspect (Untitled2). 1. Select the Zoom tool. Click the Zoom tool located on the Tool Bar along the top edge of the screen. Notice that the Zoom button illuminates, indicating that it is the active tool, and the Rectangular AOI tool is no longer active. Note: You can also position the cursor anywhere on the image and click the right mouse button. Select Zoom In from the pop-up menu. 2. Zoom-in on the Bankvid.tif image. Move the cursor to the Bankvid.tif image window. Note that the cursor appears as a magnifying glass once it is within the image. Place the magnifying glass over the suspect, then click the left mouse button once to magnify the image 2X. This action zooms-in on the region. The Bankvid.tif image window appears like this:

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Enhancing the Image

3.

Place the original image (Bankvid.tif) and the modified image (Untitled2) side by side to compare the two images. Note that the facial features of the suspect are easier to see in the enhanced and cleaned up image of Untitled2.

Continue to the next set of steps in this exercise: Printing the Image.

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Image-Pro Plus Start-Up Guide

Printing the Image

In this step you will print the working image (Untitled2) in centered form. 1. Select the Untitled2 image to print. The Untitled2 image is now active. Select the Print command from the File menu. The Print dialog box appears.

2.

3. 4.

Make sure the Fit to Page box is unchecked. Select the Position button. The Print Position dialog box appears.

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Enhancing the Image 5. Increase the Width percentage value to 200%. Notice as the Width value changes, the Height value changes too. The image thumbnail in the box on the left correspondingly increases in size. Click the Center button in the Position group box. The image thumbnail is now centered on the page.

6.

7.

Click OK. The Print dialog box reappears. Click Print. The image is sent to the printer.

8.

Continue to the next set of steps in this exercise: Saving the Image.

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Image-Pro Plus Start-Up Guide

Saving the Image

In this step you will save the working image Untitled2 to a new file, in a new file format. 1. Select the Save As... command from the File menu. The Save File As dialog box appears.

2.

Select the Windows Bitmap (BMP) option from the Save as type list box. Type: Suspect.bmp in the File name box. Click Save. Image-Pro Plus saves the modified image to the Suspect.bmp file in Windows bitmap format.

3. 4.

Continue to the next set of steps in this exercise: Closing the Image Windows and Exiting Image-Pro Plus.

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Closing the Image Windows and Exiting Image-Pro Plus

Closing the Image Windows and Exiting Image-Pro Plus

In this step you will close the Bankvid.tif, Untitled1, and Suspect.bmp image windows and exit out of Image-Pro Plus.

1.

Close Image-Pro Plus by selecting the Exit command from the File menu. You can also double-click each image windows close box.

2.

Do not save any changes. Click on the No All button when the dialog box appears asking the question Save changes to untitled1? This will close the Image-Pro Plus application.

Continue to the next exercise: Filtering, Calibrating, and Measuring.

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Image-Pro Plus Start-Up Guide

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Filtering, Calibrating, and Measuring

Filtering, Calibrating, and Measuring

The Tutorial in this section is designed to introduce you to tools used for filtering and measuring. In this lesson you will: Load an image from the Image-Pro Plus Database. Use a sharpening filter to accentuate object edges in an image. Calibrate the spatial scale to measure in microns. Measure image objects using the manual and auto-trace measurement tools. Save the measurements to an ASCII file. This exercise will take about 25 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now by

double-clicking on the Image-Pro Plus icon within the Image-Pro Plus folder. Once Image-Pro Plus is running, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 now retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

Loading an Image from the Database

.

1.

Open the image Circuit.tif from the Image-Pro Plus sample images folder, as you did in the previous exercise. The Circuit.tif image window is opened in the ImagePro Plus application window. This is an image of an etched printed circuit board (PCB).

Continue to the next set of steps in this exercise: Sharpening the Image.

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Filtering, Calibrating, and Measuring

Sharpening the Image

In this exercise you will use the Sharpen filter to accentuate the edges in the Circuit.tif image. Because the circuit board traces in this image will be measured, you will want them to have clean and distinct edges. Sharpening will accentuate the places where a trace ends and the board begins.

1.

Select the Filters... command from the Process menu. The Filter dialog box appears.

2.

Click the Sharpen radio button under the Enhancement tab label.

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Image-Pro Plus Start-Up Guide 3. Set the Strength value in the Options group box to the value of 8. This applies the sharpen filter at approximately 80% of its maximum strength, which will result in a significant, but not extreme, application of the filter. Note: When you begin typing into the Strength field, two special data buttons appear:

These buttons are used by Image-Pro Plus to enter or clear the data that is typed into a field. Clicking the Checkmark ( 4) button enters the data into the field (pressing the <Return> key will do the same thing). A limits check is performed at this time, if appropriate. If the data is not within the allowed range, the Checkmark and X buttons will not be cleared. Clicking the X button clears the contents of the field and returns the value to the original unit measurements. If the Passes option is not set at the default value of 1, set it to 1 before continuing to the next step.

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Filtering, Calibrating, and Measuring

4.

Click in the Sample window to preview the results.

Click here to preview.

Dragging in the Sample window will apply the filter to different portions of the image. 5. Click Apply. The Circuit.tif image is sharpened. Edges and details within the image are now more pronounced.

6.

Close the Filter dialog box.

Continue to the next set of steps in this exercise: Calibrating the Spatial Scale.

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Image-Pro Plus Start-Up Guide

Calibrating the Spatial Scale

This set of steps establishes the unit-of-measure associated with this image. By default, Image-Pro Plus expresses all spatial measurements in terms of pixels. You will change the spatial scale to make measurements in terms of microns. This particular image is marked with a Calibration bar identifying the length of 1 micron. This bar will be used to specify the length of 1 micrometer to Image-Pro Plus. You will use the Zoom tool to facilitate a precise measurement. 1. Select the Zoom tool from the tool bar. The cursor appears as a magnifying glass.

2.

Place the Zoom cursor (the magnifying glass) over the Calibration bar in the lower right corner.

the Calibration bar

3.

Click the left mouse button. The Circuit.tif image is magnified to 200% of its original size. The status bar shows the amount of magnification.

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Filtering, Calibrating, and Measuring

4.

Place the Zoom cursor over the Calibration bar, and click the left mouse button, again for the second time. The Circuit.tif image is magnified to 400% of its original size.

the Calibration bar

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Image-Pro Plus Start-Up Guide 5. From the Measure menu, select the Calibration submenu, and then select the Spatial... option. The Spatial Calibration - circuit.tif window appears.

6.

Click the New button. The calibration fields become active, and the Spatial Cal 0 name appears in the Name field. The Name field identifies the set of calibration values you are about to create. Calibration settings can be saved so they can be recalled when they are needed again.

7.

Delete the contents of the Name field and type: circuit x5000 This descriptive name is assigned to the set of calibrations you are about to create.

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Filtering, Calibrating, and Measuring

8.

Delete the contents of the Unit field and type: microns This instructs Image-Pro Plus to label your measurements as microns whenever a spatial measurement is reported (e.g., in a measurement data sheet or a histogram). You can also select the unit of measurement by scrolling through the Unit list box to view the available choices.

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Image-Pro Plus Start-Up Guide 9. Click the Image button in the Pixels/Unit group box. The Scaling dialog box appears, and a defining line is placed in the image. Make sure that the reference represents 1 unit (1 micron).

The Scaling dialog box appears, and a defining line is placed in the image.

the Defining line

the Calibration bar

The Image button allows you to define your spatial calibration unit from a known standard-of-measure in the image. In this case, the white mark (the bar) in Circuit.tif is 1 micron in length. In the next few steps, you will calibrate the defining line to the length of the bar. 10. Click the Zoom tool to turn Zoom off.

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Filtering, Calibrating, and Measuring 11. Place the cursor over the rightmost endpoint of the defining line. The cursor changes to the crosshairs. 12. Match the length of the defining line to the length of the Calibration bar. Click and hold the left mouse button to drag the endpoints of the defining line. Reposition the endpoints until the line is straight and exactly the same length of the Calibration bar, as shown in the following example:

13.

Click OK in the Scaling dialog box. The Spatial Calibration - circuit.tif window returns. The Pixels/Unit fields should read approximately 20, indicating that 20 pixels will represent 1 micron. Image-Pro Plus will report all spatial measurements in these terms (i.e., a measurement of 10 pixels will be expressed as .5 microns, a measurement of 40 pixels will be expressed as 2.0 microns, and so forth). Click OK in the Spatial Calibration - circuit.tif dialog box. The Spatial Calibration window closes.

14.

Continue to the next set of steps in this exercise: Measuring Length. Page- 4-11

Image-Pro Plus Start-Up Guide

Measuring Length
In this exercise you will use the Length tool to measure the width of a gap in the circuit trace. 1. Press the right mouse button when the cursor is on the Circuit.tif image. The Zoom pop-out menu appears.

2.

Select the Zoom sub-menu, and then select the Zoom 100% option. The Circuit.tif image returns to its original size. Select the Measurements command from the Measure menu. The Measurements tabbed dialog appears.

3.

4.

Click the Advanced button

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Filtering, Calibrating, and Measuring 5. Select the Best Fit Line feature. Make sure the Auto-Add checkbox is selected.

6.

The Create Best Fit Line message box appears. The Best-Fit Line feature is used to obtain a straight-line measurement.

In this case, you are measuring the length of the trace between the bends. 7. Place the cursor over the trace as shown below, then click the left mouse button. The first point of the measurement is anchored.

Place the cursor here.

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Image-Pro Plus Start-Up Guide 8. Move the cursor to start of the bend in the trace (see illustration below), and double-click the left mouse button.

Place the cursor here.

A measuring line appears connecting the two anchor points on the image.

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Filtering, Calibrating, and Measuring 9. The Measurements tab reports the length of the line as approximately 5.7 microns.

Note: You can adjust this line in the image by dragging either of its ends to a new position. Use the selection tool (arrow) and press the <Shift> key to move the ends of the line. The measurement in the Adjust Line message box will automatically update as the lines length changes. 10. 11. Image-Pro labels your measurement line as BL1. Perform the measurement of the second trace by repeating steps 4 through 9, as shown below.

The second trace

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Image-Pro Plus Start-Up Guide 12. The Measurements tab is automatically updated with data from both traces.

The new measurement is approximately 8.8 microns. Note that the measurements are labeled BL1 and BL2. The BL signifies that it is a best-fit line measurement, and the number indicates the measurement. For example, for BL1, the 1 signifies that it is the first measurement in this set of data.

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Filtering, Calibrating, and Measuring

Using the Trace Measurement Tool

You will also use the Trace measurement tool to manually outline and measure the perimeter of the terminals, and automatically measure another trace on the PCB. 1. Click the Trace button in the Measurements toolbar. The Creating Trace message box appears.

The Trace measurement tool is used to measure lines that are not straight. In the following steps, first measure the terminal using this tool's manual mode, then measure a trace in automatic mode. 2. Place the cursor at the base of the circuit terminal as shown below, and click the left mouse button to define the beginning of the measurement line.

Place the cursor here

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Image-Pro Plus Start-Up Guide Move the cursor to each of the points illustrated below, and click the left mouse button at each position. This will produce a polyline (a line made up of many consecutive segments).

3.

After you have defined each of the points above, click the right mouse button to end the polyline measurement. The terminal is highlighted.

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Filtering, Calibrating, and Measuring 4. The measurement result will appear in the Measurements window datasheet, and the measurement will be outlined and labeled in the image.

Note that the measurement is labeled T1. The T signifies that it is a Trace measurement, and the 1 signifies that it is the first trace measurement in this set of data.

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Image-Pro Plus Start-Up Guide 5. To perform an automatic trace, locate and enable the Auto-Trace checkbox in the Outline dialog box on the screen.
Enable the Auto-Trace

The Outline dialog box appears when the Trace measurement tool is activated. It controls the Image-Pro Plus automatic trace measurement feature. When the Auto-Trace checkbox is enabled, Image-Pro Plus automatically measures an edge that you specify. 6. Set the Smoothing option to 5. This option tells Image-Pro Plus to produce a smooth trace, and not follow every nook and cranny produced by minor pixel variations along the edge. Place the cursor at the beginning of the edge as shown below, and click the left mouse button once.

7.

Place the cursor here.

This action identifies the first point of the measurement line.

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Filtering, Calibrating, and Measuring 8. Place the cursor a few pixel positions further along the edge (see below) and click the left mouse button again.

Click again here.

Image-Pro Plus begins measuring the edge of this trace and continues until it reaches the edge of the image. (You can stop the Auto-Trace operation by pressing the right mouse button at the same time.) As illustrated by this exercise, Image-Pro Plus' automatic trace measurement facility requires you to define the first two points along the edge you want to measure. The first point specifies the beginning of the line (see Step 7 above) and the second point specifies the direction in which the line is to be drawn. 9. Once the line reaches the edge, click the right mouse button. The edge is highlighted.

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Image-Pro Plus Start-Up Guide 10. Click on OK in the Trace Measurement window. The measurement result (approximately 21 microns) is placed in the Measurements window datasheet, and the measurement is outlined and labeled in the image as T2.

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Filtering, Calibrating, and Measuring 11. On the Options tab, select Display Feature Statistics to view the statistics associated with the current set of measurements.

Note: Area and Angle values are zeros , as those measurements have not been performed.

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Image-Pro Plus Start-Up Guide 12. Select the Features tab. You will see the measurement statistics displayed on the lower portion of the page.

Continue to the next set of steps in this exercise: Saving the Measurements.

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Filtering, Calibrating, and Measuring

Saving the Measurements

In this step you will save the measurements that you have collected into an ASCII file. 1. Select the Input/Output tab on the Measurements tabbed dialog.

2.

Select the Measurements from the Data to Output list, and File from the Output Data To list. Click the Save button. The Save Measurements dialog box appears.

3.

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Image-Pro Plus Start-Up Guide 4. Type: CIRCUIT in the File name edit box.

5.

Click Save. This saved measurement file can be imported into another application.

Continue to the last set of steps in this exercise: Closing the Images and Exiting Image-Pro Plus.

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Filtering, Calibrating, and Measuring

Closing all the Windows and Exiting Image-Pro Plus

Close all the windows and exit Image-Pro by selecting the Exit option under the File menu. Do not save any files. Continue to the next exercise: Optical Density Analysis.

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Image-Pro Plus Start-Up Guide

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Optical Density Analysis

Optical Density Analysis

The Tutorial in this section introduces you to Image-Pro Plus Intensity calibration and Analysis tools. In this exercise you will: Load an image of a gel sample and calibrate the intensity scale in terms of optical density. Obtain density measurements from a band of pixels within the gel image. Establish a baseline measurement of incident light. Plot the density measurements of a band of DNA pixels against the incident light baseline. Capture the screen image of the plot. Store into an image file to be imported into another application. This exercise will take about 15 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now. Once
the Image-Pro Plus application window is active, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

Loading the Image

In this exercise, you will modify Image-Pro Plus intensity scale so that the intensity values of a DNA gel sample image are expressed as density values (calibrated to a standard density curve). This image was captured in a light box with a CDD camera, with the UV light source positioned behind the sample. 1. Load the DNA.tif file from the Images folder of the Image-Pro Plus folder. The DNA.tif image window is opened. The dark stripes (lanes) indicate the presence of DNA.

DNA lane

Empty lane

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Optical Density Analysis

2.

From the Measure menu, select the Calibration submenu, and then the Intensity command. The Intensity Calibration - dna.tif dialog box appears.

