EVOS™ M7000 Imaging System

For Fluorescence and Transmitted Light Applications

Catalog Number AMF7000

Doc. Part No. 710247

Publication Number MAN0018326
Revision C.0

For Research Use Only. Not for use in diagnostic procedures.

Information in this document is subject to change without notice.

DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS
DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history MAN0018326

Revision Date Description

Added “Chapter 6 – Analyze and annotate saved images”, and revised Appendix C to
C.0 16 February 2021
add the EVOS™ Analysis application section.
Update the “Create and run automated scan” chapter and “Appendix C - Automate
B.0 28 January 2020 tab” section, and the relevant screens throughout the document. Add more
information about Raw and Displayed images, and Tiled and Merged images.
A.0 17 December 2018 New user guide

Important Licensing Information

These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and
conditions of all applicable Limited Use Label Licenses.

Manufacturer: Life Technologies Corporation | 22025 20TH Ave SE | Bothell, WA 98021.

Trademarks
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
Cy is a registered trademark of GE Healthcare UK Limited. DRAQ5 is a registered trademark of Biostatus Limited. Windows is a
registered trademark of Microsoft Corporation. Kimwipes is a registered trademark of Kimberly-Clark Corporation. Hoechst is a
registered trademark of Hoechst GmbH. Swagelok is a registered trademark of Swagelok Company.

© 2021 Thermo Fisher Scientific Inc. All rights reserved.

 Contents

About this guide ...................................................................................................................... 4

1. Product information .......................................................................................................... 5
Product description ......................................................................................................................................... 5
Standard items included................................................................................................................................. 7
Instrument exterior components ................................................................................................................... 8
Computer ports and slots ............................................................................................................................. 10
Graphical user interface (GUI)..................................................................................................................... 11
2. Installation .......................................................................................................................12
Operating environment and site requirements ......................................................................................... 12
Prepare for installation ................................................................................................................................. 13
Set up............................................................................................................................................................... 14
After installation is complete ....................................................................................................................... 16
3. Capture and save images .................................................................................................19
Overview ........................................................................................................................................................ 19
Capture images .............................................................................................................................................. 20
Capture Z-Stack ............................................................................................................................................. 33
Save ................................................................................................................................................................. 40
4. Create and run automated scan .......................................................................................44
Overview ........................................................................................................................................................ 44
Create a scan protocol ................................................................................................................................... 45
Run automated scan protocol ...................................................................................................................... 69
5. Review saved images .......................................................................................................71
6. Analyze and annotate saved images .................................................................................75
EVOS™ Analysis application ...................................................................................................................... 75
Image display settings .................................................................................................................................. 79
Grid ................................................................................................................................................................. 80
Scale bar .......................................................................................................................................................... 81
Histogram ....................................................................................................................................................... 82
Measurements and Annotations ................................................................................................................. 83
Analyze cell culture ....................................................................................................................................... 85
Count cells – Auto Count ............................................................................................................................. 86
Count cells – Manual Count......................................................................................................................... 90
Measure confluence....................................................................................................................................... 92
Calculate transfection efficiency .................................................................................................................. 95

EVOS™ M7000 Imaging System User Guide 1

 Save analysis results ...................................................................................................................................... 98
Batch Analysis .............................................................................................................................................. 100
7. Care and maintenance ...................................................................................................103
General care.................................................................................................................................................. 103
Objective lens care ....................................................................................................................................... 103
Stage care ...................................................................................................................................................... 104
Decontamination procedures..................................................................................................................... 104
Calibrate the stage ....................................................................................................................................... 105
Calibrate vessel ............................................................................................................................................ 107
Change EVOS™ light cubes ........................................................................................................................ 111
Change the objectives ................................................................................................................................. 112
Calibrate the objectives ............................................................................................................................... 114
Install the shipping restraints .................................................................................................................... 118
Appendix A: Troubleshooting ...............................................................................................120
Image quality issues .................................................................................................................................... 120
Software interface issues ............................................................................................................................ 121
Mechanical issues ........................................................................................................................................ 121
Appendix B: System overview ..............................................................................................122
Technical specifications .............................................................................................................................. 122
Operation principles and technical overview.......................................................................................... 124
Image capture and save formats ............................................................................................................... 125
Appendix C: Graphical user interface (GUI) ..........................................................................126
Viewing area ................................................................................................................................................ 127
Capture tab ................................................................................................................................................... 139
Automate tab................................................................................................................................................ 157
Review tab .................................................................................................................................................... 192
Settings .......................................................................................................................................................... 196
EVOS™ Analysis Application ................................................................................................................... 215
Appendix D: EVOS™ Onstage Incubator .................................................................................224
Technical specifications .............................................................................................................................. 225
EVOS™ Onstage Incubator components ................................................................................................... 226
Set up the EVOS™ Onstage Incubator ....................................................................................................... 228
Use the EVOS™ Onstage Incubator ........................................................................................................... 233

2 EVOS™ M7000 Imaging System User Guide

 Appendix E: Safety ...............................................................................................................236
Safety conventions used in this document............................................................................................... 236
Symbols on instruments ............................................................................................................................. 237
Safety labels on instruments ...................................................................................................................... 239
General instrument safety .......................................................................................................................... 240
Safety requirements for EVOS™ Onstage Incubator ............................................................................... 241
Chemical safety ............................................................................................................................................ 242
Chemical waste safety................................................................................................................................. 243
Electrical safety ............................................................................................................................................ 244
Physical hazard safety ................................................................................................................................ 245
Biological hazard safety .............................................................................................................................. 245
Safety and electromagnetic compatibility (EMC) standards ................................................................. 246
Documentation and support .................................................................................................247
Obtaining support ....................................................................................................................................... 247

EVOS™ M7000 Imaging System User Guide 3

 About this guide

Audience This user guide is for laboratory staff operating, maintaining, and analyzing data
using the Invitrogen™ EVOS™ M7000 Imaging System.

User attention Two user attention words appear in this document. Each word implies a specific
words level of observation or action as described below.

Note: Provides information that may be of interest or help but is not critical to the
use of the product.

IMPORTANT! Provides information that is necessary for proper instrument

operation, accurate installation, or safe use of a chemical.

Safety alert words Three safety alert words appear in this document at points where you need to be
aware of relevant hazards. Each alert word—CAUTION, WARNING, DANGER—
implies a particular level of observation or action, as defined below:

CAUTION! – Indicates a potentially hazardous situation that, if not avoided,

may result in minor or moderate injury. It may also be used to alert against
unsafe practices.

WARNING! – Indicates a potentially hazardous situation that, if not

avoided, could result in death or serious injury.

DANGER! – Indicates an imminently hazardous situation that, if not

avoided, will result in death or serious injury. This signal word is to be
limited to the most extreme situations.

4 EVOS™ M7000 Imaging System User Guide

 1. Product information

Product description
EVOS™ M7000 The Invitrogen™ EVOS™ M7000 Imaging System (Cat. No. AMF7000) is a fully
Imaging System automated, digital, inverted multi-channel fluorescence and transmitted light
imaging system. The system is designed for a broad range of applications including,
but not limited to, multi-channel fluorescence imaging, multiple-position vessel
scanning, area scanning with montage or tile stitching, and time-lapse imaging.

EVOS™ M7000 The EVOS™ M7000 Imaging System is controlled by the integrated Invitrogen™
Software EVOS™ M7000 Software accessed by the touchscreen monitor or the computer
mouse and keyboard. The software comes pre-installed with the software shortcut
placed on the desktop and under StartAll Programs ThermoM7000.
Key features of the EVOS™ M7000 Software include:
• Capture: Allows control over every aspect of the system for image capture
through a simple user interface. All images acquired can be saved in TIFF,
PNG, C01, DIB, and JPEG formats, or compiled into a video sequence in AVI or
WMV formats.
• Automate: Allows the creation, saving, and running of user-defined routines to
automate image collection.
• Autofocus: Can be set up in five different modes to optimize speed and
accuracy.
• Tiled images and Image stitching: Allow the scanning of an area to acquire
multiple images to build tiled and stitched images. The Review tool allows
zooming in/out and panning of the composite image. The entire scan or only
regions of interest can be exported.
• Z-stacking: Captures a series of images along the z-axis that can be saved
individually or combined into a Z-stack projection with a greater depth of field
than any of the individual source images.
• Time lapse: Creates and runs time lapse movies based on user specifications.
• Review: Allows you to review captured images and analyze them with the
EVOS™ Analysis application that is included in the EVOS™ M7000 Software
package (page 6).

Note: For a detailed description of the EVOS™ M7000 Software controls, see
“Appendix C: Graphical user interface (GUI)” (page 126).

EVOS™ M7000 Imaging System User Guide 5

 EVOS™ Analysis The EVOS™ Analysis application is a quantitative image analysis and annotation
application tool that runs independently from the EVOS™ M7000 software used for controlling
the instrument. The application is included in the EVOS™ M7000 Software package.
Key features for the EVOS™ Analysis application include:
• Analysis and annotation: Allow you to change image display settings, toggle
the display of grid and scale bar, display pixel intensity histogram, and
annotate the image with basic geometric shapes and display dimensions, area,
or perimeter information for the annotations.
• Cell count: Allows you to perform automatic or manual cell count in
fluorescence mode post-acquisition.
• Confluence: Allows you calculate the percentage confluence of your culture
based on selected reference objects and background.
• Transfection efficiency: Allows you to calculate the percentage of cells that
express a fluorescence marker compared to the entire population.
• Batch Analysis: Allows you to save and apply the analysis parameters set in
Auto Count, Confluence, and Transfection Efficiency tools to other images that
you have collected and saved in an image folder

Note: For a detailed description of the EVOS™ Analysis application controls, see
“EVOS™ Analysis Application” in “Appendix C: Graphical user interface (GUI)”
(page 215).

6 EVOS™ M7000 Imaging System User Guide

 Standard items included
Before the EVOS™ M7000 Imaging System is installed (page 12), unpack the unit
and accessories and verify all parts are present. Examine the instrument carefully
for damage incurred during transit. Contact your distributor if anything is
missing. Damage claims must be filed with the carrier; the warranty does not
cover in-transit damage.

Note: If you do not have your distributor information, contact Technical Support
(page 247).

• EVOS™ M7000 Imaging System

• Computer
• Touchscreen monitor
• Keyboard
• Mouse
• Accessories box (located in the instrument box), containing:
- Power cable*
*For use with North American outlets; instruments intended for use
outside North America must order a separate power cord.
- Power supply
- Display Port to Display Port cable
- Mini-Display Port adapter
- USB 3.0 type A to B cable
- Dust cover
• Light cubes, as ordered
• Light cube tool
• Objectives, as ordered
• Vessel holder(s) for Well Plates (Cat. No. AMEP-VH022) and Double Slides
(Cat. No. AMEP-VH021) and as ordered (located in the accessories box)
• Light box with cover (located in the accessories box)
• Sliders: Block slider, Diffuser slider
• USB flash drive (includes User Guide and Quick Start Guide)

IMPORTANT! Wiping the computer supplied with the EVOS™ M7000 Imaging
System (i.e., erasing the hard drive to remove all programs, files, and the
operating system) voids the product warranty. Do not install third party software
or update the Windows™ operating system.