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Image-Pro Plus Start-Up Guide

3.

Click the New button to create a new calibration. The intensity curve appears and the Intensity Cal 0 name appears in the Name field.

The y-axis depicts the calibrated value.

The x-axis depicts the original intensity value.

The Name field identifies the set of calibration values you are about to create. Calibration settings can be saved within ImagePro Plus so they can be recalled when they are needed again. The intensity curve illustrates the effect of a calibration change upon the intensity scale. The x-axis describes the original intensity value (i.e., the value read from the image), and the y-axis reflects the way in which the value will be interpreted per the calibration changes. 4. 5. Enter UV Light as the Name of the new calibration. Enter OD as the Unit Name for optical density.

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Optical Density Analysis 6. Click the Standard Optical Density radio button. The intensity curve changes to reflect the density/intensity relationship.

The x-axis depicts intensity. The y-axis depicts density.

The optical density scale reflects an exponential decay of light within a transmitting material. As you can see, the optical density curve is inversely related to the intensity scale. This means: Small intensity values reflect large density values. Large intensity values reflect small density values i.e., the darker the pixel, the denser the material.

Continue to the next set of steps in this exercise: Specifying the Black and Incident Levels.

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Image-Pro Plus Start-Up Guide

Specifying the Black and Incident Levels

In this exercise you will establish the levels that represent black (indicating that no light is present) and incident or white (indicating that no material is present). The black circular object (black spot) in the upper-left corner of the image is the calibration standard, which will be used to establish the black level. This object represents the intensity recorded by the camera when no light was passed to it (it was created by affixing a solid object to the sample when the image was captured in the light box). The empty area along the left side will be used to establish the incident level or white level. This area represents the intensity recorded when no material was present, and all possible light was passed to the camera. You can now enter values for the levels or click on the corresponding Image buttons to interactively set the levels. 1. Click the Options button in the Intensity Calibration dialog box. The Optical Density Calibration dialog box appears.

2.

Click the Image button in the Black Level group box. The Black Level message box appears, and the cursor appears as crosshairs over the image.

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Optical Density Analysis 3. Move the crosshairs to the center of the black spot in the upper-left corner. Click the left mouse button when the center of the cursor is positioned over the center of the dark circle, and the Black Level message box reads 8. The Optical Density Calibration dialog box reappears, and the Black Level field reads 8. Click the Image button in the Incident Level group box. The Incident Level message box appears. The cursor appears as crosshairs.

4.

5.

Move the crosshairs to the empty area along the left side of the image. Click the left mouse button when the Incident Level message box reads 121. The Optical Density Calibration dialog box reappears, and the Incident Level field reads 121.

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Image-Pro Plus Start-Up Guide 6. Click OK. The Intensity Calibration - dna.tif dialog box appears. The end points of the density curve reflect the black and incident levels you have just entered.

7.

Click OK. The Intensity Calibration - dna.tif dialog box closes.

Continue to the next set of steps in this exercise: Plotting the Density of a Lane.

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Optical Density Analysis

Plotting the Density of a Lane

In this exercise you will collect the density measurements for one of the DNA lanes in the gel, and plot its density using the Line Profile command. A Line Profile plots the values of each pixel in a line in the image. In this exercise, you will define a column of pixels (instead of a single line), and plot the average density value along the length of the column. 1. Select the Line Profile command from the Measure menu. The Line Profile - dna.tif dialog box appears, and a diagonal defining line is placed in the upper-left corner of the DNA.tif image window.

Note: Some of the example images used in this exercise show the profile's statistic values along with the graph. On your system, these statistics might not appear. Do not be concerned; this difference will not affect the objectives of this exercise.

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Image-Pro Plus Start-Up Guide 2. From the Line Profile-dna.tif dialog box, select the Report menu, and then select the Thick Vert command. A pair of defining lines appear in the upper-left corner of the image.

3.

From the same Report menu, make sure the Intensity Cal and Range/Area options are selected. Deselect the Full Scale option in the Report sub-menu. The Y-axis of the graph is scaled to the minimum and maximum values of the profile line.

4.

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Optical Density Analysis

5.

Match the width of the pair of lines to the second DNA lane in the image. Position the pair of lines over the second DNA lane and drag the rightmost defining line to the right edge of the second DNA lane.

Using the same procedure, drag the leftmost defining line to the left edge o the second DNA lane. 6. Stretch the pair of lines the length of the lane. Position your cursor between the pair of lines at the top end of the column. When the vertical double-arrow appears, drag the endpoints to the top edge of the DNA lane.

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Image-Pro Plus Start-Up Guide Using the same procedure, drag the bottom endpoints to the bottom of the DNA lane. When complete, your defining pair should look as follows:

Image-Pro Plus measures, and plots, the average density down the length of the lane. You can see that the graph reflects the bands of material in the lane. The darkest (densest) bands produce the highest peaks.

Continue to the next set of steps in this exercise: Plotting the Density Against a Baseline.

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Optical Density Analysis

Plotting the Density Against a Baseline

In this exercise you will define an empty column as a baseline, and plot the differences between it and the sample lane. 1. Move the defining pair to an empty lane. Position your cursor anywhere between the defining lines. When the 4way cursor appears, drag the lines to the empty lane to the left of the current DNA lane.

Note: If necessary, adjust the lanes individually to ensure both are entirely within the empty lane. You do not want them touching the DNA lanes on either side.

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Image-Pro Plus Start-Up Guide 2. Click the Reference checkbox in the Line Profile - dna.tif window. The defining-line pair appears as dashed lines, and Image-Pro Plus plots their entire length as 0. This creates a baseline against which other profiles can be measured. The graph changes to reflect the new (empty) band of data.

Checkbox to create a reference pair.

The defining pair of lines on an empty lane.

Graph reflecting the baseline.

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Optical Density Analysis 3. Place a defining pair over the second DNA lane. Place your cursor between the reference lines. When the 4-way cursor appears, click the left mouse button. A new defining pair will be cloned from the reference pair and placed directly over the initial pair. Drag the new pair over the second lane as shown.

Now, two pairs of lines appear in the image. The dashed pair in the empty lane represents the baseline, and the solid pair in the DNA line represents the measured pair. The graph shows the variance of the measured pair from the baseline pair. In the next set of steps, you will copy the graph to an image file.

Continue to the next set of steps in this exercise: Storing the Plot to an Image File.

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Image-Pro Plus Start-Up Guide

Storing the Plot to an Image File

In this exercise you will save the line plot to a TIFF file using Image-Pro Plus Screen Capture utility. Once your plot has been saved in TIFF form, it can be pasted or imported into a word processor or desktop publishing program. 1. Select the Screen Capture command from the Image-Pro Plus File menu. The Screen Capture Options dialog box appears.

2.

Select the Client option in the Selection list box under the Capture Options group This option lets you copy the content of the active window to an image file (just those pixels inside the window; not the border and title bar). The other choices in this list box let you capture the entire screen, an entire window, or just a particular area of the screen that you specify.

3.

Select the c:\ipwin5\images folder. This directs Image-Pro Plus to store the captured screen to this directory. By default, Image-Pro Plus stores captured screens to files prefixed with GRAB. You will not change this naming convention in this exercise, so Image-Pro Plus will store the screen to a file called GRAB0000.tif in the c:\ipwin5\images directory.

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Optical Density Analysis 4. Click OK. The Screen Capture Options dialog box closes. Press <F7>. The Select Window/Client dialog appears. Click and drag the Window Selector tool over the Line Profiledna.tif window . Use the mouse to click and drag the Window Selector tool . Release the left mouse button after selecting the Line Profiledna.tif window. Image-Pro Plus reads the contents of the Line Profiledna.tif window and creates an image from the pixels that it contains. A camera icon appears, and a progress line runs down the length of the window as Image-Pro Plus captures the window. The image is stored to GRAB0000.tif in the ipwin4\images folder. Note: You have just stored an image of the profile graph. The data used to generate the graph are not a part of this file. To store the data associated with this graph, use the Save command on the File menu in the Line Profiledna.tif window.

5.

6.

More about... Screen Capture You could now use this captured image just as you would any image file. You could read the file into Image-Pro Plus to edit it, enhance it, or combine it with another image. Moreover, you could read (or paste) it into a word processing or desktop publishing document. In this exercise, Image-Pro Plus stored the image to a file called GRAB0000.tif. Subsequent screen images stored to this directory would be assigned the same prefix GRAB, but a new consecutive number (e.g., GRAB0001.tif, GRAB0002.tif, etc.) would be assigned. You also have the option to change the GRAB prefix to something else, for example IPP. You may store a captured screen data in any file format supported by Image-Pro Plus. If your application does not accept TIFF, select another format with the Screen Capture command. Page 5-17

Image-Pro Plus Start-Up Guide By default, <F7> is assigned as the hot key to invoke the Screen Capture facility. You may change this assignment with the Screen Capture command.

Continue to the next set of steps in this exercise: Closing all the Windows and Exiting Image-Pro Plus.

Closing all the Windows and Exiting Image-Pro Plus

Close all the windows and exit Image-Pro Plus by selecting the Exit option under the File menu. Continue to the next exercise: Filtering with Fast Fourier Transforms.

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Filtering with Fast Fourier Transforms

Filtering with Fast Fourier Transforms

The Tutorial in this section introduces you to the Fast Fourier Transform (FFT) command. The FFT command transforms an image into a frequency spectrum. Image noise appears as a repetitive pattern which is difficult to remove using standard spatialfiltering 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. In this exercise you will: Load an image containing pattern noise and transform that image into its energy spectrum using a Fast Fourier Transform. Edit the spectrum to remove the pattern frequency. Regenerate the edited image through an Inverse Transform. Send the file as an e-mail attachment. This exercise will take about 25 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now by

double-clicking on the Image-Pro Plus icon in your Image-Pro Plus folder. Once the Image-Pro Plus application is active, you may begin following the steps below.

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Image-Pro Plus Start-Up Guide

Transforming the Image

In this exercise, you will use an image embedded with a strong diagonal interference signal and transform that image into its frequency spectrum using the Image-Pro Plus FFT command. 1. Load the Fftprint.tif file from the Images folder. The Fftprint.tif image window appears. Notice the strong diagonal noise patterns. .

2.

Select the FFT command from the Process menu. The FFT window appears.

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Filtering with Fast Fourier Transforms 3. Transform the image to its frequency spectrum. Click the Forward button in the FFT window to transform the image. This process may take a few moments. You can monitor its progress by observing the message bar at the bottom of the screen. When the transformation process is complete, the FFT1fftprint.tif image window appears, containing the spectrum.

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. You will remove these points in the next few steps.

Continue to the next set of steps in this exercise: Editing the Spectrum.

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Image-Pro Plus Start-Up Guide

Editing the Spectrum

In this exercise, you will eliminate the anomalous frequencies (the 4 outside bright points) by defining an AOI around them and cutting the frequencies from the spectrum. 1. Click the Spike-Cut radio button in the Filter group box in the FFT window.

2.

Select the AOI Rectangle tool from the Tool Bar.

3.

Draw a small, rectangular AOI around the outermost bright point in the upper-right quadrant of the FFT1fftprint.tif window.

Make the AOI as small as you can, to enclose just the bright point. If you can't remember how to define an AOI, refer to that exercise in the Learning the Basics section of this tutorial.

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Filtering with Fast Fourier Transforms 4. Click the Apply button in the FFT window. The spectrum is filtered, and the selected frequencies are removed from it. This process may take a few moments. Notice that the bright point, and its counterpoint (diagonally opposite) are gone. The frequencies represented by these points have been removed from the spectrum.

5.

Click the New AOI button from the tool bar along the top edge of your screen. The AOI you drew around the upper-right bright spot is cleared from the screen. The New AOI tool removes the active AOI from the screen and allows you to define a new one.

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Image-Pro Plus Start-Up Guide 6. Repeat steps 3 and 4 on the innermost bright point in the upper-right quadrant (i.e., define an AOI around it and cut it from the spectrum). When this step is complete, the last pair of bright points will be gone.

The majority of the anomalous frequencies have now been removed from the image. Note: The remainder of the cross-shaped distribution around the points you just cut represent edge effects of the noise. As an additional exercise, you can go back and cut more of these edge effects using the AOI tools. 7. Click on the AOI Rectangle tool to turn it off.

Continue to the next set of steps in this exercise: Returning the Spectrum to Spatial Form.

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Filtering with Fast Fourier Transforms

Returning the Spectrum to Spatial Form

In this exercise, you will inversely transform the spectrum back into its spatial form. This will generate the original image, minus the frequencies that were edited-out in the last step. 1. Click the Options button in the FFT window. The Inverse FFT & Filter Options dialog box appears.

2.

Select the New Image option from the Write Inverse FFT Into combo box.

This directs Image-Pro Plus to write the results of the Inverse Transformation to a new image window. The original image window can also be selected here. 3. Click OK. The Inverse FFT & Filter Options dialog box closes.

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Image-Pro Plus Start-Up Guide 4. Click the Inverse button in the FFT window. The FFT data is inversely transformed back into spatial form and placed in an untitled image window. This process may take a few moments.

Nearly every trace of the diagonal pattern has now been removed. The new image is the same size as your original, although the FFT image is larger. An FFT transformation requires sizes of powers of 2 (e.g., 16, 32, 64, 128, 256, 512...) for efficiency, and images that are not powers of 2 in size are expanded to the next larger power of 2 for processing. 5. Click on Close to close the FFT window.

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Filtering with Fast Fourier Transforms

More about... FFT filtering Image-Pro Plus will transform an AOI, instead of the entire image, if an AOI is active when the Transform button is clicked. The spectrum window remains open even after you have performed an inverse transformation. You can now make additional edits to the spectrum if you are not satisfied with the results. In addition to the Spike Cut option, which was used in this exercise to eliminate a specific range of frequencies, the Hi-Pass and LoPass options let you eliminate or attenuate all frequencies below or above a specified point. See the FFT command in the Reference Manual for more information about these options. Image-Pro Plus will write the results of an Inverse Transform into an AOI if one is active in the designated destination window when the Inverse button is clicked.

Continue to the next set of steps in this exercise: Sending as an E-mail Attachment.

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Image-Pro Plus Start-Up Guide

Sending as an E-mail Attachment

In this exercise, you will learn how to send the modified image as an e-mail attachment. 1. Select the Send Mail option from the File menu. The Send Mail dialog box appears.

2.

Type the e-mail address you are sending the file to in the To combo box. In this example, type the Media Cybernetics Technical Support Internet address: [email protected] in the To field. For this example, type Tax fraud suspect #1234ABCD in the Subject combo box.

3.

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Filtering with Fast Fourier Transforms 4. For this example, type Joe, Here is the suspects fingerprint after a noise removal using Image-Pro Plus. in the message area.

5.

Click the Attach button. The Attach dialog box appears.

9.

Click on OK. The Attach dialog box closes and the Send Mail dialog box contains the e-mail information, including the new attachment.

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Image-Pro Plus Start-Up Guide 10. Prepare to send the e-mail message by clicking on Send. For the purposes of this exercise, click Cancel and do not send this e-mail. Normally, however, to send an e-mail, you would click OK. Image-Pro would then directly connect to your e-mail software package so you could log in. After sending the file as an attachment, Image-Pro would immediately return back to the application window and close the Send Mail dialog box.

Continue to the last set of steps in this exercise: Closing all the Windows and Exiting Image-Pro Plus.

Closing all the Windows and Exiting Image-Pro Plus

Close all the windows and exit Image-Pro by selecting the Exit option under the File menu. Continue to the next exercise: Counting, Measuring, and Classifying.

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Counting, Measuring, and Classifying

Counting, Measuring, and Classifying

The Tutorial in this section is designed to introduce you to Image-Pro Plus' automatic Object-counting and Measuring features. An Object is considered to be any contiguous group of pixels that share a specified range of intensity values. With the Count/Size command, these objects can be identified, counted, and measured. In this exercise you will: Perform manual gray scale thresholding of metal grains. Count the grains in an image and measure each grains pore area, pore ratio, and the number of pores within each object. See the visual effects of filtering out (graphically excluding) objects from the count. Split clustered objects. Classify the measurement data. Color-code the objects by class. Store the measurement results to an Excel worksheet. Create a customized report using Image-Pro Plus and the Image-Pro Report Generator, as well as the template feature. This exercise will take about 40 minutes to complete.

Setup: Start Image-Pro Plus by double-clicking on the Image-Pro

Plus icon in your Image-Pro Plus folder. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

Perform Gray Scale Thresholding

In this exercise you will use an image of a metal sample (captured from a microscope at 50x magnification) to analyze the grains. First, youll use the manual thresholding feature to segment the image into objects and background. Image-Pro can detect objects automatically as well; however, this exercise will demonstrate the manual thresholding feature. 1. Load the Exm50x.tif file from the Images folder. The Exm50x.tif image window is opened.

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Counting, Measuring, and Classifying 2. Select the Count/Size command from the Measure menu. The Count/Size dialog box appears.

3.

Click the Manual radio button in the Intensity Range Selection group box.

The Select Ranges button is now accessible.

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Image-Pro Plus Start-Up Guide 4. Click the Select Ranges button. The Segmentation - exm50x.tif dialog box appears.

5.

Change the threshholds range from 182 to 255 to select the bright objects (holes) in the image. First change the upper threshhold from 130 to 255 and then change the lower threshhold to 182. You can use the spin boxes ( and ) to change the values (type the values or use the spin arrows). - Or you can drag the range limit bars ( and ) to the appropriate values, which are reflected in the spin boxes (see the diagram in the next step).

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Counting, Measuring, and Classifying

Or, you can drag both limits with this bar.

Or drag this upper range limit bar to 255. Change the value in this spinbox to 255 Then drag this lower range limit bar to 182.

Then change the value in this spinbox to 182.

As you change the thresholding values, you will notice an overlay on the image. This overlay defines the area you are thresholding, but does not effect or change your image data. 6. Click Close. Image-Pro selects the intensity range of the objects to be counted and measured, and the Segmentation - exm50x.tif dialog box closes.

Continue to the next set of steps in this exercise: Select Measurements.

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Image-Pro Plus Start-Up Guide

Select Measurements
In this exercise, you will choose which types of measurement data for ImagePro Plus to record when the objects are counted. 1. From the Measure menu in the Count/Size dialog box, select the Select Measurements command.

The Select Measurement dialog box appears. The Area measurement is the default.

In the following steps, add the measurement data relevant for the pores (Hole Area, Hole Ratio, Holes, and Class).

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Counting, Measuring, and Classifying 2. Add the Hole Area measurement to be included in the measurement statistics. Scroll down the Measurements list box on the left and then click on Hole Area. Hole Area calculates the area of the holes within the object. The new measurement is included in the Filter Ranges list box on the right.

3.

Add the Hole Ratio measurement. Repeat the same procedure from step #2. Hole Ratio is the ratio of the object area (excluding holes) to the total area of the object.

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Image-Pro Plus Start-Up Guide 4. Add the Holes measurement. Repeat the same procedure from step #2. Holes calculates the number of holes within an object.

5.

Add the Class measurement. Repeat the same procedure from step #2. Class groups counted objects by their class.

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Counting, Measuring, and Classifying

6.

Click OK. The Select Measurement dialog box closes.

Continue to the next set of steps in this exercise: Counting the Objects.

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Image-Pro Plus Start-Up Guide

Counting the Objects

In this exercise, you will count the grains and then modify the options relating to the way counted objects are identified and outlined. 1. Click the Count button in the Count/Size command window. The image is analyzed, grains identified, counted, and measured. Object outlines and labels appear in the Exm50x.tif window.

Note that the pores or holes within the metal grains are not outlined in this image after the initial count.

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Counting, Measuring, and Classifying 2. Click the Options button. The Count/Size Options dialog box appears.

3.

Select the With Holes option from the Outline Style drop down list. This option tells Image-Pro Plus to highlight counted objects around their perimeter and around any holes that are within the object. (Holes are defined as pixels within an object that are not of the specified intensity).

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Image-Pro Plus Start-Up Guide 4. Make sure that the Fill Holes box is unchecked. At this point, doublecheck to make sure that your settings for ImagePro match the ones in the previous dialog box to continue with this exercise. (Image-Pro retains the settings from previous experiments, which may not be the same as the ones needed for this exercise.) 5 Click OK. The pores are now highlighted.

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Counting, Measuring, and Classifying 6 Double-click on any object. The Object Attributes window appears.

Measurements for a single object can be viewed by simply doubleclicking on that object. The Object Attributes window presents all recorded measurements for the selected object. 6. Click on OK to close the Object Attributes window.

Continue to the next set of steps in this exercise: Graphical Exclusion of Ranges.

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Image-Pro Plus Start-Up Guide

Graphical Exclusion of Ranges

In this exercise, we will demonstrate a new method (graphical exclusion of ranges) in Image-Pro Plus 6.0 for visualizing the effect of your filter ranges. 1. From the Measure menu in the Count/Size dialog box, select the Select Measurements command.

The Select Measurement dialog box appears.

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Counting, Measuring, and Classifying

2.

Highlight the Area filter range. The Edit Range button is now undimmed.

3.

Press the Edit Range button. The Edit Measurement Range - Area graph appears. This is a graphical representation of the Area range shown in the Select Measurements dialog box in Step #1.

The x-axis represents the Area values for the individual objects, and the y-axis the number of objects with that area. The From and To fields in this Edit Measurement Range dialog box correspond with the Start and End fields in the Select Measurements dialog box.

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Image-Pro Plus Start-Up Guide 4. Drag the left-hand vertical marker to the right to view the outlines of the smaller areas disappearing in your image. The smaller area outlines that are disappearing are the objects that you are filtering out as you drag the vertical marker (i.e., what you are graphically excluding as you increase the area values on the x-axis).

In this way, you can visually see the numerical values you are setting for the Filter Ranges in the Select Measurements dialog box. 5. To see the outlines of the larger areas disappear, drag the right-hand vertical marker to the left. The larger area outlines that are disappearing are the objects that you are filtering out as you drag the vertical marker (i.e., what you are graphically excluding as you decrease the area values on the x-axis). 6. Click Close to return back to the Select Measurements dialog box.

Continue to the next set of steps in this exercise: Splitting Objects.

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Counting, Measuring, and Classifying

Splitting Objects
If you examine the image, you will see a problem with the results of the count: Grain #61 (located on the left side of the image) is not a single object, but actually two individual objects counted as one. In the next few steps, you will correct this problem. 1. From the Edit menu in the Count/Size dialog box, select the Split Objects option. The Split Objects message box appears.

2.

Place the outline cursor above object #61, as shown in the following illustration. Then click the left mouse button to begin drawing the line. Be sure that the cursor is not touching the object when you click the mouse the first point of the dividing line must lie outside of the object being split.

Place the cursor here.

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Image-Pro Plus Start-Up Guide

3.

Move the cursor below the object, as shown below, and then click the left mouse button again to end the line. Be sure that the cursor is well below the object when you click the endpoint of the dividing line must be outside of the object being split.
Place the cursor here, and press the left mouse button again.

4.

Once the line is completed, click the right mouse button. A dividing line is drawn between the two objects.

Note: Your cursor must be within the Exm50x.tif image window when you press the right mouse button; otherwise this action will have no effect.

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Counting, Measuring, and Classifying 5. Click OK in the Split dialog box. Image-Pro Plus reanalyzes the image, and splits object #61 into two new objects, now numbered #61 and #64.

Continue to the next set of steps in this exercise: Obtaining Measurement Distributions.

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Image-Pro Plus Start-Up Guide

Obtaining Measurement Distributions

Image-Pro Plus lets you obtain a size distribution curve for the measured objects. In this exercise, you will obtain a graphical representation of the area and hole ratio distribution.. 1. Go back to the Count/Size dialog box. From the View menu, select the Histogram command. The Histogram dialog box appears, with Area as the default.

2.

Change the Area option to Hole Ratio in the list box. This displays the range of values the grains have for Hole Ratio.

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Counting, Measuring, and Classifying 3. Set the Number of Bins to 25. The default value of 10 is quite coarse. Raising the number of histogram bins will give a more detailed view of the data.

Clustering above 0.8391096

Look at the data for clustering. Note that there appears to be a cluster of objects with a hole ratio of 0.91955485 and above, a small gap in the data at 0.8391096, and a scattering of objects with lower hole ratios. For the purpose of this exercise, you will divide the objects at 0.875, based on the clustering observed in the histogram. 4. Close the Histogram dialog box.

Continue to the next set of steps in this exercise: Classifying the Measurement Data.

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Image-Pro Plus Start-Up Guide

Classifying the Measurement Data

Image-Pro Plus lets you take the measurement data you have collected about objects, and categorize them into ranges. In this exercise you will categorize the grains by the pore (hole) ratio. You will use Image-Pro Plus' single variable classification tool to define the classes. 1. Go back to the Count/Size dialog box. From the Measure menu, select the Single Variable Class command. The Classification dialog box appears.

Make sure that Hole Ratio is displayed in the list box. This tells Image-Pro Plus that you want the objects classified by their Hole Ratio measurements. 2. Make sure the Number of Bins field is equal to 2. This instructs Image-Pro Plus to divide the hole ratio range (from the minimum value to the maximum value) into 2 intervals. The beginning value of each interval is indicated by the first 2 values in the Bins Start At box. The 3rd value in the box reflects the maximum value in the range.

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Counting, Measuring, and Classifying 3. Change the second bin interval value to .875. This is the value obtained from the previous histogram exercise.

4.

Click OK. The Classification data window appears. This window lists the number of objects in each class, the percentage of objects in each class, the range values, and hole ratio measurements for each class.

Note: You may need to enlarge the Classification window to view all of the measurement data.

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Image-Pro Plus Start-Up Guide

5.

From the View menu in the Count/Size dialog box, select the Measurement Data command.

Note: You may need to enlarge the Measurement Data window to view all of the measurement data.

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Counting, Measuring, and Classifying 6. Click on the Sort Down radio button to sort the list in descending order and change the drop list from Area to sort on Hole Ratio.

Image-Pro reorders the format of the Measurement Data table to correspond to the sort button.

Continue to the next set of steps in this exercise: Color-coding the Objects.

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Image-Pro Plus Start-Up Guide

Color-coding the Objects

In this exercise you will color-code the grains by their hole ratio class. Colorcoding is accomplished using the Class Filled option in the Count/Size Options dialog box. 1. Go back to the Count/Size dialog box and click on the Options button. The Count/Size Options dialog box appears.

2.

Make sure the Class Filled option is selected in the Outline Style drop-down list box. This directs Image-Pro Plus to fill the objects with color, according to their class. In this exercise, Image-Pro Plus uses two colors, each representing one of the classifications you defined in the previous step.

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Counting, Measuring, and Classifying 3. Select the Class option in the Label Style list box. This directs Image-Pro Plus to label the objects according to the class to which they belong, e.g., objects falling into class 1, will be marked with a 1, those falling into class 2 will be marked with a 2, and so forth.

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Image-Pro Plus Start-Up Guide 4. Click OK. The grains appear color-coded and labeled according to their class.

Note: Move the Measurement Data window if it is obstructing your view of the image window

Continue to the next set of steps in this exercise: Saving the Measurement Data.

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Counting, Measuring, and Classifying

Saving the Measurement Data

In this exercise you will store the measurement results to a file. Important: Be sure the Measurement Data dialog box is active before beginning the steps below. 1. From the File menu in the Measurement Data dialog box, select the Data to File command. The Save Data dialog box appears.

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Image-Pro Plus Start-Up Guide 2. Select EXCEL (*.XLS) from the Save as type list box. This directs Image-Pro Plus to store the measurements as a Microsoft Excel spreadsheet file. You will notice that you can also choose to store the data in ASCII or Lotus 1-2-3 formats.

3. 4.

Type: Sample1 in the File name text box. Click Save. The measurement data is saved to file Sample1.XLS in your Image-Pro Plus Images folder. You can now read this worksheet file into Microsoft Excel.

5.

Close the Measurement Data window.

Continue to the next set of steps in this exercise: Creating a Report.

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Counting, Measuring, and Classifying

Creating a Report
In this exercise, you will use the Report Generator to create a customized report based on the current Image-Pro Plus session: the Exm50x.tif image and the existing Classification table. 1. From Image-Pros Measure menu, select the Report command. The Report Generator window appears.

2.

From the File menu, select the New command. The New Report dialog box appears.

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Image-Pro Plus Start-Up Guide 3. Click on the New Template button. The blank Untitled1 window appears.

4.

From the Insert menu, select the Image command. The original Exm50x.tif image appears.

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Counting, Measuring, and Classifying 5. Reduce and then reposition the image to fit on the New Template window, as shown below. Shrink the image by clicking and dragging the corner handles of the image window. Move the image to the new location in the top, middle portion of the New Template window by placing the cursor over the image and holding the left mouse button down.

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Image-Pro Plus Start-Up Guide 6. From the Insert menu, select the Image w/Overlay command. Resize and reposition that image as well to match below. The current Exm50x.tif image appears. Follow the instructions in Step #5 to resize and reposition this image, so that it is the same size as the first image and appears next to it (for comparison purposes), as shown below.

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Counting, Measuring, and Classifying 7. From the same Insert menu, select the Count/Size sub-menu, and then select the Classification Table option. The Classification table appears overlaid on the image.

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Image-Pro Plus Start-Up Guide 8. Move the Classification table to the bottom of the image. Drag the Classification table by placing the cursor over the image and holding the left mouse button down. Enlarge it a bit so that it's easier to read.

Continue to the next set of steps in this exercise: Using Templates.

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Counting, Measuring, and Classifying

Using Templates
In this exercise, you will use the template feature in Report Generator. The template feature is useful if you are creating reports with the same type of data being entered for each report. For other examples of using templates to generate standardized reports in ImagePro Plus, open and run Gear.tmpl in the Report Generator. 1. From the Insert menu, select the Insert sub-menu, and then select the Prompted Text option. The Prompted Text dialog box appears.

2.

Enter "Sample" for the Prompted title, and then double-click to enter "Enter Sample Type" for the Prompt to display line. "Sample" will be the name of the dialog box, with "Enter Sample Type" as the text for this template. Make sure that the Default Text line is blank (delete the existing test).

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Image-Pro Plus Start-Up Guide 3. Click OK. The newly created Sample dialog box with the text you typed above appears.

4.

Enter Aluminum as the Sample Type.

5.

Click OK. The information you were prompted for appears on the report.

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Counting, Measuring, and Classifying 6. Next, from the Insert menu, select the Insert submenu, and then select the Selection Text option. The Selected Text dialog box appears.

7.

Enter "Results" for the Prompted title and doubleclick to enter "Please select results" for the Prompt to display line.