EVOS™ M7000 Imaging System User Guide 7

 Instrument exterior components
Top view

Condenser slider slot

Condenser
Automatic X-Y axis stage
Light cube tool
Objective turret (accomodates up to 5 objectives)
X-Y stage shipping restraint
Light cube shipping restraint
Camera shipping restraint
Phase annuli selector

8 EVOS™ M7000 Imaging System User Guide

 Side view

Condenser
Automatic X-Y axis stage
Handholds

Rear view

Power switch
4-pin power input port (24 VDC, 5 A)
USB 3.1 Type B port

EVOS™ M7000 Imaging System User Guide 9

 Computer ports and slots

Power button Display port (2×)

Media card reader USB 3.1 Gen 1 Type A port (4×)
Universal audio jack USB 2.0 Type A ports (2×)
USB 2.0 Type A port (2×) Mini Display port (4×)
(1 with PowerShare) Release latch
USB 3.1 Gen 1 Type A port Serial port (9-pin D)
USB 3.1 Gen 2 Type C port Ethernet port (RJ45)
(with PowerShare) Power input
Line-out Power supply diagnostic button
PS/2 (6-pin mini DIN) port (2×)

10 EVOS™ M7000 Imaging System User Guide

 Graphical user interface (GUI)
The functions of the EVOS™ M7000 Imaging System is controlled by the integrated
Invitrogen™ EVOS™ M7000 Software through a graphical user interface (GUI),
which is accessed by the touchscreen monitor or the computer mouse and
keyboard.

GUI layout The GUI of the system consists of the Viewing area on the left and a series of tabs
representing the main functions of the software (Capture, Automate, Review, and
Settings) on the right. Each tab contains the controls necessary to execute the
selected function. The Locations button, Area/Field View toggle, and the Zoom
slider are located above the Viewing area.

Locations: Opens the Locations controls, which allows you to select location for
automated scan protocols.
Area View/Field View toggle: Switches between Area and Field Views.
Zoom slider: Zooms in and out of the Viewing area.
Capture tab: Contains the controls for the manual capture of images.
Automate tab: Allows you to create and run automated scan protocols and time
lapse experiments.
Review tab: Allows you to review and annotate captured images.
Settings: Contains controls to select and adjust basic and advanced system
options and instrument functions.
Viewing area: Displays the sample in Area View or Field View modes.

Note: Click the View Help Content icon to open the Help window, which
provides EVOS™ M7000 Imaging System Help.
Position your pointer over the Tooltip icon without clicking it to view
additional information.

Note: For more information and detailed descriptions of software controls, see
“Appendix C: Graphical user interface (GUI)”, page 126.

EVOS™ M7000 Imaging System User Guide 11

 2. Installation

IMPORTANT! Do not perform the initial installation of the EVOS™ M7000 Imaging
System yourself. A Thermo Fisher Scientific representative will contact you to
schedule the installation.
When the installation is scheduled:
1. Receive and inspect the system.
2. Move the crated instrument to the installation site.

Operating environment and site requirements

• The EVOS™ M7000 Imaging System has a footprint of approximately
92 cm × 92 cm (36 in × 36 in); entire system includes the instrument, computer,
and 23-inch touchscreen, high-resolution LCD monitor.
• The dimensions (L × W × H) of the EVOS™ M7000 instrument are
45.7 × 33.0 ×35.6 cm (18 ×14 × 13 in).
• If the system includes the optional EVOS™ Onstage Incubator (Cat. No.
AMC1000), then add 40 cm (15.8 in) to the width of the bench, for a total
bench width of ~132 cm (51.8 in).
• The EVOS™ M7000 Imaging System should be placed on a level surface away
from vibrations from other pieces of equipment. Tabletop centrifuges, vortex
mixers and other laboratory equipment can vibrate the instrument during a
run that can cause a decrease of instrument performance.
• Allow at least 5 cm (2 in) free space at the back of the instrument to allow for
proper ventilation and prevent overheating of electronic components.
• The EVOS™ M7000 Imaging System should be installed away from direct light
sources, such as windows. Ambient room lighting can enter the imaging path
and affect the image quality.
• Operating temperature range: 4°–32°C (40°–90°F).
• Relative humidity range: 30–90%.
• Operating power: 100–240 VAC, 1.8 A
• Frequency: 50–60 Hz
• Electrical input: 24 VDC, 5 A

IMPORTANT! Do not position the instrument so that it is difficult to turn off the
main power switch located on the back of the instrument base (page 9). In case of an
instrument malfunction, turn the main power switch to the OFF position and
disconnect the instrument from the wall outlet.

12 EVOS™ M7000 Imaging System User Guide

 Prepare for installation
Receive and inspect 1. Verify that the items shown on the shipping list are the same items that you
the shipment ordered at the time of purchase.
2. Carefully inspect the shipping containers and report any damage to the
Thermo Fisher Scientific service representative. Record any damage or
mishandling on the shipping documents.
3. Unpack the instrument and accessories and verify all parts are present.
See “Set up”, page 14, for detailed instructions on how to unpack the
instrument and accessories.
See page 7 for the list of standard items included in the shipment.
4. Examine the instrument carefully for damage incurred during transit. Contact
your distributor if anything is missing. Damage claims must be filed with the
carrier; the warranty does not cover in-transit damage.

IMPORTANT! Do not lift the EVOS™ M7000 Imaging System by stage or condenser
arm. Lift the instrument by using the handholds in the base.

Move the 1. Clear the installation site of all unnecessary materials.

instrument to the 2. If possible, move the crated instrument and other shipping containers to the
installation site installation site.

CAUTION! PHYSICAL INJURY HAZARD. Do not attempt to lift or move the

instrument without the assistance of others, the use of appropriate moving
equipment, and proper lifting techniques. Improper lifting can cause
painful and permanent back injury. Depending on the weight, moving or
lifting an instrument may require two or more persons

IMPORTANT! Do not subject the EVOS™ M7000 Imaging System to sudden impact
or excessive vibration. Handle the instrument with care to prevent damage.

EVOS™ M7000 Imaging System User Guide 13

 Set up
IMPORTANT! The following set-up instructions are provided for informational
purposes only. Do not attempt the initial set-up of the instrument on your own.
The initial set-up of the EVOS™ M7000 Imaging System will be performed by your
Thermo Fisher Scientific service representative, who will also provide some basic
operator training.

Unpack and 1. Open the case and remove the monitor and accessories.
connect the 2. If a VGA cable is attached to the monitor, take it off.
monitor 3. Remove protective covering from monitor.
4. Plug the USB touch screen cable into the USB port on the monitor.
5. Plug power cord into monitor.

Unpack and 1. Open the box and remove the keyboard.

connect the 2. Unpack the keyboard from its box.
computer 3. Unpack the computer.
4. Unpack the mouse and power cord from the accessory holder.
5. Plug in the mouse and keyboard into USB 3.1 Type A ports (blue) on the back
of the computer (page 10).
6. Plug the USB cord already connected to the monitor into a USB 3.1 Type A
port (blue) on the back of the computer (page 10).
7. Plug in the power cord into the computer.

IMPORTANT! Wiping the computer supplied with the EVOS™ M7000 Imaging
System (i.e., erasing the hard drive to remove all programs, files, and the
operating system) voids the product warranty.

Unpack and connect 1. Open the box and remove the accessory box.
the instrument 2. Carefully lift the instrument out of the box, holding it by two of the four
handholds in the base (page 9).
IMPORTANT! Do not lift the EVOS™ M7000 Imaging System by stage or
condenser arm. Lift the instrument by using the handholds in the base.
3. Place the instrument on a flat, level surface that will be free from vibration
and leave enough room around it for the stage to move freely.
4. Remove the following from the accessory box (located in the instrument box):
• Power cable (for North America and as ordered)
• Power supply
• Display Port to Display Port cable
• Mini-Display Port adapter
• USB 3.1 type A to B cable
• White cardboard box (contains the light box and vessel holders)
• Dust cover
5. Confirm that the power switch is OFF (located on the back; page 9).

14 EVOS™ M7000 Imaging System User Guide

 6. Attach the Mini-Display Port adapter to the Display Port-to-Display Port
cable, then connect the cable to the Mini Display Port output on the computer
(page 10) and the DVI input on the monitor.
7. Use the USB 3.1 type A to B cable to connect the blue USB 3.1 Type B port on
the back of the instrument (page 9) to a USB 3.1 Type A port (blue) on the
back of the computer (page 10).
8. Plug the power cable into the power supply and check for the light on the
power supply.
9. Plug the power supply connector into the instrument.

Note: At this point, everything should be plugged in and OFF. Save the packaging
for future shipping/storage of the instrument.

EVOS™ M7000 Imaging System User Guide 15

 After installation is complete
Training When the installation of the EVOS™ M7000 Imaging System is complete, the
Thermo Fisher Scientific service representative will perform installation tests.
During and/or after installation, the Thermo Fisher Scientific service
representative will provide basic operator training. For additional training and
reference information, see the user documents provided with the instrument

Remove shipping The EVOS™ M7000 Imaging System is equipped with three shipping restraints
restraints (X-Y stage, light cube, and camera shipping restraints), which prevent damage to
the instrument from shock and vibration during transport. The shipping restraints
must be removed before the EVOS™ M7000 Imaging System is powered on.

Light cube tool Light cube shipping restraint

X-Y stage shipping restraint Camera shipping restraint

1. Unscrew the three thumb screws on the X-Y stage shipping restraint until they
spin freely. You do not need to remove them from the restraint altogether.
2. Gently pull the X-Y stage shipping restraint forward, away from the unit.

16 EVOS™ M7000 Imaging System User Guide

 3. Unscrew the light cube tool (the metal cylinder) and remove the light cube
shipping restraint (the white block) through the opening on the X-Y stage.

4. Unscrew the camera shipping restraint and pull it up to remove it from the
X-Y stage.

Note: Store the shipping restraints and the light cube tool in the accessories box
for future use. To reinstall the shipping restraints, page 118.

IMPORTANT! Do not subject the EVOS™ M7000 Imaging System to sudden impact
or excessive vibration. Handle the instrument with care to prevent damage.

EVOS™ M7000 Imaging System User Guide 17

 Turn ON the EVOS™ 1. Turn the instrument power switch located in the back of the instrument base
M7000 Imaging to the ON position.
System 2. Turn the computer and monitor ON.
3. When the computer shows the Windows™ desktop and the X-Y stage
of the instrument has stopped moving, click the M7000 icon on the
desktop to start the EVOS™ M7000 software.
4. When the Capture tab is displayed, the EVOS™ M7000 Imaging System is
ready to use.

IMPORTANT! All shipping restraints must be removed before turning on the

EVOS™ M7000 Imaging System to prevent damage (page 16).

18 EVOS™ M7000 Imaging System User Guide

 3. Capture and save images

Overview
Capture tab The basic functions of the EVOS™ M7000 Imaging System, such as viewing the
sample, setting optimal focus, and capturing and saving images are performed in
the Capture tab, which is the first screen after start-up.

Note: For a detailed description of the Capture tab controls, see “Capture tab” in
“Appendix C: Graphical user interface (GUI)” (page 139).