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Image-Pro Plus Start-Up Guide 8. Double-click to enter "Pass" for the first entry of the List of selections to make available.

8.

Double-click to enter "Fail" for the second entry of the List of selections to make available.

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Counting, Measuring, and Classifying 9. Double-click to enter "Retest" for the third and final entry of the List of selections to make available.

10.

Click OK. The newly created Results dialog box with the Fail option appearing in the drop down list box. Image-Pro lists the options you entered in alphabetical order.

11.

Select Pass from the drop-down list. Click on the drop-down list to see the other choices (Pass and Retype), and select Pass.

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Image-Pro Plus Start-Up Guide 12. Click OK. The newly created Sample dialog box with the text you typed above appears.

Unfortunately, the Pass text box overlaps the Aluminum text box.

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Counting, Measuring, and Classifying 13. Reposition the Pass text box. Click and drag the Pass text box and move it below the text box created earlier, so that you can read both.

Note: For other examples of using templates to generate standardized reports in Image-Pro Plus, open and run Gear.tmpl in the Report Generator. We will now save this report.

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Image-Pro Plus Start-Up Guide 14. From the File menu in the Report Generator window, select the Save command. The Save As dialog box appears.

15.

Type: Sample1 in the File name text box.

16.

Click Save. Image-Pro Plus saves your customized report as Sample1.rptin the Images folder.

Note: For other examples of using templates to generate standardized reports in Image-Pro Plus, open and run Gear.tmpl in the Report Generator. Continue to the last set of steps in this exercise: Closing All the Windows.

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Counting, Measuring, and Classifying

Closing All the Windows and Exiting Image-Pro Plus

Close all the windows and exit Image-Pro by selecting the Exit option under the File menu. Do not save any changes. Continue to the next exercise: Color Segmentation.

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Image-Pro Plus Start-Up Guide

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Color Segmentation

Color Segmentation
The Tutorial in this section introduces you to the color segmentation options within Image-Pro Plus. Color segmentation separates objects or features from the background, based upon their color characteristics. In this exercise you will: Load an image of a cross-section of a tissue sample off a microscope. Enhance the image. Perform a color cube color segmentation. Count the nuclei contained within an AOI. Measure the percent area and view the corresponding statistical data. This exercise will take about 25 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now by

double-clicking on the Image-Pro Plus icon within the Image-Pro Plus folder. Once Image-Pro Plus is running, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

Contrast Enhancement
In this exercise, you will use contrast stretching to enhance an image of a cross-section of a tissue sample from a microscopic slide. 1. Load the Tumor_cells.tif file from the Images folder. The Tumor_cells.tif image window appears.

2.

From the Enhance menu, select the Equalize submenu, and then select the Best Fit command. Image-Pro enhances the contrast of the Tumor_cells.tif.

This operation maximizes the image contrast, taking the darkest portion of the image to black and the lightest portion of the image to white.

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Contrast Enhancement 3. Apply the contrast changes to the image. The contrast enhancement performed in the preceding step was recorded into a Lookup Table (LUT), through which the original image is interpreted. To permanently modify the image, the LUT must be applied to the image and then saved. Select the Apply Contrast command from the Enhance menu. The Apply Contrast dialog box appears.

4.

Click on OK. Clicking OK uses the contrast enhancement to permanently modify your image bitmap.

Continue to the next set of steps in this exercise: Counting the Nuclei Using Color Segmentation.

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Image-Pro Plus Start-Up Guide

Performing Color Segmentation

In this step you will count the nuclei by using the color cube method of separation. 1. Select the Count/Size option from the Measure menu. Image-Pro Plus opens the Count/Size dialog box.

Doublecheck to make sure that your settings for Image-Pro match the ones in this dialog box; e.g., Accumulate Count should be unchecked. (Image-Pro retains the settings from previous experiments, which may not be the same as the ones needed for this exercise.) 2. Click the Manual radio button. For this color segmentation, you will be defining the color range manually. The Select Colors button is now accessible.

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Contrast Enhancement 3. Click on the Select Colors button. Now, specify the range of colors that define the objects in the active object. The Segmentation - tumor_cells.tif tagged dialog box appears.

4.

Click on the Color Cube Based tab. For this exercise, you will use the color cube model to separate the objects that are to be counted and measured.

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Image-Pro Plus Start-Up Guide

Note: The Histogram model provides an alternative color segmentation method; the best method to use will vary with the data. 5. Click on the Eyedropper button in the Select Image group box. Move the cursor on to the area in the open image where you want to perform segmentation. You will see the selected area in the box under the eyedropper. Move the cursor to pixel location 544,338 on the image and then click the left mouse button. Here, you are selecting a specific color (corresponding to a 3x3 are around a pixel location) to segment. The exact location of the cursor is displayed on the bottom of the window in the status bar.

6.

Place the cursor here.

The cursor positions coordinates are shown here

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Contrast Enhancement As you highlight areas to segment, Image-Pro Plus separates the selected color by overlaying it in red on your image. You are now adding additional colors to the range of segmented colors. The eyedropper tool adds colors, while the eraser tool

subtracts them. Use these tools to define the desired colors for segmentation in the next couple of steps. 7.
Place the cursor here.

Repeat step #6 for pixel location 90, 118.

8.

Repeat step #6 for pixel location 722,165.

Place the cursor here.

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Image-Pro Plus Start-Up Guide 9. Click Close. The Segmentation-tumor_cells.tif tagged dialog box closes. At this point, you have defined a color range for segmentation. In the next section, you will apply that range to separate or measure the nuclei.

Continue to the next set of steps in this exercise: Counting the Nuclei.

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Contrast Enhancement

Counting the Spots

In this step you will define an Area of Interest (AOI) around portion of the tissue to count the nuclei using the color range you just defined, and view the resulting measurement statistics. 1. Select the Rectangular AOI tool.

2.

Define the AOI to cover most of the tissue section, as shown below in the Tumor_cells.tif image window. The AOI is thickly outlined for emphasis in the image below. (It does not appear as such on your screen.)

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Image-Pro Plus Start-Up Guide

3.

Click on Count in the Count/Size dialog box. Image-Pro Plus counts the nuclei contained in the area defined by the AOI, outlining them in red.

4.

From the Measure menu in the Count/Size dialog box, select the Select Measurements command. The Select Measurements dialog box appears, with Area as the default.

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Contrast Enhancement 5. Add the Roundness measurement to be recorded in the measurement statistics. Scroll down the Measurements list box on the left and then click on Roundness. The new measurement is included in the Filter Ranges list box on the right.

Roundness is a measure of how circular an object is (the expected perimeter of a circular object divided by the actual perimeter). A circular object will have a roundness of 1.0, while more irregular objects will have larger values. 6. Click on Measure. Roundness is added to the measurement statistics.

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Image-Pro Plus Start-Up Guide 7. From the View menu in the Count/Size dialog box, select the Statistics command. The Statistics window appears with the statistic data sheet for the image, including the Area and Percent Area measurements. You may need to expand the size of the window to view all of the data in this table.

The following statistics in the Statistics data sheet are listed by row for each measurement you have made:

Min: The smallest value for each measurement . The object possessing the minimum measurement is identified by number (Obj.#), below the stated minimum. Max: The largest value for each measurement. The object possessing the maximum measurement is identified by number (Obj.#), below the stated maximum. Range: The difference between the maximum and minimum values for each specified measurement. Mean: The average (mean ) for each measurement. Std. Dev: The standard deviation for each measurement.

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Contrast Enhancement

Sum: The sum of the measurements for each measurement type (e.g., area, perimeter, etc.) you have specified. Samples: The total number of objects that have been included in the count from which these statistics were derived.

Note that the Locate the object check box is turned on. Image-Pro Plus can identify on your image any object that is the Max or Min object for a given statistic.

8.

Click on the minimum (Min) value in the Statistics data table to see where that object is located on the image. Click on the object number in the Obj.# row for the Min value. (Obj.# rows are flagged with a symbol .) Image-Pro Plus uses the radiating signal to identify the object with the corresponding maximum values on the actual image.
Click on the Min value here.

Image-Pro locates the object here.

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Image-Pro Plus Start-Up Guide 9. Double-click on any individual object to see its statistical information. Make sure that the AOI tool is turned off to see the statistical data. The Object Attributes dialog box appears with the statistical data corresponding to the individual object selected.

Continue to the next set of steps in this exercise: Closing the Image Windows and Exiting Image-Pro Plus.

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Contrast Enhancement

Closing all the Windows and Exiting Image-Pro Plus

Close all the windows and exit Image-Pro by selecting the Exit option under the File menu. Do not save any changes. Continue to the next exercise: Calculating Population Density.

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Image-Pro Plus Start-Up Guide

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Calculating Population Density

Calculating Population Density

The Tutorial in this section introduces you to the Population Density features of Image-Pro Plus. Using Population Density analysis, you can measure the number of objects per unit of area. This feature is often utilized in cell analysis to determine the number of grains within a cell (grain-counting applications). In this exercise you will use the Population Density command to calculate density of grains in a cell. You will: Load an image containing silver grains. Locate cell sites. Save site outlines to a file. Filter the grains in the original image to separate clustered grains. Count the grains. Load the cell site outlines over the grains and calculate their density. This exercise will take about 25 minutes to complete.

Setup: Start Image-Pro Plus by double-clicking the Image-Pro Plus

icon in your Image-Pro Plus folder. Once the Image-Pro Plus application window is active, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

Copying the Image to a New Window

In this exercise you will use a modified image of silver grains (attached to cells via a chemical process), and then copy the image into a new window. 1. Load the Grains.tif file from the Images folder. The Grains.tif image window is opened.

2.

Select the Copy command from the Edit menu. The contents of the Grains.tif window are copied to the Clipboard. Note: Image-Pro Plus copies the contents of an AOI to the clipboard if one is active when the Copy command is selected. If no AOI is active, Image-Pro Plus copies the entire active image to the Clipboard (which is what you did in this step).

3.

Select the Paste New command from the Edit menu. The copy of Grains.tif will be pasted into a new window. The new image is called Untitled1.

4.

Save the Untitled1 image as IPpop1.tif. Select the Save As command from the File menu and type IPpop1.tif for the filename.

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Calculating Population Density

Identifying Cell Sites

In this image, the individual grains are distinct, but there is no way to determine where the cell boundaries are located. There are actually 11 cells in this image. In this step you will identify the location of these cells by dilating the grains (using a morphological filter) until they form large clumps. The clumps will indicate where the cell sites are located. Important: Be sure the IPpop1.tif image window is active before beginning the steps below. 1. Select the Filters command from the Process menu. The Filter tabbed dialog box appears.

2.

Click the Morphological tab. In the following steps, Image-Pro will fill the gaps and enlarge protrusions to connect the objects that are close. Click the Close radio button in the Filters group box. Click the 7 x 7 Circle radio button in the Options group box. Make sure that the number of Passes in the Option group box is 1. Page 9-3

3. 4.

5.

Image-Pro Plus Start-Up Guide

6.

Click the Apply button. The Close filter is applied to the IPpop1.tif image. The image appears as follows:

The Close filter fills gaps between closely spaced bright spots. Because a rather large filtering kernel (7 x 7) was used, the filtered results are very coarse. However, the image is beginning to show concentrations marking where its cell locations are.

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Calculating Population Density

Removing Extraneous Grains

You will note 8 large, and fairly distinct patches of grains with many, many small spots surrounding them. In this step, you will eliminate these small spots using another morphological filter. 1. Click the Open radio button in the Filters group box of the Morphological tab. Click the Apply button. The Open filter is applied to the IPpop1.tif image. The image appears as follows:

2.

The Open filter smoothes the contours of bright objects and removes minor protrusions. Because a rather large filtering kernel (7 x 7) was used, the filter also eliminated all small bright spots in the image. Now the cell sites, as distinguished by concentrations of grains, can easily be identified. You'll note that while only 8 sites were apparent initially, a 9th site has been located with this filter.

Continue to the next set of steps in this exercise: Outlining the Cell Sites.

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Image-Pro Plus Start-Up Guide

Outlining the Cell Sites

In this exercise, you will count the cell sites using the Count/Size command, and outline each cell site with an ellipse. You will ultimately superimpose these elliptical outlines onto the Ippop1.tif image and use them to calculate grain density. 1. From the Calibration sub-menu under the Measure menu, select the Spatial command and reset the calibrations, if necessary. The Spatial Calibration - Ippop1.tif dialog box appears. Make sure the Name field selection is (none).

Since this image has not been spatially calibrated, marking none applies no calibrations. 2. Close the Spatial Calibration - Ippop1.tif dialog box.

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Calculating Population Density 3. Select the Count/Size command from the Measure menu. The Count/Size dialog box appears.

4.

Click the Automatic Bright Objects radio button in the Intensity Range Selection group box. Click Count.

5.

Image-Pro segments 35 objects. The next step is to select a subset of these objects, defining a valid size range.

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Image-Pro Plus Start-Up Guide 6. From the Measure menu in the Count/Size dialog box, select the Select Measurements command. The Select Measurements dialog box appears.

7.

Select the Area selection. Use your cursor to highlight the Area measurement in the Filter Ranges group box. Delete the old value in the Start field and type:

8.

275 and click the checkmark.

This instructs Image-Pro Plus to ignore all objects with areas less than 275. Note: The actual value you select is dependent upon your own data. In this case, 275 was selected to isolate large clusters of grains in this image.

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Calculating Population Density 9. Click Measure. The image is analyzed, and bright objects with areas greater than 275 are extracted, counted, and measured. Object outlines and labels appear within the IPpop1.tif image window.

Continue to the next set of steps in this exercise: Saving the Cell Outlines.

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Image-Pro Plus Start-Up Guide

Saving the Cell Outlines

In this step, you will change the outlines to Ellipses. Because the patches merely suggest the location of the cells and do not actually represent cell boundaries, you will, in this step, convert the outlines to elliptical form. This will more closely resemble the actual shape of the cells to which the grains were attached. Then, you will save the elliptical outlines to a file. 1. Click the Options button. The Count/Size Options dialog box appears.

2.

Select the Ellipse option in the Outline Style list box. This instructs Image-Pro Plus to change the object outlines to ellipses with the same major and minor axes as the object outline.

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Calculating Population Density

3.

Click OK in the Count/Size Options dialog box. The object outlines appear as ellipses.

4.

From the File menu in the Count/Size dialog box, select the Save Outlines command. The Save Outlines dialog box appears.

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Image-Pro Plus Start-Up Guide

5.

Type: Cells in the Save Outlines list box. Image-Pro Plus will automatically assign the .OUT extension when the file is saved.

6.

Click Save. The outlines will be saved to file Cells.OUT in the Image-Pro Plus program directory. Outlines are saved in binary files containing a list of the polygons comprising the outlines.

Continue to the next set of steps in this exercise: Preparing the Grain Image.

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Calculating Population Density

Preparing the Grain Image

In this exercise you will again work on the Grains.tif image window and filter it to make sure that any clustered grains are separated. 1. Click on the Grains.tif window to reactivate it as the working image. The Grains.tif image window is selected.

Page 9-13

Image-Pro Plus Start-Up Guide 2. Activate the Filter dialog box. Note: The Filter dialog box was opened earlier in this exercise, and should still be open on the screen. It may be hidden behind other open image or command windows, so you may have to move a few windows to locate it.

3.

Click the Erode radio button in the Morphological filters group box. The Erode filter reduces the size of bright objects. This will shrink the grains in our image. Click the 2 x 2 Square radio button in the Options group box. Because the objects in the image are quite small, the 2 x 2 kernel is used to produce a subtle effect.

4.

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Calculating Population Density 5. Click Apply. The Erode filter is applied to the Grains.tif image. This filtering ensures that the grains get counted as individual objects. The grains in the image appear smaller.

Continue to the next set of steps in this exercise: Counting the Grains.

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Image-Pro Plus Start-Up Guide

Counting the Grains

In this exercise you will count the grains in this image. Because the grains are clearly distinguished against the dark background, you will use Image-Pro Plus' automatic object selection method. Since there are so many small objects, you will disable the object labeling feature. You will also eliminate all size selection criteria to ensure that all grains are included in the count. 1. 2. Activate the Count/Size dialog box. Click the Automatic Bright Objects radio button in the Intensity Range Selection group box. Click the Options button. The Count/Size Options dialog box appears.

3.

4.

Select Outline for the Outline Style list box. This directs Image-Pro Plus to highlight the counted grains with outlines, not the ellipses used in the previous exercise. Select None for the Label Style list box. This directs Image-Pro Plus to suppress labeling of the grains. Since there are so many small objects clustered together, labeling them would simply clutter the screen.

5.

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Calculating Population Density 6. Click on the Choose Color button. The Color dialog box appears.

The cyan color block

7.

Select the cyan color block. The cyan block is on the second row down, fifth block from the left. This directs Image-Pro Plus to highlight the counted grains in cyan. Note: When measuring data, you can choose what color contrasts best with your image.

8.

Click OK. The Color dialog box closes. Click OK. The Count/Size Options dialog box closes. Click the Apply Filter Ranges check box to remove the checkmark from the box. This action instructs Image-Pro Plus to ignore any selection criteria specified in the Select Measurements dialog box, and simply count all objects in the image.

9.

10.

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Image-Pro Plus Start-Up Guide 11. Click the Count button. The image is analyzed, and its bright objects (the grains) extracted, counted, and measured. Grains are colored cyan in the Grains.tif image window.

If you look in the lower right corner of the Count/Size dialog box, you will see that 1239 objects have been counted.

Continue to the next set of steps in this exercise: Calculating Population Density.

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Calculating Population Density

Calculating Population Density

In this exercise you will perform the population density analysis. This process will request the Cells.out file that you created earlier in this exercise. Those cell outlines will be superimposed upon Grains.tif, and the grain density within each site will be calculated. 1. From the Measure menu in the Count/Size dialog box, select the Population Density command. The Sites Outline File dialog box appears.

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Image-Pro Plus Start-Up Guide 2. Open the Cells.OUT file. The yellow cell outlines appear in the Grains.tif image window overlaying the cyan-colored grains, and the Population Density data window appears.

The Population Density data window contains the density calculations for each cell and other statistics. This data can be stored to a file using the Data to File option on the File menu in the Population Density window. Page 9-20

Calculating Population Density

Summary of The Population Density process The population density process involves three essential steps: 1) First, create a file that contains the outlines of the areas to be measured. In this exercise, you created the outlines by counting objects that were generated using morphological filters. In real life, you might create these outlines by counting objects that actually define the areas you want to measure. Or, by drawing the outlines using the Draw/Merge Objects or Measurements tools. 2) Next, count the objects making up the population for which you want to measure density. In this exercise, you counted the grains in the Grains.tif image. 3) Finally, use the Population Density command to load the outlines (created in Step 1), and calculate the object density within each. See the Population Density Analysis discussion in the Image-Pro Plus Reference Manual for more information about this subject.

Continue to the next set of steps in this exercise: Closing all the Windows.

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Image-Pro Plus Start-Up Guide

Closing all the Windows

Close all of the open image and command windows, and if you want, exit Image-Pro Plus.

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Advanced Exercises

Advanced Exercises
This section contains some additional exercises developed to demonstrate the new and improved features found in Image-Pro Plus version 6.0.

Using the Calibration Wizard

The Calibration Wizard is a new feature of Image-Pro Plus v. 6.0. It is designed to make it easier for you to calibrate a single image, or to develop a set of reference calibrations to be used when you are capturing images, or with Stage-Pro and Scope-Pro.

The first part of this exercise shows you how to create a reference
calibration.

The second part shows you the two modified steps necessary to calibrate
an active image

This exercise will take about 25 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now by

double-clicking on the Image-Pro Plus icon within the Image-Pro Plus folder. Once Image-Pro Plus is running, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 now retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

Creating a Reference Calibration

In this exercise, you will create a reference calibration for your Image-Pro Plus v. 6.0 system. When you first select the Calibration Wizard from the Measure menu, you will have a choice:

Create a reference calibration Calibrate the active image.

The following table indicates the steps required by both actions: Step 1 2 3 4 5 6 7 Calibrate the active image Welcome page Name and Units page Image Calibration page Finish Image page Create reference calibrations Welcome page Reference Process page Capture Setup page Magnifier page Objective page Capture Preview page Reference Calibration page Finish Reference page

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Advanced Exercises

To create a reference calibration, follow the steps below:

1.

Select the Calibration option from the Measure menu. Then choose the Spatial Calibration Wizard from the Calibration sub-menu. You will see the Welcome screen.

2.

3.

Click the radio button next to Create Reference Calibrations. Click Next.

4.

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Image-Pro Plus Start-Up Guide

5.

You will see the Reference Process page.

The Reference process page discusses the process of defining reference calibrations, what will be required to proceed, etc. It includes an option to skip this page in the future (in which case the Wizard will go to the Capture Setup page after the Welcome page). The Back button can still be used from the Capture Setup page to display the Reference process page. Check the Skipcheckbox. 6. Click the Next button to continue.

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Advanced Exercises

7.

The next page is the Capture Setup Page.

Click the Test Capture button. The Test Capture page is designed to ensure that your image capture settings are configured correctly. Clicking Test Capture snaps an image using the current capture settings.

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Image-Pro Plus Start-Up Guide

When your Capture setup is ready to go, you will see the following message:

8.

Click Next to continue.

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Advanced Exercises

9.

You will see the Magnifier page.

The Magnifier page allows you to use a magnifier that does not have any 1.0 magnification settings. If your magnification changer does not have a 1.0 magnification setting, check the Use Magnifier box, and then enter the correct magnification setting. 10. Click Next to continue.

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Image-Pro Plus Start-Up Guide

11.

The next page is the Objective page. You will need to select the microscope objective by the name of its lens file, or by entering the objective name.

If you have already defined your lens using the Edit Lens List feature on the Edit menu, you can select the lens you want to calibrate from that list. Otherwise, you can type the name of your objective into the space, or click the Objective button to select the correct Lens Information File. The name that you enter here will be used as the name of the Reference Calibration that your are currently creating. 12. Click Next to continue. Image-Pro will check to make sure that a calibration with the same name does not already exist.

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Advanced Exercises

The next page is the Capture Preview page.

13.

Click the Capture button. The goal of the Capture Preview page is to get the stage positioned so as to capture the largest area of the micrometer possible, and to focus the image for the calibration capture. A live preview is started automatically. The Stop Preview button can be used to stop the preview, at which time the button changes to Start Preview, so the preview can be restarted. When the Capture button is pressed, the preview is stopped, and an image is captured and displayed in an image workspace.

14.

When you have captured a clear image of the micrometer, click Next to continue. You will see the Reference Calibration page.

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Image-Pro Plus Start-Up Guide

15.

You will be asked to draw a calibration line on the image that you just captured. Click the Color button to select a color. 16. Click the Draw Reference Line button.

17.

Position your reference line on the image, and indicate the number of calibrated units that it represents, then Click OK.

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Advanced Exercises

18.

You can click on the magnifier button to open a Local Zoom window that displays the portion of the image that is under the cursor. This may help you to position the reference line more accurately. You may draw more than one calibration reference line. As each reference line is drawn, the Calibration Report section is updated to indicate the calibration so far, including the average calibration and the calibration from the last reference line. When you click Next, a reference calibration is created, using the name from objective selected on the Objective page, the calibration from this step, and using um as the units. The calibration is added to the reference calibration list, it is set as the system calibration, and it is applied to the active image

19. 20.

Click Next to continue. You will see the Finish page.

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Image-Pro Plus Start-Up Guide

You have successfully created a Reference Calibration. The Finish page verifies the creation of the calibration, and permits you to create more calibrations if you choose. All of the navigation buttons are enabled on this page. The Create Another button will switch the wizard to the Objective page where the user can select the next objective to calibration. To calibrate the active image, please follow the instructions in the next section.

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Advanced Exercises

Calibrating the Active Image

In this exercise, you will calibrate an image in the Image-Pro Plus 6.0 workspace. 1. 2. Open an image in the Image-Pro Plus workspace. Select the Calibration option from the Measure menu. Then choose the Spatial Calibration Wizard from the Calibration sub-menu. You will see the Welcome screen.

3.

4.

Click the radio button next to Calibrate the active image. Click Next.

5.

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Image-Pro Plus Start-Up Guide

6.

The next step names your calibration.

7.

Click Next to continue.

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Advanced Exercises

8.

You will see the Reference Calibration page.

You will be asked to draw a calibration line on the image that you just captured. Click the Color button to select a color. 9. 10. Click the Draw Reference Line button. Position your reference line on the image, and indicate the number of calibrated units that it represents, then Click OK. Click Next to continue. When you click Next, a calibration is created using a name derived from the active images name, the calibration from this step, and using your selected spatial reference units. The calibration is added to the reference calibration list, it is set as the system calibration, and it is applied to the active image 12. You will see the Finish page.

11.

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Image-Pro Plus Start-Up Guide

Now you have finished calibrating an image in Image-Pro Plus.

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Advanced Exercises

Tracking Objects
The Tracking feature of Image-Pro Plus 6.0 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. In this part of the Tracking exercise, you will learn to add tracks to an image manually. You will also learn to save the tracking data and send it to an Excel spreadsheet. This exercise will take about 45 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now by double-clicking on
the Image-Pro Plus icon within the Image-Pro Plus folder. Once Image-Pro Plus is running, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 now retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

1.

Manual Tracking
Open the sample image Trackseq.seq from the Images folder in IPP 6.0. Open the entire sequence (all 8 frames).

The image workspace will look something like this:

2. 3.

Click the Play button on the Sequence toolbar to see how the objects move. Click the Stop button.

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Advanced Exercises 4.

Select Track Objects from the Measure menu. You will see the Tracking Data Table.

5.

Click the Add Track Manually button. You will see a message asking you to click on the points to be tracked in the active image.

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Image-Pro Plus Start-Up Guide 6.

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.
7.

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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Advanced Exercises 8.

Click the Add Track Manually button another track. This will be Track 2.

again to add

9.

Click Close Track to stop adding points. The image in your workspace should look something like this:

Track 1 Track 2

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 .

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Image-Pro Plus Start-Up Guide

The Tracking Data Table displays information about the tracks recorded from your image, as shown here:

10.

To display the statistics for each track, click the Show Tracking Statistics button. The tracking statistics are shown in the bottom half of the Tracking Data Table, as shown here:

11.

Click the Tracking Options button Tracking Options dialog.

. You will see the

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Advanced Exercises

You can edit the list of measurements displayed in the Tracking Data Table by choosing items from the Measurements list box, and adding them to the Selected Measurements list.

12.

Use your cursor to highlight the Or. Distance measurement. Then click the Add button. The Or. Distance will appear in the list of Selected Measurements.

13.

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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Image-Pro Plus Start-Up Guide

14. 15.

Use the Select Track button

to select Track 2. button to hide Track

Click the Hide Unselected Tracks 1. Only Track 2 will be displayed on the image.

16.

Click The Show All Tracks appear on the image. Click the Save Tracks button

button. Now both tracks will

17.

You will see the Save Tracks dialog box.

18. 19.

Enter the file name as Mytracks.trc. Click the Save button.

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Advanced Exercises

Your tracking information is now saved in the Documents and Settings folder in Image-Pro Plus 6.0.
20.

Click the Show Graph button. Graph displayed, as shown here:

You will see the Tracking

You can choose to display different values on the graph using the dropdown list box.
21.

Click the Send to Excel button

Your tracking information will be saved in an Excel spreadsheet.

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Image-Pro Plus Start-Up Guide

Automatic Tracking
In this part of the tracking exercise, you will learn to use the automatic tracking feature in Image-Pro Plus version 6.0.

1.

Open the sample image Fish.seq from the Images folder.

2.

Open the entire sequence (all 300 frames).

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Advanced Exercises

Your image workspace will look something like this:

The fish in the image are not easy to see, so we will have to adjust the image.

3.

Select Difference from the Sequence menu. A new (untitled) image will appear in the workspace. This image has two fish, one dark and one light. We are going to track the lighter fish.

4. 5.

Select Track Objects from the Measure menu. Click the Find Tracks Automatically button on the Tracking Data Table toolbar. You will see the Count/Size dialog box.
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Image-Pro Plus Start-Up Guide

6. 7.

Click the Manual radio button. Click the Select Ranges button.

8.

Adjust the slider bars on the Segmentation dialog to show the one light fish as red against the background.

9.

Click Continue on the Tracking dialog.

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Advanced Exercises

10.

Click Yes when Image-Pro asks if the outlines are correct.

You will see the image with the one track highlighted.

11.

Click the Show Graph button. Graph displayed, as shown here:

You will see the Tracking

This graph shows the accumulated distance for the fish track.

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Image-Pro Plus Start-Up Guide 12.

Use the drop-down list box to display a different measurement, such as the Distance.

13.

Continue to the last set of steps in this exercise: Closing the Images and Exiting Image-Pro Plus.

Closing all the Windows and Exiting Image-Pro Plus

Close all the windows and exit Image-Pro by selecting the Close All option under the Window menu. Do not save any files. Note: For more Tracking exercises, see the demo macros included in your Image-Pro Plus installation.

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Advanced Exercises

Creating a Workflow Toolbar

The Workflow Toolbar is a way of saving and storing features of Image-Pro Plus that you use on a regular basis. You can create one toolbar, or several, to make the features that you use most often more easily accessible. This exercise will take about 20 minutes to complete.

Setup: If you have not yet started Image-Pro Plus, do so now by doubleclicking on the Image-Pro Plus icon within the Image-Pro Plus folder. Once Image-Pro Plus is running, you may begin following the steps below. Important: Please check that your Image-Pro settings for this exercise match those in the dialog boxes shown in the tutorial. Image-Pro 6.0 now retains the settings from previous experiments, which may not be the same as the ones used in this exercise.

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Image-Pro Plus Start-Up Guide

To create a new workflow toolbar, follow the steps below: 1. Select the Edit Workflow Toolbar option from the Window menu. You will see the Edit Toolbar dialog.

2.

Click Add button You will see the list of available buttons on the Add/Edit button dialog.

3.

Use your cursor to highlight Capture and doubleclick it. A capture button will be added to the Workflow Toolbar.

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Advanced Exercises

4.

Now, we need to add more buttons to the Workflow Toolbar. Some of the operations are not already saved. Click the Record button before continuing.

5.

You will see the Macro Recording dialog. Name your new macro Enhance, and click OK.

Now, all your subsequent actions will be recorded for the Workflow Toolbar.

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Image-Pro Plus Start-Up Guide

6.

Capture (or open) the image called Spots.tif.

Some of the spots look a little fuzzy. We will enhance the contrast using the Best Fit display range. 7. Choose Display Range from the Enhance menu. Click the Best Fit button. The image contrast will be automatically adjusted, and the histogram will reflect this:

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Advanced Exercises

Note that there is now an Enhance button in the Workflow Toolbar.