Workflow Select sample vessel


Select objective and light source

Set brightness and camera options

Set image display

Adjust brightness

Focus on the sample

Optional: Set Z-Offsets

Find the region of interest in the Live mode

Select fields and capture images

Save captured images

EVOS™ M7000 Imaging System User Guide 19

 Capture images
Select sample 2. Place the vessel containing your sample on the stage using the appropriate
vessel vessel holder.
Note: For the types of vessel holders available for the EVOS™ M7000 Imaging
System, visit thermofisher.com/evos or contact Technical Support (page 247).
2. On the Capture tab, click the Vessel button to open the Vessel
Selection dialog.
3. Select the Vessel category that corresponds to your sample vessel.
Available options are:
- Well Plates
- Flasks
- Dishes
- Slides
4. Select the appropriate Holder and
Vessel type from the available
dropdown menus.
Note: The Vessel Selection dialog is
contextual; the dropdown menus
display only the options available for
the selected Vessel category.

5. Click Done to complete your selection and close the dialog. The Vessel map
on the Capture tab displays your selected vessel.
By default, the first well on the sample vessel is selected and indicated in blue
on the Vessel map. The Viewing area displays the selected well.

6. To select another well, click on the desired well on the Vessel map.

20 EVOS™ M7000 Imaging System User Guide

 Select objective and 1. Click the desired Objective button to select the corresponding magnification.
light source You can select only one objective at a time. In the example below, 10X objective
has been selected.

2. Select the desired Light source from the available options for which you want
to adjust brightness and set focus. In the example below, the DAPI channel
has been selected.

Note: You can select only one light source at a time to adjust brightness and
set focus. However, you can display and capture multiple channels
simultaneously (see “Capture multiple channels simultaneously”, page 32).

Set brightness and 1. Click the Brightness and camera settings button to expand the controls for
camera options setting mode, camera, and phase options.

2. For Mode, select Simple or Actual.

- Simple mode allows you to control Brightness as a single parameter.
- Actual mode allows you to adjust Light (i.e., LED intensity), Exposure,
and Gain parameters individually.
3. For Camera, select Mono (monochrome) or Color.
- Mono is used for image capture in fluorescence and transmitted light
(brightfield) channels
- Color is used for image capture in the brightfield channel only
4. Optional: Choose Phase options. Available options are:
- Small Ring: Used for objectives with low magnification (i.e., Olympus™
4× PH)
- Medium Ring: Used for objectives with medium magnification (i.e.,
EVOS™ 4×/10× PH)
- Large Ring: Used for objectives with high magnification (i.e., EVOS™
20×/40× PH)
- Brightfield (phase contrast off)

Note: Phase options are available only for the transmitted light channel; they
are not available for fluorescence channels. The phase contrast option does
not require phase contrast objectives. However, to obtain a phase image, you
must first install a phase contrast objective.

5. Click the Brightness and camera settings button again to collapse the
controls.

EVOS™ M7000 Imaging System User Guide 21

 Adjust brightness 1. Click the Light button to turn on the excitation light for the
currently selected light source and enter the instrument in the
Live mode.
In the Live mode, the Viewing Area shows the sample illuminated with the
selected light source and the image is displayed in pseudo-color in the default
emission color.
2. Adjust brightness using the Brightness controls:
- In the Simple mode, adjust the brightness by moving the Brightness
slider in the desired direction.
Alternatively, double-click the slider handle to activate the brightness text
box and directly enter the desired brightness value (0%−100%).

- In the Actual mode, adjust the brightness parameters individually by

moving the Light (LED intensity), Exposure, and Gain sliders in the
desired direction.
Alternatively, double-click the slider handle to activate the corresponding
text box and directly enter the desired value for the selected parameter
(0%−100% for Light, 0−4.00 seconds for Exposure, or 1.0−8.0 for Gain).

Note: For best results, optimize the brightness parameters as follows:

• When searching for sample: Increase Gain for a brighter signal and decrease
Exposure for faster frame rate during navigation around the vessel.
• When capturing image: Decrease Gain to reduce background noise and
increase Exposure to regain signal intensity, as needed.
• For brighter signal: Increase Light intensity for brighter illumination. If
needed, follow by increasing Gain.
• For time lapse imaging: Increase Gain and Exposure, decrease Light intensity
to reduce photobleaching and phototoxicity.
For example, for overnight time lapse experiments, capture one image every
30 minutes or less, limit the use of autofocus, and use a channel other than
DAPI for autofocus.

22 EVOS™ M7000 Imaging System User Guide

 Set image display 1. Select the channels you want display in the Viewing Area by clicking on the
options small circular checkboxes on the upper left of the corresponding Light source
buttons.
You can select multiple channels to display in the Viewing Area. In the
example below, DAPI, RFP, and transmitted light (brightfield) channels have
been selected.

2. Click Image display settings button to expand the controls for

adjusting the image display parameters (brightness, contrast, gamma)
for the selected channels.

Note: The controls for image display settings are contextual; only the controls
for the selected channels will be available. In the example above, only the
controls for the selected DAPI, RFP, and transmitted light (brightfield)
channels are displayed.
3. Optional: To remove a channel from displaying in the Viewing Area, unselect
the corresponding Display checkbox. To display an available channel not
shown in the Viewing Area, re-select the checkbox.
4. Adjust the Brightness , Contrast , and Gamma settings for each of
the selected channels using the corresponding sliders.
5. Click the Image color display button to display the sample pseudo-
colored in the default emission color of the selected channels. By default, color
display option is on.
6. Click the Image display settings button again to collapse the controls.

Note: Adjustments made to Image display settings only affect how the image is
displayed in the Capture tab; they do not change how the image is captured .

EVOS™ M7000 Imaging System User Guide 23

 Optional: Display 1. Click the Scale Bar button to superimpose a scale bar over the Viewing
scale bar area.

The size the scale bar represents depends on the magnification of the selected
objective and it is displayed below the scale bar. After an image captured, the
size of the scale bar automatically changes as you zoom on a captured image

No zoom Zooming in on captured image

2. Click the Scale Bar button again to remove the scale bar from the Viewing area.

Note: The scale bar displayed in the Capture tab is not saved with your captured
images. However, you can add a scale bar to your saved images with the EVOS™
Analysis application (page 75).

Optional: Display 1. Click the Grid button to superimpose a grid over the Viewing area.
grid

The scale of the grid depends on the magnification of the

selected objective. The grid scale is displayed in the top
left unit square.
2. Click the Grid button again to remove the grid from the
Viewing area.

Note: To include a grid when you save your captured images, check the Include
Grid option in the Save window and select the grid size (page 42). You can also
add a grid to your saved images with the EVOS™ Analysis application (page 75).

24 EVOS™ M7000 Imaging System User Guide

 Focus on the 1. Click Autofocus to run the autofocus procedure to find the
sample best focal plane for the current channel and sample.
Alternatively, use the Coarse and Fine focus sliders to
manually find the best focal position for the current channel and sample.

Note: If you run the autofocus procedure with the light off, the autofocused
image will be captured and stored in the image cache for that channel.
If you run the autofocus procedure with the light on (i.e., with the Light
button pressed), the instrument will simply find the optimal focus, but will
not capture the autofocused image.

2. To choose a different autofocus method, click Advanced focus settings

button located to the left of the Autofocus button, then select from the
AutoFocus Method dropdown.

Available options are:

- Fluorescence Optimized: The focal plane is derived from the highest ratio
between detailed, high-contrast objects against the background. This
option is recommended for fluorescence imaging.
- Transmitted Optimized: The optimal focal plane is derived through
statistics-based edge detection over 9 different regions to determine the
highest ratio of edge-to-background. This option is recommended for
transmitted-light imaging.
- Small Structure: This option is best for samples with many fine, hair-like
structures (e.g., filaments or structural stains). This method computes the
energy according to the size of image features and can measure the
presence or absence of small size image features.
- Large Structure: Choose this option when your sample contains large
structures (e.g., whole cell stains). This method is statistics-based and looks
for large changes in image content as parts of the image go in and out of
focus.
- Small Bright Objects: Choose this option when capturing samples with
localized staining (e.g., nuclei). This method looks for brightness changes
of the center of the cell and optimizes the focus on cells that have a bright
center with a dark surrounding.

Note: To learn more about the autofocus strategy, click Advanced focus settings
(the gear icon next to the AutoFocus button in the Capture tab), then click the
View help content icon to open the Help window. In the Help window, click
How do I set Software Autofocus?

EVOS™ M7000 Imaging System User Guide 25

 Optional: Set Z-Offsets allow you to specify the optimal focus position in each channel relative
Z-Offsets to the focus position in other channels. Setting the correct Z-Offsets is especially
important when the fluorescent markers in different channels are in different focal
planes due to their normal localization in different cellular compartments.
1. Verify that the Lock Z-Offsets option is checked in the Advanced focus
settings window, then click the Advanced focus settings button again to close
the window.

2. Select the Objective and Light source (i.e., channel) you want to capture.

Note: To learn more about locked and unlocked Z-Offset states and how to set the
correct Z-Offsets, click Advanced focus settings (the gear icon next to the
AutoFocus button in the Capture tab), then click the View help content icon to
open the Help window. In the Help window, click How do I set Z-Offsets?

26 EVOS™ M7000 Imaging System User Guide

 3. Click the Light button to turn the light on and enter the Live mode.

4. Focus on the sample manually using the Coarse focus and Fine focus sliders
or automatically by clicking the AutoFocus button.
You can also double-click on the handle of the focus slider to activate the text
box and enter the desired value for the focus position.

5. After you have found the optimal focus position in the first channel, click the
Advanced focus settings button, then uncheck the Lock Z-Offsets option.

6. Without changing the objective, select the next Light source (i.e., channel) you
want to capture.
7. If the light is off, click the Light button to turn it on, then focus on the sample
manually using the Coarse focus and Fine focus sliders or automatically by
clicking AutoFocus.
8. Repeat this procedure for every channel you want to capture.
9. When you have found the optimal focus position in all the additional channels
you want to capture, click the Advanced focus settings button, then check the
Lock Z-Offsets option.

When you have completed this procedure, the focus position in each selected
channel will be offset relative to each other.

EVOS™ M7000 Imaging System User Guide 27

 Find the region of 1. While in Field View, click the Light button and enter the instrument in the
interest in the Live Live mode.
mode 2. To go to a specific location on the sample vessel,
click and drag the crosshair to the corresponding
location on the virtual vessel.

3. If needed, click on the Zoom button to open a larger view of the

Vessel map for easier navigation to the desired location.

Click the Zoom button again to close the zoom window.

4. While in the Live mode, use the Jog Control to move the stage at an
intermediate pace to the desired location as you view the sample
until you find the field of view you want to capture.
Alternatively, click on the navigation arrows (up, down, left, right)
on the Viewing area to move in the corresponding direction exactly
one field of view. You can also click and drag the field of view itself
to move around the sample vessel.

5. Click the Light button again to exit the Live mode in Field View.

28 EVOS™ M7000 Imaging System User Guide

 Select fields and 1. In Area View, position the capture crosshair over the region of interest and
capture images click to select the field you want to capture.
Alternatively, find the field you want to capture in the Live mode.