8.

Click Stop Recording to end the macro recording.

Now we will add the button called Show Filters to the Workflow Toolbar. 9. Use your cursor to highlight Show Filters on the Add/Edit button dialog, and click OK.

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Image-Pro Plus Start-Up Guide

10.

Use the Filters dialog to apply a Sharpen filter to the spots image.

11.

Next, we want to count all the spots in the image. Use the Add/Edit button dialog to add the Count and Size button to the Workflow Toolbar.

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Advanced Exercises

12.

Choose Automatic Dark Objects for your count.

13.

Use the Add/Edit button dialog to add the Save to File button to the Toolbar.

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Image-Pro Plus Start-Up Guide

14.

Save your count in a file called spots.csv

15.

Now, you need to save your Workflow Toolbar. Click the Save As button on the Edit Toolbar dialog.

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Advanced Exercises

16.

Name your Workflow toolbar MyTools.ipt.

17.

Click the Save button. Your toolbar is now saved in Image-Pro Plus 6.0.

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Image-Pro Plus Start-Up Guide

Closing all the Windows and Exiting Image-Pro Plus

Close all the windows and exit Image-Pro by selecting the Close All option under the Window menu. Do not save any files. Note: For more exercises, see the demo macros included in your Image-Pro Plus installation.

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Glossary

Glossary: Imaging Terms

Active Window
The image window that is currently selected. If only one image window is open, it will always be the active window. If more than one image window is open, the active window is the frontmost window. Its title bar is highlighted. A technique for representing gray tones through dots of varying sizes placed at regular intervals on a grid. The distance between the dots on the grid is the resolution in dots per inch (DPI). The positions of the dots on the grid fall on two sets of imaginary lines at 90 degrees to one another. The angle of these imaginary lines to the horizontal is 45 degrees, which is the conventional angle in printing processes using the screen technique. Area of Interest. A contiguous subset of pixels defined within an image, which may be arranged in any polygonal shape. They are used to isolate the subset from the rest of the image. Application Program Interface Brightness, Contrast, and Gamma The simplest way to represent an image in digital terms, with each pixel represented by a single bit that is either on or off. Also referred to as Black and White, Line art or Halftone. A scaling technique that interpolates a pixels rescaled value based upon its relationship to surrounding pixels. This technique creates smoother scaled output than traditional factor or pixel replication scaling methods. The smallest unit of information recognized by a computer. A pixel is represented by one or more computer bits. The number of bits per pixel directly determines the number of colors or gray shades that can be represented. See Bit Depth.

Angle dot screen

AOI or Area of Interest

API BCG Bilevel

Bilinear Scaling

Bit

GL-1

Glossary Bit Depth Bitmap

The number of pixels used to represent one pixel value. Also referred to as Pixel Depth and bits-per-pixel (BPP). A two-dimensional array used to represent an image. Each cell in the array contains a value which describes a sample of the image in terms of its color. Contrast this with Vector graphics, where an image is created by describing its elements as geometric objects.

BMP BPP Brightness

Image bit-mapped format used by Windows and OS/2. Bits Per Pixel. Describes the depth of an image. Bilevel images are only 1 BPP, True Color images are 24 BPP. The amount of white in an image. The brighter the image, the more white it contains, and as brightness is increased, each color in the image is shifted more toward white. The process of setting Color Map attributes to compensate for the variations in quality of images due to differences in printers, scanners, and the materials associated with them. You can calibrate your printer(s) or scanner(s), save the calibration(s), then load them each time you use a different scanner or printer, or as your printer toner or ribbon age. The process of redefining the intensity scale associated with an image, to express intensity values in another unit of measure. For example, intensity values can calibrated to reflect density, or temperature. The process of redefining the spatial scale in an image to express distance between pixels in a unit of measure other than pixels. For example, you might calibrate the spatial scale to measure microns or kilometers. Consultative Committee for International Telegraph and Telephone. The committee developed the CCITT compression methods for FAX data, which are used to compress Bilevel image files. The luminance and color components associated with a color image. An RGB image contains 4 channels: Red, Green, Blue, and Luminance. The Luminance channel represents intensity without regard to color.

Calibration, Device

Calibration, Intensity

Calibration, Spatial

CCITT

Channel

GL-2

Glossary Chunk, RGB Chunk

The 3-byte (24-bit) group that represents the red, green and blue values for a pixel. A chunky image data line is made up of consecutive chunks, with no gaps or fillers between chunks. See Image Class. Color model in which color is expressed in varying amounts of Cyan, Magenta, and Yellow (CMY). CMY is the standard color model in printing. Making changes to brightness, contrast, color, highlight, shadows in an image to compensate for perceived deficiencies. A mathematical model describing color. There are several Color Models are used today. Image-Pro supports the popular models. See the Color Model discussion in Section 1 of the Image-Pro Reference Guide, and CMY, HSI, HSV, RGB, and YIQ entries in this Glossary. See Plane. Optimization or reduction of the number of colors in a color image. Accomplished using the Convert To menu's Palette command. Often performed to reduce the amount of space require to store a color image. Mathematical technique that allows an image to be stored with less memory. Redundancies in the internal representation of the image are identified and given a code; the data in the redundancies is then replaced by the code. The sharpness of an image. The higher the contrast in an image the larger the difference between white and black in the image, or (in color images) the more spread out the color range is. As contrast is increased, all the colors or gray shades in the image spread apart. Windows Dynamic Link Library

Class CMY

Color Correction

Color Model

Color Plane Color Reduction

Compression

Contrast

DLL

GL-3

Glossary Document ID
A document ID (DocId) is assigned to an image window when it is opened. It retains this ID for the duration of its existence. IDs are assigned consecutively, in the order in which images are opened. The next higher ID number is used when a new window is created e.g., if image 4 is already open, the next image is assigned an ID of 5. Because of the dynamic nature of DocId (the mix and sequence of images on your desktop varies from session to session), macros involving multiple images should be recorded and played back from an empty imaging area (i.e., one in which there are no images open), or images should be selected relatively using the DOCSEL_NEXTID and DOCSEL_PREVID options described below. These measures will ensure that the recorded image numbers select the intended images on playback.

DPI Drag

Dots Per Inch To press and hold down a button on your pointing device while you move the cursor on the screen. You drag something when you want to move it to a new location on the screen, or when you want to resize it. A halftoning technique where the difference between the image pixel value, and the targeted halftoned pixel is added to the value of the next image pixel. A scaling technique that uses a zoom factor. The zoom factor allows scaling to be accomplished quickly. The method with which an image is stored to disk; based on image class, compression type, and halftone pattern. TIFF and BMP are examples of file formats. A nonlinear logarithmic contrast correction factor which is used to adjust the contrast in dark areas of the image. In gray scale images, the brightness value assigned to a pixel. In an 8-bit image, this value ranges from 0 to 255 (from black, through shades of gray, to white). A method of simulating a gray scale image using patterns of very small black and white dots.

Error Diffusion

Factor Scaling File Format

Gamma Gray Level

Halftoning

GL-4

Glossary Horizontal Differencing

Technique used for enhancing LZW compression. Horizontal differencing takes advantage of the fact that, in many images, the difference between adjacent pixels if frequently 0 or a small number. Instead of compressing the actual value, LZW then compresses the differencing values of the adjacent pixels. Color model in which color is expressed in varying amounts of Hue, Saturation and Intensity. The color space for the HSI model is normally represented by a double hexcone, with Intensity forming the primary axis as shown in the drawing below.
1.0 White

HSI

Green Cyan Blue

Yellow Red Magenta

Hue 0.0 Black Saturation

HSV

Color model in which color is expressed in varying amounts of Hue, Saturation and Value. HSV differs from HSI in the way Intensity is derived. See HSI above. See Modified Huffman. A symbol that represents a tool or item. For example, the symbols on the Ribbon are icons of the available tools. The document that Image-Pro works upon. Also may refer to the original artwork, graphics, or photo that you scan or import. Image category, determined by bit depth. Image classes supported by Image-Pro include Bilevel (1-bit), Palette (8bit), Gray Scale (8-bit), Gray Scale 12 (12-bit) and True Color (24-bit).

Huffman Icon Image

Image Class

GL-5

Glossary Image Conversion IMG Instance, Image Instance

The process of changing an image from one class and/or format and/or compression type to another. Image class developed by Digital Research, and used extensively by Ventura Publisher. A rectangular view of a virtual image. An instance may encompass the entire virtual image, or define just part of it. Instances provide access to virtual image data since virtual images cannot be accessed directly. (Imaging) A collection of image information stored in a database. (Bio) A collection of clones which partially or completely represent the complexity of genomic DNA or cDNA from a defined biological source. Type of compression that allows full recovery of the original image and is fully reversible. Type of compression that degenerates an image during compression and is not fully reversible. Intensity irrespective of color. Usually derived from the mean of the RGB color values. A table in which BCG adjustments are maintained, and through which the image bitmap is rendered. This allows you to experiment with the BCG qualities without affecting your actual image data. Also lets you improve the image for viewing purposes without affecting the integrity of your data for analytical purposes. A method of converting a 24 bit color image to a 8 bit palette image. The MColor technique, developed by Media Cybernetics, uses a fixed 256 color palette, representing a range of values across the color spectrum. During MColoring, the 24 bit image is reduced to 8 bits using a dithering technique to convert the pixel values into MColor palette indicies.. Compression type which is a variation on RLE. Modified Huffman uses a standard table of patterns that represent typical patterns in an image. These tables are usually tuned to the type of data they are used to compress. Image file format developed by Microsoft Paint.

Library

Lossless Lossy Luminance LUT Lookup Table

MColor

Modified Huffman

MSP

GL-6

Glossary Normal Scaling Normalized Image Data

Scaling is accomplished using the following formula:
Dest(x)=Source(x * SourceWidth/DestWidth)

A method of representing image data utilizing 1 byte per color channel, regardless of image class. For Bilevel Gray Scale and Palette images, normalized data is arranged as 1 byte per pixel. Normalized Bilevel pixel values are either 0 or 255. For RGB images, normalized data is arranged as 3 bytes per pixel, representing the 3 color channels. For a Palette class image, the 256 element array used to specify the RGB values associated with that image. Each pixel in the image contains an index to the palette, which contains the RGB value for that pixel. See Appendix C for additional discussion of palette images.

Palette

PColor PCX Pixel Pixel Depth Plane, Color Plane

A method of converting a 24-bit color image to an 8-bit palette image using a palette defined by the user. Image file format developed by ZSoft Inc. Picture element. The smallest element in a digitized image. The number of pixels used to represent one pixel value. Also referred to as Bit Depth and bits-per-pixel (BPP). An arrangement of RGB image data such that all Red values are located in one array, all Green values in a second array, and all Blue values in a third. Contrast with Chunk above. A series of lines connecting pairs of points (vertices). The process of assigning RGB color values to replace gray levels for display purposes. Usually used to highlight certain intensity ranges in a Gray Scale image, with color. Three arrays of 256 lookup values for the Red, Green, and Blue channels. This table is produce by converging the BCGM controls into it. See Spatial Resolution. Color model in which color is expressed in varying amounts of Red, Green, and Blue (RGB). RGB is used by most digital imaging devices. See Chunk.

Polyline Pseudo-color

Response Lookup Table Resolution RGB

RGB Chunk

GL-7

Glossary Ribbon
The graphic bar along the left side of the Image-Pro application window that displays icon controls for AOI creation, zooming, selected commands, and adjustments to brightness, contrast, and gamma. Run Length Encoding. A compression technique that encodes data runs (lengths of identical information).

RLE or Run Length Encoding Sharpen

A filtering process that intensifies edges and details in an image by increasing the difference between gray values of neighboring pixels. Sharpen produces a crisper image. Bilinear scaling option that may be used during printing and resizing. This is a form of resizing that takes into account adjacent pixel values to generate in-between values for the resized image, so that the new image does not look as if pixels were arbitrarily removed or added. When this option is not selected, scaling is done using pixel replication (bigger) and decimation (smaller). Image attribute defined by a two-dimensional (width and height) grid of pixels. A value used to separate gray values into 2 values. Gray values less than the threshold will be set to 0. Gray values greater or equal to the threshold will be set to 255. Typically the threshold is set to 128. Tagged Image File Format. A general-purpose file format recognized as a standard for image files. One page panel in a multi-page poster-size print. TARGA image file format developed by TrueVision Inc. 24-bit color. Image class in which each pixel contains 256 shades each of red, green, and blue, therefore the image can contain up to 16.7 million colors. A component analog color model wherein Y represents luminance value, and I and Q represent the color components. YIQ is the standard in broadcast television.

Smooth

Spatial Resolution Threshold

TIFF Tile TGA True Color

YIQ

GL-8

Glossary Virtual Image

The memory-based copy of an image. It contains the image data and the image attributes. Image attributes can be accessed using the HilImControl and HilImControlInst commands; Image data can only be accessed through an image instance. A zoom factor is the factor used to rescale an image. Positive factors are used to zoom in (expand), and negative factors are used to zoom out (shrink).

Zoom Factor

GL-9

Glossary

GL-10

Index

Index
2
2 x 2 Square Option, 9-14

3
3D VIEWER Button Bitmap, 1-123

7
7 x 7 Circle Option, 9-3

A
Accumulated Histogram, 1-50, 1-67 Active Window, GL-1 Add Track Manually, 10-19, 10-21 Add/Edit button, 10-32, 10-35, 10-36, 10-37 Adding A Macro To A Menu, 1-102, 1-119 Adding A Macro To The Toolbar, 1-105 Adding Your Plug-In To A Menu, 1-118 Advanced Button Bitmap, 4-11 Angle dot screen Defined, GL-1 ANNOTATE Button Bitmap, 1-123 AOI, 3-9, 3-10, 5-17, 5-18, 6-4, 6-9, 9-2 Defined, GL-1 AOI Tools, 1-6 API, GL-1 Application Area, 8 Apply Button, 6, 3-7 Apply Contrast Dialog Box, 8-3 Apply Ranges Checkbox, 9-17 Area Measurement, 7-23, 7-24, 7-25, 8-10, 9-8 Aspect Ratio, 1-74 Attach Image Dialog Box, 6-11 Automatic Bright Objects Option, 7-3 Automatic Dark Objects, 10-37 Automatic Intensity Selection, 9-16 Automatic Option, 9-16 Automatic Tracking, 10-26 Auto-Split Objects, 1-88 Auto-Trace measurement, 4-19 Ave. Dia. Measurement, 7-23, 7-25

Band of Lines Thick Line Profile, 1-69 Bankvid.tif, 3-3, 3-12 BCG Controls, 1-41 BCGM Structure, GL-1 Bell Equalization of Histograms, 1-51 Best Fit Command, 8-2 Best Fit display range, 10-34 Best Fit Equalization of Histograms, 1-52 BESTLUT Button Bitmap, 1-123, 3 Bilevel, 1-35 Bilevel Class, GL-1 Bilinear Scaling, GL-1 Binarizing an image, 1-43 Bins, 7-20, 7-21, 7-22 Bins Start At Field, 7-22 BIOS, 1-14 Bit Defined, GL-1 Bit Depth, 1-34, 1-35 Defined, GL-2 Bit Flag Options, 1-120 Bitmap, GL-2 Analysis, 1-62, 1-66, 1-71 Save File, 1-71 Bitmaps, For Command Buttons, 1-106, 1-123 Black Level Group Box, 5-6 BMP, GL-2 BMP File Type, 3-16 BPP, GL-2 Branch/Endpoints Filter, 1-60 Brightness, 1-42, GL-2 Brightness control, 5 Brightness Control, 3-5 Brightness, Contrast Gamma, 5 Button Bitmaps, 1-106, 1-123 button Statement, 1-103, 1-111 Buttons, Customizing, 1-103