2. Click Capture to acquire an image of the selected field using

the current capture settings.
A thumbnail of the captured image is displayed above the Light
source button for the channel in which the image was captured
(in this example, DAPI).
The Viewing area displays the captured image at the location
of its capture when the zoom level is greater than one field of
view.

IMPORTANT! Captured images are stored in the memory buffer. If unsaved,

newly captured images will overwrite the previously captured image in the
selected channel for the selected field. Images captured in other fields and
channels will not be affected.

EVOS™ M7000 Imaging System User Guide 29

 3. To capture the same field in another channel, select the desired channel using
the corresponding Light source button.

4. If needed, readjust brightness and focus, then click Capture.

A thumbnail of the captured image is displayed above the Light
source button for the new channel in which the image was
captured (in this example, RFP).
The Viewing area displays a multicolor overlay of the images
captured in multiple channels at the location of their capture.
In this example, the second image was captured in the RFP
channel, and the Viewing area displays a multicolor overlay of the images in
the DAPI and RFP channels.

5. To zoom in on the Viewing area, use the Zoom

slider located above the Viewing area.

30 EVOS™ M7000 Imaging System User Guide

 6. To capture another field, position the capture crosshair over the new region of
interest, and click to select the new field.
Alternatively, find the new field you want to capture in the Live mode.
7. Click the Light source button to select the corresponding channel, readjust
brightness and focus (if needed), then click Capture.
In the example below, a second field was captured in the DAPI channel to the
left of the first field, which was captured in DAPI and RFP channels.

8. To capture a field using a different magnification, select the desired Objective.

9. Click the Light source button to select the corresponding channel. If needed,
readjust brightness and focus.
10. Position the capture crosshair over the new field of interest, click to select the
new field, then click Capture.
In the example below, a third field located was captured in the DAPI channel
using the 4X objective.

11. To save your captured images, click Save…. For more information, see “Save”
(page 40).

EVOS™ M7000 Imaging System User Guide 31

 Capture multiple 1. To capture a selected field in multiple channels simultaneously, check the
channels Toggle channel display option for the channels you want to capture.
simultaneously The Toggle channel display checkboxes are located on the top left corner of
the corresponding light source buttons. In the example below, DAPI and RFP
channels have been selected.

2. Click the desired Objective button to select the corresponding magnification.

You can select only one objective at a time. In the example below, 4X objective
has been selected.

3. Adjust brightness and focus for each of the selected channels as described
before.
4. Position the capture crosshair over the new region of interest, and click to
select the new field. Alternatively, navigate to the region of interest you want
to capture in the Live mode.
5. Click Capture Channels. The instrument captures an image
of the selected field in each of the selected channels using the current capture
settings.
A thumbnail of the captured image is displayed above the Light source button
for each channel in which it was captured.
The Viewing area displays a multicolor overlay of the images captured in the
selected channels at the location of their capture.
In the example below, the selected field was captured in the DAPI and RFP
channels using the 4X objective, and the Viewing area displays the merged
image captured in these channels.

6. To save your captured images, click Save…. For more information, see “Save”
(page 40).

32 EVOS™ M7000 Imaging System User Guide

 Capture Z-Stack
Capture Z-Stack Capture Z-Stack allows you to capture multiple images of a selected field at
different focal planes along the z-axis and combine them to generate a final image
with a greater depth of field than any of the individual source images.
1. Select the Objective and Light source (i.e., channel) you want to capture.

Note: You can capture z-stack images in only one channel at a time; however,
you can save the z-stacks images captured individually in separate channels
into a single merged video (see “Save Z-Stack as a video”, page 38).

2. Position the capture crosshair over the region of interest, then click to select the
new field. Alternatively, navigate to the region of interest in the Live mode.
3. Adjust brightness and focus using the corresponding sliders on the Capture
tab. The focus position you set in this step is the Default Focus position.

4. Click Capture Z-Stack to open the Z-Stack Settings window. You can move
the Z-Stack Settings window anywhere on the screen.

EVOS™ M7000 Imaging System User Guide 33

 5. If not already on, click the Light button to illuminate the sample as you locate
the top and bottom boundaries of the z-stack.
6. Using the focus sliders on the Capture tab, locate the top position of the
z-stack, then click Set Top Position.
7. Repeat the procedure for the bottom position, then click Set Bottom Position.
This sets the top and bottom boundaries of the z-stack image set.
8. Enter the Step Size (in µm) or the Number of planes, or define the z-stack as a
multiple of Depth of Field.
These parameters determine the number of “optical sections” captured in the
z-stack.
9. Select the Projection Method from the dropdown menu.
This is the mathematical algorithm used to extract the most in-focus pixels
from the images captured at different focal planes when generating the z-stack
projection. Available options are:
- Maximum: For fluorescent images that are bright on dark background.
- Standard deviation (StDev): For unstained transmitted images where the
objects are identified by the contrast instead of the intensity.
- Average: For fluorescent or histochemically-stained transmitted images
when assessing concentration of the stain/marker.
10. To save the z-stack parameters you have set up, click Save. This allows you to
capture z-stack images in other channels using the same specifications.

34 EVOS™ M7000 Imaging System User Guide

 11. To capture the z-stack, click Capture Z-Stack in the Z-Stack
Settings window. The instrument captures multiple images
of the sample along the z-axis based on your specifications.
A thumbnail of the z-stack projection is displayed above the
Light source button for the channel in which the z-stack images
were captured.
The z-stack icon on the top left corner of the thumbnail
image identifies it as a z-stack projection.

12. The Area View displays the z-stack projection at the location of its capture.
The z-stack icon on the top left corner of the captured field identifies it as a
z-stack projection.

13. To view the individual planes that make up the z-stack image set,
double-click the field that shows the z-stack projection in Area View.
Alternatively, click the Field View icon. The Field View displays the z-planes
and the z-stack controls appear to the left of the Viewing area.

EVOS™ M7000 Imaging System User Guide 35

 14. Move the z-stack slider up or down to view the z-planes (i.e., the
“optical sections”) in the sequence they were captured.
• Move the slider up to display the z-planes towards the top
position of the z-stack.
• Move the slider down to display the z-planes closer to the
bottom position.
• The z-slice number that is displayed above the slider handle as
you move it up or down indicates the order of the plane within
the z-stack.

15. To view the the z-stack projection, click the Toggle Projection Image
button.
The Viewing area displays the z-stack projection, which is generated by
combining the most in-focus pixels from each z-plane. By default, the
projection image is on.
16. To view the z-planes in sequence as a movie, click the Start z-stack
playback button located below the z-stack slider.
The Viewing area displays the z-planes in sequence, starting at the
bottom slice (Z-plane: 1) and stopping at the top.

17. To play the z-stack movie in a loop, click the Repeat playback button.
The Viewing area displays the z-planes in a continuous loop, starting at
the bottom slice and looping back to beginning after displaying the top
slide.
• To change the playback speed, move the Playback speed slider up
(faster) or down (slower). The playback speed is expressed in
frames per second (FPS).

• To stop the z-stack movie, click Stop z-stack playback.

Note: During playback, z-planes that make up the z-stack are shown
sequentially in the channel in which they were captured. The projection image
is not displayed (i.e., the Toggle Projection Image is unselected).

36 EVOS™ M7000 Imaging System User Guide

 18. To limit the z-stack movie to specific z-planes, drag the z-stack
calipers to the desired z-planes to set upper and lower boundaries.
When replaying the z-stack movie or saving it as a separate video file,
only the z-planes that fall within the z-stack calipers are included in
the video file.

19. To capture z-stack images in another channel, select the Light source (i.e.,
channel) you want to capture, then repeat the procedure as described.

Note: To view the z-stack images captured in multiple channels as an overlay,

check the Toggle channel display option for the channels you want to include
in the overlay.
The Toggle channel display checkbox is located on the top left corner of the
corresponding light source button. In the example below, GFP and RFP
channels have been selected.

20. To save the z-stack movie as a separate file, see “Save Z-Stack as a video”
(page 38).
21. To save the z-stack projection and individual z-planes for each field and each
channel as separate images, click Save… to open the Save dialog. For more
information, see “Save”, page 40.

EVOS™ M7000 Imaging System User Guide 37

 Save Z-Stack as a 1. Check the Toggle channel display option for the channels you want to
video include in your z-stack video. In the example below, GFP and RFP channels
contain z-stacks and both have been selected for the z-stack video.

Note: Selected channels must contain captured z-stack images to be included

in the z-stack video. If multiple channels with z-stacks are selected, the z-stack
video displays an overlay of the z-stack images captured in different channels.

2. Click Save as Video icon to save the z-stack images as a video sequence.
The Save Z-Stack Videos dialog opens.

3. Click Browse, navigate to the folder in which you want to save your z-stack
video, then click Select.

Note: We recommend that you save your captured images to an external

hard drive.

38 EVOS™ M7000 Imaging System User Guide

 Alternatively, click New to create a new folder at the desired location, type in
the name of the newly created folder, then click Select.

4. If desired, enter a new name for the z-stack video file in the File name textbox.

5. Confirm that the Selected Z Planes text box

displays the correct z-indices for the planes that
you want to include in your z-stack video.

Note: When saving the z-stack movie as a separate video file, only the
z-planes that fall within the z-stack calipers are included in the movie.
To change the z-planes you want to include in the z-stack movie, click Cancel
and readjust the z-stack calipers as described on page 37.

6. Select the file format for the z-stack video

from the dropdown menu. Available
options are AVI and WMV.
7. To display the z-index of each plane (i.e.,
the order within the z-stack) in the video,
check Display Z-Index on movie, then
select the desired Location, Font Size, and
Font Color from the drop-down menus.
By default, this option is not checked.
8. Confirm that the Frame rate text box displays the correct Playback speed for
your z-stack video.

Note: The Frame rate of your z-stack video sequence is set using the Playback
speed slider. To change the Frame rate for your z-stack movie, click Cancel
and readjust the Playback speed slider as described on page 37.

9. Click Save to save the z-stack images as a video sequence based on your
specifications.

EVOS™ M7000 Imaging System User Guide 39

 Save
Save captured 1. When finished capturing images, click Save… to open the Save window.
images

2. Select the captured fields you want save. Available options are:
• Currently selected field: Saves images only from the currently selected field.
• All newly captured fields: Saves images that have been captured and
stored in the image cache, but not yet saved. This option is available only if
you have previously saved images from the same session.
• All captured fields: Saves images from all captured fields that are held in
the image cache. This is typically all the images captured in an imaging
session.

Note: If the software remains open while users access the instrument back to
back, there may be several sets of images stored in the image cache. To clear
these images from the cache, click the trash can icon (see “Delete image
options”, page 130).
Saving the same images in the cache again does not overwrite the existing
files in the destination directory; newly saved files simply have a
differentiator added to the name.

40 EVOS™ M7000 Imaging System User Guide

 3. Click Browse, navigate to the folder in which you want to save your captured
images, then click Select.

To create a new folder in which to save your captured images, navigate to the
desired location, click New, type in the name of the newly created folder, then
click Select.

Note: We recommend that you save your captured images to an external

hard drive.