C
Calculating Population Density, 9-1, 9-19, 9-21 Calibrate Before Measuring, 1-73 Calibrated units, 10-10 Calibrating Black Level, 5-6 Calibrating Incident Level, 5-7

B
Background Filter, 1-60 BACKSUB Button Bitmap, 1-123, 2-3

Index-1

Index
Calibrating the Active Image, 10-13 Calibrating The Spatial Scale, 4-6 Calibration, 1-73, GL-2 Device, 1-74 Calibration Command Spatial Calibration, 4-7 Calibration Curve, 5-4 Calibration line, 10-10, 10-15 Calibration Wizard, 10-1, 10-2, 10-3, 10-13 Calibration, Device, GL-2 Calibration, Intensity, GL-2 Calibration, Spatial, GL-2 CAMERA Button Bitmap, 1-123, 2 Capture Preview page, 10-9 Capture Setup, 10-6 Capture Setup Page, 10-5 CCITT, GL-2 Centering Printed Image, 3-15 Chunky, RGB, GL-3 Circuit.tif, 4-5, 4-6, 4-7, 4-13, 4-16 Class, GL-3 Class Measurements, 7-8 Class option, 7-26 Classification Dialog Box, 7-20, 7-22 Bins Start At, 7-22 Number Of Bins Option, 7-20, 7-21, 722 Classification Window, 7-23, 7-24, 7-25 Classifying, 7-1, 9-1 Classifying The Measurement Data, 7-22 Clean Borders, 1-84 Clipboard, 3-4, 9-2 CLOCK Button Bitmap, 1-123 Closing An Image Window, 3-17, 4-26, 5-19, 6-13, 7-45, 8-15, 9-22, 10-30, 10-40 Closing Filter, 1-59, 9-3, 9-4 Closing Image-Pro, 3-17, 4-26, 5-18, 6-12, 7-45, 8-15, 9-22, 10-30, 10-40 CLUB Button Bitmap, 1-123 CMY Defined, GL-3 CMY Color Model, 1-39 Color Channel, GL-2 Color Channel Control, 6, 3-6 Color Composite Demonstration of, 12 Color Correction Defined, GL-3 Color Cube, 8-5 Color Dialog Box, 9-17 Color Model CMY, 1-39 Defined, GL-3 HSI, 1-39 HSV, 1-39, 1-40 RGB, 1-39 YIQ, 1-39, 1-40 Color Models, 1-39 Color Plane, GL-3, GL-7 Color Reduction Defined, GL-3 Color Segmentation, 8-1 Color Segmentation Dialog Box, 8-5 Color-Coding The Objects, 7-26 ColorModels, 1-1 columnbegin Statement, 1-99, 1-100, 1-107 Command Bar, Customizing, 1-99 Command Buttons Assigning A Macro To, 1-105 Customizing The Toolbar, 1-103 Command Windows, 2-10 Common Access, 2-10 Linked, 2-10 Common Access Windows, 2-10 Compensating for Variations Calibrating, GL-2 Composite_Demo demo macro, 2-13 Compression Defined, GL-3 Contrast, 1-42, GL-3 CONTRAST Button Bitmap, 1-123, 2-3 Contrast Control, 2-5, 3-6 Contrast Enhancement, 8-2 Contrast Enhancement Tool, 5, 3-5 Control Button, 7 Convolution Filtering Described, 1-53 Convolution Filters, 1-52, 1-53 Copy Command, 3-4, 9-2 Copy protection plug, 1-7 Copying An Image to a New Window, 9-2 Copying and Pasting, 3-4 Copy-Protection Plug, 1-14 Copy-protection plug problems, 1-14 Count Button, 7-4, 7-6, 7-7, 7-10, 9-18

Index-2

Index
COUNT Button Bitmap, 1-123, 4, 7-3, 8-4, 9-7 Count/Size Command, 7-3, 8-4, 9-6, 9-7, 9-16 Count/Size Dialog Box, 8-4, 8-10 Count/Size Options Dialog Box, 7-26, 9-10, 9-16 Count/Size Options Dialog Box, 7-11 Count/Size window Class option, 7-26 Count/Size Window, 7-3, 9-6, 9-7 Apply Ranges, 9-17 Automatic Intensity Selection, 9-16 Bright Objects, 7-3 Count Button, 7-10, 9-18 Edit Range Button, 7-15 Ellipse Option, 9-10 Fill Holes Checkbox, 7-11 Labels None Option, 9-16 Manual Button, 9-6, 9-7 Measure Menu, 7-6 Object Attributes, 7-13 Options Button, 7-11, 7-26, 9-10, 9-16 Outline Option, 9-16 Select Ranges Button, 7-4 CountAndClassify demo macro, 2-12 Counting, 7-1, 7-10, 9-1 and Measuring Multiple Objects, 1-72 Objects, 1-86 Counting The Grains, 9-16 Counting the Spots, 8-9 CountSmall_Cells demo macro, 2-12 Creating a Report, 7-31 Creating Outlines, 9-6 Cumulative Histogram, 1-67 Custom Calibration, 1-64 Custom installation, 1-13 Customizing Menus and Buttons, 1-99, 1103 Customizing the Command Menus and Buttons, 1-97 Defining Line For Line Profile, 5-10, 5-14, 5-16 For Spatial Calibration, 4-9 Demo.IPM demo macro, 2-12 Demonstration macros, 2-11 Density Optical Analysis, 5-1 Population, 9-1, 9-19, 9-21 Density Curve, 5-4, 5-5, 5-8 Density Plot, 5-12 Device Calibration Defined, GL-2 Device Drivers, 1-31 Dialog box Prompted Text, 7-37 Selected Text, 7-39 DIAM Button Bitmap, 1-123 Difference, 10-27 Dilation Filter, 1-58 Distance Filter, 1-60 DLL, GL-3 Dna.tif, 5-2 Document ID, GL-4 DPI, GL-4 Drag, GL-4 DRAW Button Bitmap, 1-123, 3 Draw/Merge Objects, 1-86

E
Edge Filters, 1-56 Edit Lens List, 10-8 Edit Measurements Range Dialog Box, 7-15 Edit Measurements Range graph, 7-15 Edit Range Button, 7-15 Edit Toolbar, 10-32, 10-38 Editing the .MNU file, 1-99 Editing the Spectrum, 6-4 Eliminating Object Labels, 9-16 Eliminating Objects, 7-17 Ellipse Option, 9-10 ELLIPTICAL AOI Button Bitmap, 2-3, 2-6 E-Mail, 6-10 endcolumn Statement, 1-99, 1-100, 1-107 endgroup Statement, 1-100, 1-110 Enhance menu, 10-34 Enhancement Filters, 1-52, 1-53

D
DARK MODE Button Bitmap, 1-123 DATA Button Bitmap, 1-123 DATABASE Button Bitmap, 1-123, 2 Default destination, 1-11 Defining An AOI, 3-9

Index-3

Index
Enhancing the Image, 3-5 Enhancing Your Image, 1-41 EQUALIZE Button Bitmap, 8-2 Equalize Command Best Fit, 1-52 Erosion Filter, 1-58, 9-14 Error Diffusion, GL-4 Excel Spreadsheets, 7-30 Exit Command, 3-17 Exm50.tif, 7-2, 7-10, 7-32, 7-33, 7-34 Exponential Equalization of Histograms, 1-51 Eyedropper Tool, 8-6 Closing Option, 9-3 Erode Option, 9-14 Open Option, 9-5 Filtering Discussion, 1-52 FFT, 1-61 Filtering Command, 4-3, 9-3 Filtering With FFTs, 6-1 Filtering, Calibrating, and Measuring, 4-1 Filters Background, 1-60 Branch/Endpoints, 1-60 Close, 1-59 Dilate, 1-58 Distance, 1-60 Erode, 1-58 Flatten, 1-55 Gauss, 1-54 Higauss, 1-55 Hi-Pass, 1-54 Hi-Pass FFT, 1-62 Horizontal Edge, 1-57 Laplacian, 1-57 Local Equalize, 1-55 Lo-Pass, 1-53 Lo-Pass FFT, 1-62 Median, 1-55 Open, 1-58 Phase, 1-60 Pruning, 1-60 Rank, 1-55 Reduce, 1-60 Roberts, 1-56 Scuplt, 1-60 Sharpen, 1-54 Sobel, 1-56 Spike Cut, 1-62 Thinning, 1-60 Tophat, 1-59 Variance, 1-58 Vertical Edge, 1-57 Watershed, 1-60 Well, 1-59 Filters Button Bitmap, 3-8 Filters dialog, 10-36 Filters Dialog Box, 3-8 FiltersPhase, 1-57 Find Tracks Automatically, 10-27 Fish.seq, 10-26

F
Factor Scaling, GL-4 Fast Fourier Transform, 1-61 Fast Fourier Transforms, 5-17, 6-1, 6-9 FFT, 1-1, 5-17, 6-1, 6-9 FFT Button Bitmap, 1-123, 4 FFT Command, 6-2 FFT Filtering, 1-61, 6-1 FFT Spectrum Window, 5-17, 6-9 FFT window, 2-10 FFT Window, 6-2 Filter Button, 6-5 Forward Button, 6-3 Hi-Pass Option, 5-17, 6-9 Inverse Button, 6-8 Lo-Pass Option, 5-17, 6-9 New Image Option, 6-7 Options Button, 6-7 Spike Cut Option, 5-17, 6-9 Spike-Cut Option, 6-4 FFT1 - FFTprint.tif, 6-3 FFTprint.tif, 6-2 File Name List Box, 7-30, 7-44, 9-12 File Parameter Block, GL-4 File Type, 3-16 Fill Holes, 7-11 Filter Button, 6-5 FILTER Button Bitmap, 1-123, 4, 4-3, 9-3 Filter Dialog Box, 4-3, 4-5 Filter Range Visualization of effect, 7-14 Filter Window, 9-3 2 x 2 Square, 9-14 7 x 7 Circle, 9-3 Apply Button, 9-3, 9-4, 9-5, 9-15

Index-4

Index
Flatten Filter, 1-55 Floating Point 32 BPP, 1-37 Forward Button, 6-3 Fourier Transform, 1-61 Fourier Transforms, 5-17, 6-1, 6-9 Freeform Optical Density, 1-64 FREEFORM AOI Button Bitmap, 3, 6 Frequency Filtering, 1-61 Frequency Spectrum, 6-2 Accumulated, 1-50 Analysis, 1-62, 1-66 Graphic, 1-67 Numeric, 1-67 Report, 1-67 Save File, 1-67, 1-89 Histogram Equalization, 1-51 Bell Curve, 1-51 Best Fit, 1-52 Exponential, 1-51 Linear, 1-51 Logarithmic, 1-51 Histograms, 1-44 Hole Area Measurements, 7-7, 7-8 Horizontal Differencing, GL-5 Horizontal Edge Filter, 1-57 HSI Defined, GL-5 HSI Color Model, 1-39 HSV Defined, GL-5 HSV Color Model, 1-39, 1-40 Huffman, GL-5

G
Gamma, GL-4 Gamma, 1-43 Gamma control, 2-5 Gamma Control, 3-6 Gamma Correction, 1-43 Gamma Correction Curve, 1-43 Gauss Filter, 1-54 Gear.tmpl template, 7-37 Getting Started, 1 Glossary, GL-1 Grains.tif, 9-2, 9-20 Graphical Exclusion of Ranges, 7-14 Gray Level Defined, GL-4 Gray Scale, 1-36 12 BPP, 1-36 16 BPP, 1-37 32 BPP, 1-37 8 BPP, 1-36 group Statement, 1-100, 1-110, 1-116

I
Icon Defined, GL-5 Icon, Assigning to Toolbar, 1-103 Icons, 1-29, 9-13, 9-14 Identifying Cell Sites, 9-3 Image, GL-5 Image Button, 4-9, 5-6, 5-7 Image Capture Board, 1-5 Image Class, 1-35, GL-5 Bilevel, 1-35 Floating Point, 1-37 Gray Scale, 1-36 Gray Scale 12, 1-36 Gray Scale 16, 1-37 Palette, 1-38 RGB, 1-38 Image Conversion Defined, GL-6 Image Digitization, 1-33 Image Enhancement, 1-41 Image Icon, 9-13, 9-14 Image Overlay, 7-32, 7-33, 7-34 Image Processing, 1-33

H
Halftoning, GL-4 HAND Button Bitmap, 1-124 HAPPY Button Bitmap, 1-124 Hardware and Services, 1-32 HASP Diagnostic program, 1-14, 1-15 HEART Button Bitmap, 1-124 Hide Unselected Tracks, 10-24 Hiding Objects, 1-88, 7-17 Higauss Filter, 1-55 Hi-Pass Filter, 1-54 Hi-Pass Option, 5-17, 6-9 HISTO Button Bitmap, 1-124, 4 Histogram, 1-62, 1-66

Index-5

Index
Image Thumbnail, 3-15 Image Windows, 2-7 Image-Pro Window, 2-1 IMG, GL-6 Incident Level Group Box, 5-7 INFO Button Bitmap, 1-124 Inserting Data into a Report, 7-35 Inserting prompted text into a report, 7-37 Inserting selected text into a report, 7-39 Install Drivers, 1-26 Installation type, 1-10 Installing Copy-Protection Plug, 1-14 Image Capture Support, 1-26 Image-Pro Plus on a Network, 1-15 IQbase Support, 1-28 Video Capture Support, 1-26 Installing Image-Pro Plus, 1-7 Instance, GL-6 Intensity Analysis, 1-62 Intensity Analysis Tools, 1-66 Intensity Calibration, 1-62 Defined, GL-2 Intensity Calibration - dna.tif Options Button, 5-6 Intensity Calibration - Dna.tif, 5-3, 5-8 Name field, 5-4 New Button, 5-4 Optical Density Option, 5-4, 5-5 Intensity Curve, 5-4, 5-8 Intensity Index, 1-41 Intensity Modification, 1-41 Internet Addresses, 6-10 Introducing Image-Pro Plus version 6.0, 1-1 Inverse Transformation, 5-18, 6-7, 6-9 Inverse/Filter Options Dialog Box, 6-7 IP Address, 1-21 IQbase, 1-3 IQbase Support, 1-28 IRREG Button Bitmap, 1-124 item Statement, 1-100, 1-109, 1-118 License Client, 1-15, 1-19 License Server, 1-10, 1-15, 1-16, 1-17, 1-18, 1-21 Line Profile, 1-62, 1-66, 1-69 Analysis, 1-62, 1-69 Report, 1-70 Save File, 1-70 Line Profile - Dna.tif, 5-9, 5-13, 5-16 Line Profile Command, 5-9, 5-13, 5-16 Line Profile Graph, 5-12 Linear Equalization of Histograms, 1-51 Linked Windows, 10 Load Outlines Dialog Box, 9-19 Loading an Image, 3-2 Loading An Image, 3-9, 4-2, 5-2, 6-2, 9-2 Loading an Image from the Database, 4-2 Loading Outline Files, 9-20 Local Equalize Filter, 1-55 Local Zoom, 10-11 Logarithmic Equalization of Histograms, 151 Lookup Table, 8-3 Defined, GL-6 Lookup Table (LUT), 3-7 Lo-Pass Filter, 1-53 Lo-Pass Option, 5-17, 6-9 Lossless Defined, GL-6 Lossy Defined, GL-6 Luminance Defined, GL-6 LUT Defined, GL-6