4. If desired, type the prefix you want to use for your save images in the File
name prefix textbox.

Note: For an overview of the file naming convention for saved images, see
“File naming convention”, page 191.

EVOS™ M7000 Imaging System User Guide 41

 5. Select File types to save by checking the corresponding checkboxes. You can
choose multiple file types for your captured images.
Available options for file type are Raw images and Displayed images.
• Raw images: Saves images captured in different channels individually as
16-bit Raw images. Raw images contain minimally processed data from the
image sensor and it is the recommended format for image analysis and
quantitation.
Available File format for Raw image outputs are TIFF, PNG, C01, and
DIB.

• Displayed images: Saves images in a format that can be viewed in most

image display applications. Displayed images give the best (“prettiest”)
results, especially when producing tiled and stitched images.

Available Color options for displayed images are Grayscale (16-bit) and
Pseudocolor (24-bit RGB; 8-bit per RGB channel).
Available File format for displayed images are: TIFF, PNG, and JPEG.
If desired, check the Include Grid option and select the grid size.

Note: We recommend saving captured images in both Raw and Displayed image
formats to preserve the option of using the Raw images in downstream image
analysis and quantitation where 16-bit dynamic range is required, and the
Displayed images for instances where “prettier” images are required. For more
information on Raw and Displayed images, see “Raw vs. Displayed images”,
page 125.

Note: While pseudocolors help differentiate the channels used in multi-channel

overlays, grayscale images usually show more detail. 24-bit images (8-bit per
RGB channel) are NOT recommended for image analysis as not all channels will
display in many image analysis applications.

42 EVOS™ M7000 Imaging System User Guide

 6. To see more file type options in a table format, click More options…
Full menu of save options are displayed in a convenient table format, allowing
you to save your captured images in a number of formats simultaneously.

7. Select the desired file types and save options by checking the corresponding
checkboxes. You can select multiple file types and options.
Available file type options are Raw images and Displayed images.
• Raw images:Saves each channel individually as 16-bit Raw images. Raw
image files contain the full dynamic range and metadata needed for
quantitative analysis and are the recommended format for image analysis.
- Single field, Z-Stack planes, and Tiled images can be saved as Raw
images with each channel saved individually.
- Available File format for Raw images are TIFF, PNG, C01, and DIB.
• Displayed images: Saves each channel individually or as a merged image
in a format that can be viewed in most image display applications.
- Single field and Tiled images can be saved as Displayed images from
individual channels or as a merged view.
However, Z-planes can be saved as Displayed images only from
individual channels.
- Available Color options for displayed images are Grayscale (16-bit) or
Pseudocolor (24-bit RGB; 8-bit per RGB channel).
- Available File format for displayed images are TIFF, PNG, and JPEG.
8. After you selected save options for your captured images, click Save.

Note: For more information on each save option available for Raw and Displayed
images, see the detailed descriptions in “Appendix C: Graphical user interface”
under Capture tabSave (page 154).

Note: For more information about Tiled and Merged images, see “Tiled, Stitched,
and Merged images”, page 125.

EVOS™ M7000 Imaging System User Guide 43

 4. Create and run automated scan

Overview
Automate tab The EVOS™ M7000 Imaging System allows you to create and recall scan protocols,
which are a series of automatically executed steps to capture multiple images over
an area and/or time period based on your specifications. For repeat experiments,
automated scan protocols can be saved, recalled, even edited.
The scan protocols are created using the Automate tab, which is organized into a
series of panels that contain the necessary controls that organized by functionality
(i.e., Load, Hardware, Scan Area, AutoFocus and Z Stacks, Time Lapse and
Incubator, Image Save Settings, Save, and Run).
You can access the Capture, Review, and Settings at any time by selecting the
corresponding tab.

Note: For a detailed description of Automate tab controls, see “Automate tab” in
“Appendix C: Graphical user interface (GUI)” (page 157).

Workflow Set hardware options


Select scan areas

Set Z-Offsets in Live mode

Configure AutoFocus settings

Set Time Lapse options

Set incubator options

Configure image save settings

Save automated scan protocol

Run automated scan protocol

44 EVOS™ M7000 Imaging System User Guide

 Create a scan protocol
Set hardware 1. Place the vessel containing your sample on the X-Y stage using the
options appropriate vessel holder.
For the types of vessel holders available for the EVOS™ M7000 Imaging
System, visit thermofisher.com/evos or contact Technical Support (page 247).
2. On the Capture tab, click the Vessel button, then select the sample vessel as
described on page 20.

Note: If you are using the EVOS™ Onstage Incubator, set it up for operation
as described in “Appendix D: EVOS™ Onstage Incubator”, page 228.

3. In the Automate tab, click Hardware  Edit to open the Hardware panel.

Note: For a detailed description of the controls available in the Hardware

panel, see “Hardware” in “Automate tab”, page 158.

4. Select the Objective you want to use for the scan protocol. You can select only
one objective at a time. In the example below, 10X objective is selected.

EVOS™ M7000 Imaging System User Guide 45

 5. Select the Channels to capture. You can select multiple channels. In the
example below, DAPI and RFP channels are selected.

6. To modify channel settings, click Adjust Settings to go to the

Capture tab.
7. Click Brightness and camera settings button to configure the Mode, Camera,
and Phase options as descibed in “Set brightness and camera options”, page 21.

8. Click the Light button to turn on the excitation light for the currently selected
channel (i.e., Light source) and enter the Live mode.
9. While in the Live mode, adjust the brightness settings using the Brightness
controls as described in “Adjust brightness”, page 22.
10. If in Simple mode, adjust the brightness by moving the Brightness slider.

If in Actual mode, adjust the brightness parameters individually by moving

the Light (LED intensity), Exposure, and Gain sliders.

11. While still in the Live mode, select each of the remaining channels that will be
used for the automated scan and adjust the brightness settings.
12. When finished with the channel settings, click the Automate tab to return to
the Hardware panel.
13. Click Done to save hardware options, then proceed to “Select scan areas”,
page 47.

46 EVOS™ M7000 Imaging System User Guide

 Select scan areas 1. In the Automate tab  Scan Area, click the Edit button to open the Scan Area
panel. In this panel you can define the areas and fields you want to scan and
specify the order in which they are captured.

Note: For a detailed description of the controls available in the Scan Area
panel, see “Scan Area” in “Automate tab”, page 159.

To define the areas and fields you want to scan, you have two options:
• Scan Locations tool (page 161) allows you to manually assign
scan regions using the Locations tools (page 134).
To use the Scan Locations tool to select regions for automated
scan, go to Step 2 (page 48).
• Scan Pattern tool (page 166) allows you to define the capture
regions for the scan protocol as a pattern based on defined
parameters.
To use the Scan Pattern tool to select regions for automated scan,
go to Step 8 (page 51).

EVOS™ M7000 Imaging System User Guide 47

 2. Click the Locations button to open the Scan Locations tool.
• If there are no previously defined locations, the Available
Locations table displays the message “No locations are
available for selection”.

• If you have already defined locations using the Locations tools, they will
be listed in the Available Locations table.

3. To create new locations for the scan protocol, click

Create Locations to open the Locations tools (page 134).

Locations – Area View Locations – Field View

48 EVOS™ M7000 Imaging System User Guide

 4. Using the Locations tool, define the locations as described (page 135), then
navigate back to the Automate tabScan Locations. Newly created locations
are listed in the Available Locations table.

In the following example, the first location in the list (an ellipse) is selected.
The Viewing are displays the selected location and the field group that best
fits the shape of the location.

5. To add a location to the scan protocol, click the desired location in the
Available Locations table to select, then click Add Selected.
To add multiple locations to the scan protocol simultaneously, Shift-click the
desired locations in the Available Locations table to select them, then click
Add Selected.
To add all the locations listed in the Available Locations table, click Add All.
The locations added to the scan protocol are displayed in the Scan Locations
table and removed from the Available Locations table.

EVOS™ M7000 Imaging System User Guide 49

 6. To assign a scan area for the selected scan locations, click
Assign scan area to open the Vessel map.

You have the option to individually define scan locations for scan area you
want to capture or select the scan areas on the Vessel map to which the
selected scan locations are applied.
For instructions on how to define scan areas on the Vessel map, see page 165.
7. When finished, click Done to close the Vessel map and exit the area selection
mode.

50 EVOS™ M7000 Imaging System User Guide

 8. If you want to to define the capture regions for the scan protocol as
a pattern based on defined parameters, click the Pattern button to
open the Scan Pattern tool.

• To define the capture region as a matrix, select W × H fields of view, then

enter the desired dimensions. You can only enter integers (page 166).
• To define the capture region as a percentage of area covered by scan fields,
select % of well area, enter the desired value (an integer between 1 and
100), then select From center or From edge (page 167).
9. When you are finished defining the parameters for the capture pattern, click
Create.

Note: The region you define with the Field selection tools in Area View is
replaced by a set of capture fields that best fit the drawn shape or, in the
case of the Pattern tool, satisfy the criteria set for the region (see examples
on pages 166−167).
In a multi-well plate, multi-chamber slide, or multi-vessel holder, the fields
defined in a selected well, chamber, or vessel holder also apply to the other
selected wells, chambers, or vessel holders.

10. If needed, click the Delete field group button that is displayed when
you Position over the field group to delete that field group.
To delete all field groups from Area View, click the Clear field
selections button located at the bottom right corner of Area View.

EVOS™ M7000 Imaging System User Guide 51

 11. To assign a scan area for the selected scan locations, click
Assign scan area to open the Vessel map, then select the
desired areas from the Vessel map (page 165).
If you want to view a larger version of the Vessel map, click the Zoom button.
Click the zoom button again closes the zoom window.

12. When finished defining the scan areas, click Done to exit the selection mode.

13. Click Area Acquisition Order to you

specify the order in which selected areas
are captured in the automated scan
protocol.
Available options are:
- Serpentine horizontal
- Serpentine vertical
- Random selection
14. Click Field Acquisition Order to you
specify the order in which the fields are
captured in each selected area.
Available options are:
- Spiral Outward Counterclockwise
- Spiral inward Clockwise
- Serpentine Horizontal
- Serpentine Vertical
- Random Selection

52 EVOS™ M7000 Imaging System User Guide

 15. To use stitching in your scan area, select
Stitch Images.
Stitching function applies an overlap when assembling captured images into a
mosaic image of the scan area.

16. Select Default Overlap for a faster scan or select More Overlap for a higher
image quality.

Stitching off

Stitching on - DefaultOverlap Stitching on – More Overlap

17. When finished with area selection, click Done to save your selections and
return to the Automate tab.

EVOS™ M7000 Imaging System User Guide 53

 Set Z-Offsets in Z-Offsets specify the optimal focus position in each channel relative to the focus
Live mode position in other channels. Setting the correct Z-Offsets is especially important
when the fluorescent markers in different channels are in different focal planes.

Note: To learn more about locked and unlocked Z-Offset states and how to set the
correct Z-Offsets, go to Automate tab, click the Edit button to open the AutoFocus
and Z-Stacks panel, then click the View Help Content icon to open the Help
window. In the Help window, click How do I set Z-Offsets?

For best results, optimize the Z-Offsets in the Capture tab, and then carry the
offsets over to the Automate tab.
1. In the Automate tabAutoFocus and Z Stacks panel, ensure that the Capture
Z-Stacks option is unchecked, then click the Capture tab.

Note: The Z-Offsets option is not available as one of the Focus Position
controls when the Capture Z-Stacks option has been selected. For more
information, see “Z-Stack Settings”, page 173.

2. In the Capture tab, click Advanced focus settings and verify that the
Lock Z-Offsets option is selected. By default, Lock Z-Offsets option is checked.

3. Select the Objective and Light source (i.e., channel) you want to capture.

4. Click the Light button to enter the Live mode (page 143).

54 EVOS™ M7000 Imaging System User Guide

 5. Focus on the sample manually using the Coarse focus and Fine focus sliders
or automatically by clicking AutoFocus.
You can also double-click on the handle of the focus slider to activate the text
box and enter the desired value for the focus position.

6. After you have found the optimal focus position in the first channel, click
Advanced focus settings, then uncheck the Lock Z-Offsets option.

7. Without changing the objective, select the next Light source (i.e., channel) you
want to capture.
8. If the light is off, click the Light button to enter the Live mode, then focus on
the sample manually using the Coarse focus and Fine focus sliders.
Alternatively, click AutoFocus to automatically focus on the sample.

9. Repeat this procedure for every channel you want to capture.

10. When you have found optimal focus in all of the channels you want to capture,
click the Advanced focus settings button, then check the Lock Z-Offsets
option.

11. If the light is on, click the Light button to turn off the Light and exit the Live
mode.

EVOS™ M7000 Imaging System User Guide 55

 12. In the Automate tab  AutoFocus and Z Stacks, click the Review button to
open the AutoFocus and Z Stacks panel.
You will see the new settings carried over to Z-Offsets under the Focus
Position and the focus positions will be offset relative to each other.
In the example below, DAPI channel is focused to 118.8 µm.
The Z-Offsets for the RFP channel relative to the DAPI channel is set to
20.05 µm.
Therefore, the instrument will focus to 138.85 µm (118.8 µm + 20.05 µm) in the
RFP channel.

Because the Z-Offsets are preserved, selecting the RFP channel for the default
focus position at 138.85 µm refocuses the instrument to 118.8 µm in the DAPI
channel (138.85 µm – 20.05 µm).

Note: If you need to adjust the Z-Offsets while in the Automate tab, make sure to
close out the Autofocus and Z Stacks panel by clicking the Done button before
navigating back to the Capture tab to make the adjustments.

56 EVOS™ M7000 Imaging System User Guide

 Configure 1. In the Automate tab  AutoFocus and Z Stacks, click the Edit button to open
AutoFocus settings the AutoFocus and Z Stacks panel. Note that if you have previously adjusted
any settings in the AutoFocus and Z Stacks panel, the Edit button will have
changed to Review button.

Note: The options available on the AutoFocus and Z Stacks panel are
contextual; the controls that are displayed depend on the selections made in
this panel or elsewhere in the Automate tab. For more information, see
“AutoFocus and Z Stacks” in “Automate tab”, page 172.

2. Check the Use AutoFocus box. The panel reveals the

controls to configure the autofocus settings.

3. Select Single Channel or All Channels to autofocus during the scan protocol.

- Single Channel: Autofocus procedure is run in a single channel and

applied to all other selected channels. This option preserves the Z-Offsets
between the channels (if they were set previously).
- All Channels: Autofocus procedure is run separately in all selected
channels. This option does not preserve the Z-Offsets between channels.

EVOS™ M7000 Imaging System User Guide 57

 4. If you have selected Single Channel, choose the Channel for which to use
autofocus. The focal plane identified in this channel is used for all other
channels. Note that only the channels selected in the Hardware panel
(page 46) are available to choose.

5. Click the Advanced AutoFocus Configuration

button to reveal the options for autofocus methods.
6. Select the desired AutoFocus Method for each channel from the corresponding
dropdown menu, then click Close.

Available options are:

- Fluorescence Optimized: The focal plane is derived from the highest ratio
between detailed, high-contrast objects against the background.
Recommended for fluorescence imaging.
- Transmitted Optimized: The optimal focal plane is derived through
statistics-based edge detection over nine different regions to determine the
highest ratio of edge-to-background. Recommended for transmitted-light
imaging.
- Small Structure: Choose for samples with many fine, hair-like structures
(e.g., filaments or structural stains). This method computes the energy
according to the size of image features and detects the presence or absence
of small size image features.
- Large Structure: Choose for samples with large structures (e.g., whole cell
stains). This method looks for large changes in image content as parts of
the image go in and out of focus.
- Small Bright Objects: Choose for samples with localized staining (e.g.,
nuclei). This method looks for brightness changes in the center of the cell
and optimizes the focus on cells that have a bright center with a dark
surround.

Note: The effectiveness of the autofocus method depends on the plate type,
sample type, biomarker intensity, magnification, and channel. It may be
necessary to use different autofocus methods for different channels.

58 EVOS™ M7000 Imaging System User Guide

 7. Set the AutoFocus Frequency.

Available options are:

• First field only each area: Performs the autofocus operation only on the
first field of each scan area. No further autofocus is performed on
subsequent fields in the scan area. The focus position obtained on the first
field is used for the remaining fields in the scan area.
• First field only each location: Performs the autofocus operation only on
the first field of each location in every scan area. No further autofocus is
performed on subsequent fields.
• Every field: Performs the autofocus operation on every field of the scan
area.
• Every <X> fields: Allows you to specify the distance between fields in
each scan area before the autofocus is triggered.
The distance between fields is expressed as “unit fields” (equivalent to a
field of view as determined by the objective used); it is not determined by
the order in which the fields are collected.
For example, if you want to autofocus every 3 fields, autofocus will only
occur when the stage is three fields away from another autofocused field.
If the stage is less than three fields away, autofocus will not occur.

Note: For a single culture dish or flask, the scan area corresponds to the
entire dish or flask. For a multi-well plate or a multi-chamber slide, each scan
area corresponds to a single well or chamber.
Specifying fewer fields per area to autofocus reduces the overall scan times.
Frequent autofocusing may not be necessary for fields that are close together
or for samples with consistent z-locations on very flat vessel surfaces.

8. When finished with your selections, click Done to return to the Automate tab
and proceed to “Set Time Lapse options”, page 60.

Note: To learn more about the autofocus strategy, go to Automate tab, click the
Edit button to open the AutoFocus and Z-Stacks panel, then click the View Help
Content icon to open the Help window. In the Help window, click How do I
set Software Autofocus?

EVOS™ M7000 Imaging System User Guide 59

 Set Time Lapse A time lapse routine automatically captures individual images of the fields that
options were defined in the Scan Area at given intervals over a time period based on your
specifications. The images can then be stitched together into a video.

Note: The capture parameters defined in the Automate tab (z-stacks, exposures,
channels, etc.) are applied to every time point of a time lapse routine.

1. In the Automate tab  Time Lapse and Incubator, click the Edit button to
open the Time Lapse and Incubator panel, then select Use Time Lapse.

The time lapse controls in the panel become active.

2. Enter the Duration of the run into the corresponding Hours, Minutes, and
Seconds fields.

60 EVOS™ M7000 Imaging System User Guide

 3. Optional: Under Run 1, select Delay Start, then enter the desired time interval
into the corresponding Hours, Minutes, and Seconds fields.
The delay start determines the time interval after the start of the scan protocol
in which no images are captured.

4. Set the Image capture frequency.

Available options are:

• Frequency: Enter the time period in Hours, Minutes, and Seconds that
must elapse before a new set of images are captured.
For example, in an experiment with an image capture frequency of
2 minutes and 30 seconds, the images will be captured every 2 minutes
and 30 seconds after the initial set of images are acquired at time point 0.
• Intervals: Enter the total number of time intervals between the captured
image sets for a given run duration.
Note that the images are collected at the end of an interval. For example,
in an experiment with a duration of 5 minutes and 2 intervals, the images
will be captured every 2 minutes and 30 seconds after the initial set of
images are acquired at time point 0.
• As fast as possible: Select this option to capture a new set of images
immediately after completing the previous set in the order specified
without any delay between the sets.
Note that the speed with which the images are captured depends on your
specifications for the scan protocol such as the autofocus frequency and
exposure settings.
5. Optional: Click Add run to add another run with its own
parameters (delay start, duration, image capture frequency,
incubator settings) to the time lapse routine.

Note: The runs are performed in chronological order, beginning with Run 1
and ending with Run N. You can remove a run from the time lapse routine
by clicking the corresponding Delete Run button (page 184).

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 6. Set the Autofocus Settings for the time lapse routine.

Available options are:

• First time point only: Select this option if you want to autofocus at the
selected locations only at the first time point of the time lapse routine.
• Every time point: Select this option if you want to autofocus at the
selected locations at every time point of the time lapse routine.
• Autofocus fail options: Click Yes to skip an autofocus location at
subsequent time points in the event of an autofocus failure at that location.
Click No to attempt autofocus at each selected autofocus location
regardless of previous success or failure.
7. If you want to use the EVOS™ Onstage Incubator during your time lapse
routine, see “Set incubator options”, page 63.
Otherwise, click Done to return to the Automate tab and proceed to
“Configure image save settings”, page 64.

62 EVOS™ M7000 Imaging System User Guide

 Set incubator IMPORTANT! Before using the EVOS™ Onstage Incubator in your time lapse
options experiments, make sure that:
• The EVOS™ Onstage Incubator has been set up for use (see “Set up the
EVOS™ Onstage Incubator”, page 228).
• The gas inputs have been configured (see “Configure gas inputs”, page 234).
• The oxygen sensor has been calibrated (see “Calibrate oxygen sensor”,
page 235).

1. In the Automate  Time Lapse panel, select Use Incubator.

The Incubator controls in the panel become active.

Note: Use Time Lapse option must be previously checked for the Use
Incubator option to be available.

2. Enter the target values for Temperature, CO2, and Oxygen.

3. If desired, select Use humidity.

4. Select the desired Shutdown option for the incubator.

Available options are:

- Turn off manually: The incubator remains on until the Use Incubator
option is manually deselected.
- Turn off at the end of experiment: Heat, humidity, and the flow of gas
are automatically turned off at the end of the experiment.
- Turn off after: Enter the time period in hours and minutes that must
elapse before the heat, humidity, and the flow of gas are automatically
turned off.
5. When finished with your selections, click Done to return to the Automate tab
and proceed to “Configure image save settings”, page 64.

EVOS™ M7000 Imaging System User Guide 63

 Configure image 1. In the Automate tabImage Save Settings, click the Edit button to open the
save settings Image Save Settings panel.

2. Click Browse, navigate to the folder in which you want to save your images
captured during the automated scan protocol, then click Select.

Note: We recommend that you save your captured images to an external

hard drive.

64 EVOS™ M7000 Imaging System User Guide

 3. To create a new folder for the scan protocol, navigate to the desired location,
click New, type in the name of the newly created folder, then click Select.

4. If desired, type the prefix you want to use for your saved images in the Prefix
textbox.

Note: For file naming conventions for saved images, see “File naming
convention”, page 191.