M
MACEDIT Button Bitmap, 1-124, 2-4 MACREC Button Bitmap, 1-124, 2-4 Macro Adding To A Menu, 1-119 MACRO Button Bitmap, 1-124, 2-4 Macro Recording, 10-33 macrobutton Statement, 1-102, 1-105 macroitem Statement, 1-119 Macros, 1-32 Adding To A Menu, 1-102 Assigning To A Button, 1-105 Composite_Demo, 13

L
Labels List Box, 9-16 Laplacian Filter, 1-57 Library, GL-6 License agreement, 1-9

Index-6

Index
CountAndClassify, 12 CountSmall_Cells, 12 Demo.IPM, 12 Demonstration, 11 MeasureAngles, 12 MeasureAutoradiograph, 12 MeasureIronPhases, 12 MeasureStain, 12 Separate_Fluorochromes, 12 ThroughfocusSequence, 12 Magic Wand, 1-75 Magnifier page, 10-7 Magnifying Glass, 4-6, 4-9 MAIL Button Bitmap, 1-124, 2-3, 6-10 Manual Measurements, 1-74 Tagging, 1-89 Manual Button, 9-6, 9-7 Manual Intensity Selection, 9-6, 9-7 Manual Tracking, 10-18 Max Count/Size Command, 8-12 Maximize Button, 7 MColor, GL-6 Mean Count/Size Command, 8-12 MEASURE Button Bitmap, 1-124, 2-4, 4-11 MeasureAngles demo macro, 2-12 MeasureAutoradiograph demo macro, 12 MeasureIronPhases demo macro, 2-12 Measurement Data, Saving, 1-73 Measurement Distributions, 7-20 Measurement Ranges, 9-8 Measurement Tabbed Dialog Box, 4-12 Measurement Tools, 1-76 Measurements, 7-6, 7-22 Features, 1-74, 1-76 Manual, 1-74 Tools, 1-74 Measurements Command, 4-11 Measurements Dialog Box, 8-10 Measurements Window Save Command, 7-29 Measurements Window Dialog Box, 4-18 MeasureStain demo macro, 2-12 Measuring, 4-1, 7-1, 9-1 and Counting, 1-72 and Counting Multiple Objects, 1-72 Single Objects, Manually, 1-72 Measuring Length, 4-11, 4-16 Measuring length automatically, 4-19 Measuring length manually, 4-12, 4-14, 4-16 Median Filter, 1-55 Menu Adding A Macro To, 1-119 Adding A Plug-In To, 1-118 columnbegin Statement, 1-99 Defining A Pop Out Menu, 1-116 endcolumn Statement, 1-99 group Statement, 1-116 item Statement, 1-118 macroitem Statement, 1-119 Startup items, 1-121 Menu Bar, 1 Menu Items Adding A Macro, 1-102 Customizing, 1-99 Moving, 1-101 Naming Conventions, 1-101 Removing, 1-101 Renaming, 1-101 Micron Key, 4-10 Min Count/Size Command, 8-12 Minimize Button, 7 MNU File Adding A Menu Item, 1-118 group Statement, 1-116 item Statement, 1-118 macroitem Statement, 1-119 Modal Plug-ins, 1-121 Modeless Plug-ins, 1-121 Modified Huffman Encoding, GL-6 Morphological Filters, 1-58, 1-59 Moving a Menu Item, 1-101 MSP Defined, GL-6 MULTIPLE AOIS Button Bitmap, 2-3, 2-6 MyTools.ipt, 10-39

N
Name Field, 4-8, 5-4 Naming Conventions

Index-7

Index
Menu Items, 1-101 NetHASP, 1-20 New AOI Button Bitmap, 2-6 New Button, 4-8, 5-4 New Features in Version 6.0, 1-3 New Image Option, 6-7 New Report Button, 7-31 New Report Dialog Box, 7-31 Nonconvolution Filters, 1-58 None Option, 9-16 Normal Scaling, GL-7 Normalized Data Format, GL-7 Number Of Bins, 7-20, 7-21, 7-22

P
Package Contents, 1-4 Palette, 1-38 Palette Class, GL-7 PAN Button Bitmap, 3, 7 Parallel port, 1-14 Parking Area, 9-13, 9-14 Paste Command, 3-4 Paste New Command, 9-2 PColor, GL-7 PCX Defined, GL-7 Performing Color Segmentation, 8-4 Perim. Measurement, 7-23, 7-25 Phase Filter, 1-57, 1-60 Pixel Defined, GL-7 Pixel Depth, 1-34 Defined, GL-7 Plotting Density Using a Baseline, 5-13 Plotting Measurement Data, 1-90 Plotting The Density Of A Lane, 5-9 Plug-In Adding To A Menu, 1-118 Plug-ins, 1-31 Polyline, 4-17 Defined, GL-7 Pop-out Menu Creating, 1-116 Population Density, 9-1, 9-19, 9-21 Analysis, 1-94 Population Density Command, 9-19 Population Density Data, 9-20 Population Density Window, 9-20 Positioning Image, 3-14 Pre-defined Button Bitmaps, 1-106, 1-123 Preparing The Grain Image, 9-13 PRINT Button Bitmap, 1-124, 2-2 Print Command, 3-14, 3-15 Print Dialog Box, 3-14, 3-15 Print Position Dialog Box, 3-14 Printing the Image, 3-14 Problems Copy-protection plug, 1-14 Product Features, 1-1 PROFILE Button Bitmap, 1-124, 4 progbutton Statement, 1-103, 1-110 progitem Statement, 1-100, 1-108

O
Object Labels, 1-84 Outlines, 1-84 Object Attributes Window, 7-13 Object labels, 7-27 Objective page, 10-8, 10-11 OPEN Button Bitmap, 1-124, 2, 5-2, 6-2, 7-2, 8-2 Open Command, 3-2, 8-2 Open File Dialog Box, 3-2, 8-2 Opening Filter, 1-58, 9-5 OPER Button Bitmap, 1-124, 4 Optical Density Curve, 1-63 optical density, 5-5 Optical Density, 1-62, 1-63 Custom Calibration, 1-64 Freeform, 1-64 Optical Density Analysis, 5-1 Optical Density Calibrations Dialog Box, 5-6 Black Level, 5-6 Incident Level, 5-7 Optical Density Option, 5-4, 5-5 Options Button, 5-6, 6-7, 7-11 Outline Length, 1-85 Outline dialog box, 4-19 Outline Files, 9-12, 9-20 Outline Option, 9-16 Outline style option, 7-26 Outlines List Box, 9-10, 9-16

Index-8

Index
Program Group, 1-29 program icons, 1-12 Prompted text, 7-37 Prompted Text Dialog Box, 7-37 Pruning Filter, 1-60 PSEUDO Button Bitmap, 1-124, 2-4 Pseudo-Color Defined, GL-7 Defined, GL-7 RGB Chunk Defined, GL-7 RGB Color Model, 1-39 RGB-chunky, 1-39 Ribbon Defined, GL-8 Roberts Filter, 1-56 Roundness Measurement, 8-11 Run Length Encoding, GL-8

R
Range Count/Size Command, 8-12 Ranges, 1-84 Rank Filter, 1-55 Rectanglar Tool, 3-9 Rectangle Tool, 6-4 Rectangular AOI, 8-9 RECTANGULAR AOI Button Bitmap, 2-3, 2-6, 8-9 Reducing Filter, 1-60 Reference Calibration, 10-2, 10-3, 10-8, 10-9, 10-12, 10-15 Reference Line, 10-15 Reference process page, 10-4 Removing a Menu Item, 1-101 Removing Extraneous Grains, 9-5 Removing Objects, 7-17 Renaming a Menu Item, 1-101 Report Line Profile, 1-70 Prompted text, 7-37 Save Command, 7-44 Selected text, 7-39 REPORT Button Bitmap, 1-124, 2-2, 7-31 Report Generator, 7-31 Templates, 7-37 Report Generator Command, 7-31 Report Generator Window, 7-31 Report Templates, 7-32 Reset Button, 2-6 RESIZE Button Bitmap, 1-124 Resize Image Dialog Box, 3-11 RESLUT Button Bitmap, 1-125, 3 Resolution, GL-7 Defined, GL-8 Response Lookup Table, GL-7 Restoring An Image Window, 9-13, 9-14 RGB, 1-38

S
SAD Button Bitmap, 1-125 Samples Count/Size Command, 8-13 Save As Command, 3-16 Save As Dialog Box, 3-16, 7-44 SAVE Button Bitmap, 1-125, 2 Save Command, 4-24, 7-29, 7-44 Save Data Dialog Box, 7-29 Save Files As List Box, 7-30 Save Measurements Dialog Box, 4-24 Save Outlines Command, 9-11 Save Outlines Dialog Box, 9-11 Save Tracks, 10-24 Saving Data, 1-94 Measurement Data, 1-73 Saving a Plot, 5-16 Saving An Image, 3-16 Saving Outlines, 9-10 Saving The Measurement Data, 7-29 Saving the Measurements, 4-24 SCAL Button Bitmap, 1-125 Scaling Dialog Box, 4-9 SCAN Button Bitmap, 1-125, 2-2 Scattergram, 1-91 Screen Capture Options Dialog Box, 5-16 Sculpt Filter, 1-60 SEGMENT Button Bitmap, 1-125, 2-4 Segmentation, 10-28 Segmentation Dialog Box, 7-4 Segments, 1-85 Select, 7-6, 7-14 measurement types, 1-79 Select Colors Command, 8-5 Select Line Starting Point message box, 4-12

Index-9

Index
Select Measurements Dialog Box, 7-7 Select Track, 10-24 Selected text, 7-39 Selected Text Dialog Box, 7-39 Selecting An Object, 7-13 Send Mail, 6-10 Send Mail Dialog Box, 6-10 Sending E-mail, 6-10 Sending E-Mail, 6-10 Separate_Fluorochromes demo macro, 2-12 separator Statement, 1-100, 1-110 Sequence menu, 10-27 Sequence toolbar, 10-18 Server installation, 1-17 Set Ranges Command, 9-8 Set Ranges Dialog Box, 9-8 Setting Black Level, 5-6 Setting Incident Level, 5-7 Setup program, 1-8 Setup screen, 1-12 Sharpen Defined, GL-8 Sharpen Filter, 1-54, 4-3 Sharpening An Image, 4-3 Shift+V, 3-4 Show All Tracks, 10-24 Show Graph, 10-25, 10-29 Single Line Profile, 1-69 Single Variable Class. Command, 7-20, 7-22, 7-24 Slider button, 3-5 Smooth Defined, GL-8 Smoothing, 1-84 SNAP Button Bitmap, 1-125, 2-4 Snap Command Button, 1-125 Sobel Filter, 1-56 Solutions Zone, 1-31 Sorting Multiple Objects, 1-77 SPAD Button Bitmap, 1-125 SPATIAL Button Bitmap, 4 Spatial Calibration, 4-1, 4-6, 4-7 Spatial Calibration - Circuit.tif, 4-7 Image Button, 4-9 Name Field, 4-8 New Button, 4-8 Unit field, 4-8 Spatial Calibration, Defined, GL-2 Spatial Filtering, 1-52 Spatial Form, 6-7 Spatial Resolution Defined, GL-8 Special Filters, 1-60 Specifying Black And Incident Levels, 5-6 Specifying Ranges, 9-8 Spike-Cut Option, 6-4 Split Objects, 1-87, 7-17 Splitting Objects, 7-17 spots.csv, 10-38 Spots.tif, 10-34 Spreadsheet Format, 7-30 Standard Optical Density Curve, 1-63 Formula, 1-63 Standard-of-Measure, 4-9 Starting Image-Pro, 1-29 STARTUP Bit Option, 1-121 Statistics, 4-22, 8-12 Status Bar, 8 Std. Dev Count/Size Command, 8-12 Strength Option, 4-4, 4-5 Sum Count/Size Command, 8-13 SUM Button Bitmap, 1-125 SYNC Button Bitmap, 3 Syntax,*.MNUFile, 1-106 System Requirements, 1-5

T
Taking And Viewing Measurements, 7-22 Technical Support, 1-29, 1-30 Template Demo_Time_Lapse for macros, 2-12 Templates Gear.tmpl, 7-37 Report Generator, 7-37 Test Capture, 10-5 Test Capture page, 10-5 TGA Defined, GL-8 Thick Horz, 1-69 Vert, 1-69 Thick Profile Line Profile, 1-69

Index-10

Index
Thick Vert Command, 5-10, 5-14, 5-16 Thinning Filter, 1-60 Threshold, GL-8 Threshold Ranges, 7-4 Thresholding, 7-2 binarizing an image, 1-43 ThroughfocusSequence demo macro, 2-12 Thumbnail, 3-15 TIFF Defined, GL-8 Tile Defined, GL-8 Title Bar, 7 Tool Bar, 1, 2 macrobutton Statement, 1-102, 1-105 Tool Bar, Customizing, 1-103 Tophat Filter, 1-59 Trace measurement, 4-14, 4-16 Track Objects, 10-19, 10-27 Tracking Data Table, 10-19, 10-20, 10-22, 10-23, 10-27 Tracking Options, 10-21, 10-22 Tracking Statistics, 10-22 Trackseq.seq, 10-18 Transforming An Image, 6-2 Transforms, 6-1 True Color Defined, GL-8 Tumor_cells.tif, 8-2 Tutorial Basics, 3-1 Calculating Population Density, 9-1 Counting, Measuring & Classifying, 7-1, 9-1 Filtering With Fast Fourier Transforms, 6-1 Optical Density Analysis, 5-1 Typical installation, 1-16, 1-19 Upgrading your software, 1-22 USER1 Button Bitmap, 1-125 USER2 Button Bitmap, 1-125 USER3 Button Bitmap, 1-125 Using Outlines, 1-89 Using templates, 7-37

V
Variance Filter, 1-58 Vertical Edge Filter, 1-57 Viewing Measurement Data, 1-90 Viewing A Single Object, 7-13 Viewing Measurements, 7-22 Virtual Image, GL-9

W
Watershed Split Objects, 1-88 Watershed Filter, 1-60 Web Update, 1-27 Welcome screen, 1-8, 1-16, 1-19 Well Filter, 1-59 Window Overview, 2-1 Workflow Toolbar, 10-31, 10-32, 10-33, 10-35, 10-36, 10-38 Working with Filters, 3-8 Worksheet Format, 7-30

X
X-axis, 5-4 XLS Format, 7-30

Y
Y-axis, 5-4 YIQ Defined, GL-8 YIQ Color Model, 1-39, 1-40

U
UAC, 1-22, 1-23, 1-25 UNDO Button Bitmap, 2-3 Unit Field, 4-8 Up One Level button, 8-2 Upgrade Authorization Code, 1-22, 1-24, 125 Upgrading, 1-6

Z
ZOOM Button Bitmap, 3, 7, 3-12, 4-6, 4-9 Zoom Command, 4-11 Zoom Factor, GL-9 Zoom Pop-Out Menu, 4-11 Zoom Tool, 3-12, 4-6, 4-9 Zooming In On The AOI, 3-12

Index-11

Index

Index-12