5. Select File types to save by checking the corresponding checkboxes. You can
choose multiple file types for your images captured during the automated
scan protocol.
Available file type options are Raw images and Displayed images.
• Raw images: Saves images captured in different channels individually as
16-bit Raw images. Raw images contain minimally processed data from the
image sensor and it is the recommended format for image analysis and
quantitation.

- Available Raw image options are:

Single field, individual channels: Saves images captured in each field
and each channel individually.
Z-stack planes, individual channels: Saves the z-stack projection and
each z-plane for each field and each channel as a separate image.
- Available File format for Raw images are TIFF, PNG, C01, and DIB.

EVOS™ M7000 Imaging System User Guide 65

 • Displayed images: Saves images in a format that can be viewed in most
image display applications. Displayed images give the best (“prettiest”)
results, especially when producing tiled and stitched images

- Available Displayed image options are:

Single field, individual channels: Saves images captured in each
field and each channel individually.
Merged image: Saves the images of a field captured in different
channels as a multi-channel overlay.
Tiled image, merged channels: Merges the images captured in each
channel, then aligns them close together into a tiled format.
You can create tiled images with one of the following dimensions:
Small (2000 × 2000 pixels), Medium (4000 × 4000 pixels),
Large (10,000 × 10,000 pixels), or Maximum (26,000 × 26,000 pixels).
- Available Color options for displayed images are:
Grayscale: 16-bit.
Pseudocolor: 24-bit RGB (8-bit per RGB channel).
- Available File format for Displayed images are: TIFF, PNG, and JPEG.

Note: We recommend saving captured images as both Raw and Displayed

images to preserve the option of using the Raw images in downstream image
analysis and quantitation, and the Displayed images for instances where
“prettier” images are required. For more information on Raw and Displayed
images, see “Raw vs. Displayed images”, page 125.

Note: For more information about when to use tiled or stitched images, see
“Tiled, Stitched, and Merged images”, page 125.

Note: While pseudocolors help differentiate the channels used in multi-

channel overlays, grayscale images usually show more detail.
24-bit pseudocolor images (8-bit per RGB channel) are NOT recommended
for image analysis because not all channels will display in many image
analysis applications.

6. Check the Include Grid option to superimpose a grid on the displayed images.
You have the following options for the grid size (in pixels):
Auto, 10 × 10, 50 × 50, 100 × 100, 200 × 200, and 500 × 500.
7. Select Tiled per location or Tile all locations together.

66 EVOS™ M7000 Imaging System User Guide

 8. To see more file type options in a table format, click More options…
Full menu of save options is displayed in a convenient table format, allowing
you to save your captured images in multiple formats simultaneously.

9. Select the desired file types and options by checking the corresponding
checkboxes. You can select multiple file types and options.
Available file type options are Raw images and Displayed images.
• Raw images: Saves each channel individually. This is the recommended
format for image analysis and quantitation.
- Single fields, Z-planes, and Tiled images can be saved as Raw
images.
- Available File format for Raw images are TIFF, PNG, C01, and DIB.
• Displayed images: Saves each channel individually or as a merged image
in a format that can be viewed in most image display applications.
- Single fields, Z-planes, and Tiled images from individual channels
can be saved as Displayed images.
However, you can only save Single fields and Tiled images as a
Displayed image in merged view; Z-planes can only be saved from
individual channels.
- Available Color options for Displayed images are Grayscale (16-bit) or
Pseudocolor (24-bit RGB; 8-bit per RGB channel).
- Available Brightness options for Displayed images are High and Full.
- Available File format for Displayed images are TIFF, PNG, and JPEG.
10. After you have configured Image Save Settings for your automated scan
protocol, click Done to return to the Automate tab.

EVOS™ M7000 Imaging System User Guide 67

 Save automated When you have finished creating your automated scan protocol, the total number
scan protocol of images to be captured in the scan, estimated scan file size, estimated temp file
size, and the drive space available for the scans are displayed above the Save and
Load buttons in the Automate tab.

At this point, we recommend saving the automated scan protocol for future recall
in repeat experiments. For convenience, you can also edit recalled scan protocols
to create new routines rather than creating them from the very beginning.
1. In the Automate tab, click Save to open the Save As dialog, then navigate to
the location in which you want to save your scan protocol.

2. In the File name text box, type in the name of your new routine.
The default name for new scan protocols is “New Protocol.scanprotocol”.
3. Click Save to save your scan protocol and close the Save As dialog.
4. To run your automated scan protocol, proceed to “Run newly created scan
protocol”, page 69.

68 EVOS™ M7000 Imaging System User Guide

 Run automated scan protocol
Run newly created 1. In the Automate tab, click Run to run the automated scan protocol you have
scan protocol just created.
The EVOS™ M7000 Imaging System starts executing the steps specified in your
automated scan protocol to capture multiple images over an area and/or time
period.
The Viewing area displays the fields as they are being captured during the
scan, while the Vessel map indicates the area being captured.

The Scan Settings, Run Sequence Settings, and Run Settings specified in your
scan protocol are displayed below the Vessel map, and the Experiment
Progress bar tracks the progress of the run.

2. Click Pause to pause the automated scan protocol anytime during a run.
Click Resume to restart the scan protocol from the step where it was paused.
3. If you want to abort the scan protocol before it is completed, click Stop.
Click Save to save the images acquired during the aborted scan protocol in the
location specified in Image Save Settings, or click Discard to delete them.

EVOS™ M7000 Imaging System User Guide 69

 4. When the scan protocol is completed, click Done.

Load and run a For repeat experiments, you can recall and run a saved scan protocol. If desired,
saved scan protocol you can also edit the settings of the recalled scan protocol.
1. On the Automate tab, click Load, then navigate to the folder containing the
saved scan protocol you want to recall.

2. Click the saved scan protocol of interest to select it, then click Open.
3. If you want to edit the recalled scan protocol, click Review to open the
Automate tab panel, adjust the settings you want to change, then click Done
to return to the Automate tab.
4. Click Run to run the recalled routine.
5. When the scan protocol is completed, click Done.

Note: Saved scan protocols retain the information about the scan areas as well as
the camera and lighting options set when creating the protocol. To change these
settings for a new experiment, click Review after loading the saved scan protocol
and make the desired changes.

70 EVOS™ M7000 Imaging System User Guide

 5. Review saved images

Review tab The Review tab allows you to review saved images, including Z-Stack images and
images captured during automated scans, and scan and field metadata associated with
the images. It also allows you to launch the EVOS™ Analysis application to analyze
and annotate saved images (see “Analyze and annotate saved images”, page 75).
For the descriptions of the controls available in the Review tab, see page 192.

Note: For a detailed description of the Review tab controls, see “Review tab” in
“Appendix C: Graphical user interface (GUI)” (page 192).

Review saved 1. Click the Review tab.

images 2. In the Folders panel, navigate to the folder containing your saved images, then
click to select it. The contents of the selected folder are displayed in the Image
preview/Image list area.

EVOS™ M7000 Imaging System User Guide 71

 3. To display the folder/image preview in list format, click the List view
button.

4. To review an image and associated metadata, double-click the image in the

Image preview/Image list area.
The selected image is displayed in the Viewing area and the Folders/Image preview
panel changes to Image metadata/Browse panel.

The Vessel map shows the location of the imaged field in the original sample vessel.

72 EVOS™ M7000 Imaging System User Guide

 The Scan Metadata panel shows metadata about the scan and the Field
Metadata panel shows the metadata concerning the imaged field displayed.

5. Optional: Adjust the display options as desired.

• Click Scale bar to display the scale bar over the Viewing area.
This option is available only in Field View.
• Click Grid to superimpose a grid over the Viewing area.
This option is available only in Field View.
• Click Image display settings to adjust brightness, contrast, and
gamma parameters for the displayed image. This option is available
in Area and Field Views.
• Click Toggle field borders to switch the display of the borders
around the captured fields on and off. This option is available only
in Area View.
• Click Toggle locations display to switch the display of locations
(defined with the Location tools) on and off.
• Click Center on selected field to center the Viewing area on the
selected field. This option is available only in Area View.
6. To review another image, click Browse or Load, then select another image.
7. To save the changes you have made to the image in the Review tab, click Save.
8. To annotate and analyze the image using the
EVOS™ Analysis application, click
Open in EVOS Analysis.
The EVOS™ Analysis application allows annotations and measurements of your
image, and provides tools for quantitative analysis (cell count, confluence, and
transfection efficiency). For more information, see “Analyze and annotate saved
images”, page 75.

Note: The Open in EVOS Analysis button becomes available in the Review
tab only after you have opened the image you wish to analyze in the Viewing
area.

EVOS™ M7000 Imaging System User Guide 73

 9. Optional: To annotate and analyze the image using the
Celleste™ Image Analysis Software, click Open in Celleste.

Note: The Open in Celleste button is available only if you have the Celleste™
Image Analysis Software (available separately from Thermo Fisher Scientific)
installed on the computer running the EVOS™ M7000 Software. The button
becomes visible in the Review tab after you have opened the image you wish
to analyze.

Celleste™ Image The Celleste™ Image Analysis Software (available separately from Thermo Fisher
Analysis Software Scientific) is a full-feature image analysis suite designed for a range of biological
applications, from image adjustments and processing with manual and automatic
measurements over multiple channels, to segmentation and classification tools that
help you transform images into quantitative data in a streamlined and
customizable workflow.
For more information on the Celleste™ Image Analysis Software, go to
thermofisher.com/celleste. For instructions on how to analyze your images with the
Celleste™ Image Analysis Software, refer to the Celleste™ Image Analysis Software
user guide (Pub. No. MAN0018003), availabel for download at thermofisher.com.

74 EVOS™ M7000 Imaging System User Guide

 6. Analyze and annotate saved images

EVOS™ Analysis application

The EVOS™ Analysis application is a quantitative image analysis and annotation
tool that runs independently from the EVOS™ M7000 software used for controlling
the instrument.
The EVOS™ Analysis application allows you to perform the following tasks:
• Adjust image display settings (page 79)
• Display grid (page 80)
• Display scale bar (page 81)
• Display histogram (page 82)
• Add and show measurements and annotations (page 83)
• Analyze cell culture (page 85):
- Count cells – Auto Count (page 86)
- Count cells – Manual Count (page 90)
- Measure confluence (page 92)
- Calculate transfection efficiency (page 95)
• Save analysis results (page 98)
• Batch Analysis (page 100)

Note: For a detailed description of the EVOS™ Analysis application controls, see
“EVOS™ Analysis Application” in “Appendix C: Graphical user interface (GUI)”
(page 215).

Launch the There are two ways to launch the EVOS™ Analysis application:
EVOS™ Analysis • Launch the application from the Review tab when there is a saved image file
application open in the Review tab (page 76).
• Launch the application directly from your desktop, then open the image you
wish to analyze using the application (page 77).

EVOS™ M7000 Imaging System User Guide 75

 Launch 1. In the Review tab (page 71), double-click on the image that you wish to analyze
EVOS™ Analysis to open it in the Viewing area, then click Open in EVOS Analysis.
from the Review
tab

The image opens in the EVOS™ Analysis application.

Note: The Open in EVOS Analysis button becomes available only after you
have opened the image you wish to analyze in the Viewing area.

2. Hover the pointer over the Viewing area to reveal Display Settings and
Analysis Tools, then click to open the desired tool.

3. To analyze another image, click on the image in the you wish to view it in the
Viewing area.

76 EVOS™ M7000 Imaging System User Guide

 Launch 1. Double-click on the Evos Analysis icon on your desktop or select it from the
EVOS™ Analysis Start menu.
from the desktop 2. After the application has opened, navigate to the folder containing images you
want to analyze, then double-click to select it.

The contents of the selected folder are displayed in the File list/Image preview
panel.

3. To change the layout from File list to Image preview or to sort the files
by name, file type, or creation date, click the Display Settings button,
then make your selections:
• To toggle between file list and images preview formats, use the
Thumbnails and List buttons.
• To change the size of the image thumbnails, use the Zoom buttons.
• To sort the image files by Name, File Type, or Date Created, use the
Sort controls.

EVOS™ M7000 Imaging System User Guide 77

 4. Click on the image you wish to analyze to open it in the Viewing area.

5. Hover the pointer over the Viewing area to reveal the buttons for Display
Settings and Analysis Tools.

6. Click a button to open the corresponding tool; click the button again to close it.

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 Image display settings
Adjust image 1. Click the Image Display Settings button to expand the controls for
display settings image display settings (brightness, contrast, gamma).

Note: The controls for image display settings are available only for captured
channels. In the example above, the controls are available for the TGBFP
(TagBFP), GFP, RFP, CY5, and TRANS (transmitted light or brightfield)
channels. The image that is displayed in the Viewing area is captured in the
TGBFP channel, as indicated by the check mark.

2. Optional: To display an additional channel, select the corresponding checkbox.

To remove a channel from displaying in the Viewing Area, unselect the
corresponding checkbox.
In the example below, TGBFP and GFP channels are displayed.

3. Adjust the Brightness , Contrast , and Gamma settings for each of

the selected channels using the corresponding sliders.
4. Click the Reset button to return the image display settings to their default
values.
5. Click the Image Display settings button again to collapse the controls.

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 Grid
Display grid 1. Click the Grid button to superimpose a grid over the Viewing area.

2. To change the grid size, click the Grid Settings button (arrow on the
Grid split button) to open the Grid Settings tool.

3. Select the Size for the grid. Available grid sizes depend on the magnification of
the selected objective.
4. Click the Grid Settings button again to save your settings and close the tool.

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 Scale bar
Display scale bar 1. Click the Scale Bar button to superimpose a scale bar over the
Viewing area.

2. To move the scale bar, hover your pointer over the scale bar until a bounding
box appears, then click within the box and drag the scale bar to the desired
location within the Viewing area.

3. To adjust the length of the scale bar, hover your pointer over the scale bar until
a bounding box appears, then the click left or right side of the box and drag the
box to the desired length.
You can adjust the length by pre-fixed increments based on the objective
magnification.

4. To change scale bar settings, click the Scale Bar Settings button
(arrow on the Scale Bar split button) to open the Grid Settings tool.

5. Select Show End Bars to display the scale bar with the end bars.
6. Select the Color for the scale bar.
7. Click Scale Bar Settings button again to save your settings and close the tool.

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 Histogram
Display histogram 1. Click the Histogram button to open the Pixel Intensity histogram.

2. Pixel Intensity histogram shows the Pixel count vs. Intensity data of the image
displayed in the Viewing area as well as the minimum, mean, and maximum
pixel intensities.

3. To move the histogram, click within the plot heading area and drag the plot to
the desired location.
4. To resize the histogram, click the grey triange at the lower right corner of the
plot, then drag the plot to the desired size.
5. Click the Histogram button again to close the Pixel Intensity histogram.
Alternatively, click the X on the plot to close the histogram.

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 Measurements and Annotations
Add and show 1. Click the Measurement and Annotations button to open the controls
measurements and to add measurements and annotations.
annotations

2. Using the Annotations tools, draw a rectangle, line, ellipse, polygon, or a

free-form shape over the region of interest on the Viewing area. You can draw
multiple shapes of different type.

3. If needed, change the Color and Thickness of the annotation to make it more
visible over the image.

EVOS™ M7000 Imaging System User Guide 83

 4. If desired, select to display the Dimensions, Area, or Perimeter information for
the selected annotation from the dropdown menu.

5. To delete a selected annotation, click the X on the shape that appears when you
hover your pointer over it.
To delete all annotations, click Reset, then click OK in the dialog that opens.

6. Click the Measurement and Annotations button again to close the tools.
7. After you have added measurements and annotations to your image:
• Click Show Measurements and Annotations button (main part of the split
button) to turn the display on and off.
• Click Measurement and Annotations Controls button (the arrow on split
button) to display the controls to add new measurement and annotations
or to delete existing ones.

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 Analyze cell culture
Analysis tools Hover the pointer over the Viewing area in the EVOS™ Analysis application to
reveal the analysis tools, then click the Show Cell Count button to display Auto
Count, Manual Count, and Cell Culture options in the tabs area.

• Auto Count: Automatically counts the objects displayed in the Viewing area
based on your specifications (page 86). With Auto Count, you can count objects
only in a single fluorescence channel (nuclear stain channel).
• Manual Count: Allows you to tag objects in the Viewing area with up to six labels.
As you tag objects, the system keeps a running tally of the counts with percentages
for each label assigned (page 90). With Manual Count, you can count objects in
multiple channels simultaneously.
• Confluence: Allows you to select up to five reference objects for the target (i.e.,
cells) and one background reference in your image, then automatically
calculates the percentage confluence of your culture (page 92).
• Transfection Efficiency: Allows you to estimate the transfection efficiency of your
culture by calculating the ratio of fluorescence area (i.e., cells expressing the
fluorescence marker) to the total cell area in your culture (page 95).
• Batch Analysis: Allows you to save and apply the analysis parameters set in
Auto Count, Confluence, and Transfection Efficiency tools to other images that
you have collected and saved an image folder (page 100). Batch Analysis is not
available for Manual Count.

IMPORTANT! For analysis, only use 16-bit Raw image files, not Display or Merged
image files. Raw image files contain the full dynamic range and metadata needed
for quantitative analysis, whereas Display and Merged image files do not.

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 Count cells – Auto Count
Perform Auto 1. Click the Show Cell Count button, then select Auto Count.
Count

2. Select the nuclear stain channel in which to count objects.

Available options depend on the channels used when the image was captured.
In this example, TGBFP (TagBFP), GFP, RFP, CY5, and TRANS (transmitted
light) channels contain captured images, and TGBFP is selected for auto count.

3. To identify the target objects to include in your count, click Target, then click
and drag to draw a circle (blue) around a nucleus.

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 4. If desired, click Target again to identify other nuclei (for example, nuclei that
might appear different) to improve the accuracy of your count.

Note: For best results, follow these guidelines when identifying target objects:
- When selecting objects, circle the entire object and include a slight border
around it.
- To include objects of lower intensity in your count, select dimmer objects
during identification.
- Circle only one object at a time to help define object size for segmentation.

5. To distinguish the target from background, click Backround, then click and
drag to draw a circle (orange) in a background area.

6. After you define the target and background areas, the software automatically
counts the objects based on your criteria.

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 Viewing area identifies the objects that were counted with colored circles (in
this example, yellow) and the Object Count field displays the number of objects
included in the count.

Note: Depending on the quality of the image and your selection of

representative target cells and background, the auto count algorithm can
overcount or undercount the cells in the image.

Undercount – 3 cells counted as 1 Overcount – 1 cell counted as 2

To obtain a more accurate count:
- Split cells by shape or intensity.
- Refine your count by intensity, area, or circularity.

7. To count closely grouped cells that are touching or overlapping as distinct

objects, select from the Split Cells options:

• None: Touching or overlapping objects are not counted separately.

• Shape: Distinct objects are identified and counted based on shape.
• Intensity: Distinct objects are identified and counted based on pixel
intensity.

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 8. The Refine section displays a histogram that shows Count versus Intensity,
Area, or Circularity. In this example, Area is selected.

9. To refine your count, select Intensity, Area, or Circularity, move the gate
handles to set the upper or the lower boundary for the selected parameter.
You can refine the count by a single parameter or by multiple parameters.
The software applies the selected boundaries and recalculates the count.

10. When finished with the count, save your count results (see “Save analysis
results”, page 96).
11. To save your count settings for Batch Analysis, see “Save current analysis
settings”, page 100.

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 Count cells – Manual Count
Perform Manual 1. Click the Show Cell Count button, then select Manual Count.
Count

2. Select the Channels to display in the Viewing area for manual count. You can
select multiple channels that contain captured images.
In this example, TGBFP (TagBFP) and RFP channels are selected for the manual
count.

3. Click in an Object Name field to enter a name for that label. You can use up to
six labels for the manual count.

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 4. Click on the Object # to select a label, then left-click on the objects in the
Viewing area to tag them with that label. You can switch labels as desired.

As you tag the objects onscreen with the selected label, the system keeps a
running tally of the counts with percentages for each label assigned.

5. To delete a tag, right-click on the tag you wish to delete.

6. To delete all tags for a label, check the Delete box for the label, then
click the Trash button.
7. To delete all tags for all labels, check the Delete boxes for each label, then
click the Trash button.
8. When finished with the count, save your count results (see “Save analysis
results”, page 96).

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 Measure confluence
Confluence tool Confluence is a measure of how densely cells are distributed in culture. When all
available growth area is utilized in a culture vessel and the cells make close contact
with one another, the culture is at 100% confluence.
The Confluence tool measures the percent area covered by cells in the image, which
is required to calculate transfection efficiency.

Guidelines for • We recommend that you visualize your cells using transmitted light and a
confluence phase objective with 4X to 10X magnification. Set the phase ring to Oly 4× (for
Olympus™ 4X phase objective) or 4×/10× (for EVOS™ phase objectives) using
measurements
the phase annuli selector (page 8).
• For analysis, only use 16-bit Raw image files, which contain the full dynamic
range and metadata needed for quantitative analysis.
• In the analysis tool, increasing the number of targets and background areas
improves accuracy. You can select up to 5 target areas and 5 background areas.
• The Confluence tool uses a texture and intensity-based algorithm. The
sensitivity slider adjusts the algorithm sensitivity to pixel intensity (higher
intensity = more pixels included). Decreasing the sensitivity reduces the
confluence value.
• Different cell types have different confluence “patterns”, and variability in
morphology and contrast can influence the absolute confluence measurement
between different cell types. However, within a given cell type, you can
optimize the reproducibility of your measurements. Reproducibility in
confluence measurements is more important than absolute percentages.

Measure 1. In the EVOS™ Analysis application, open the image captured in the transmitted
confluence light channel.

2. Move the pointer to the Viewing area to reveal Display Settings and Analysis
Tools, then click the Show Cell Count button.

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 3. In the tabs area, select Cell Culture to display the Confluence tool.

4. Click Target, then click and drag to draw a circle around

an area that contains cells (blue circle).
5. Click Background, then click and drag to draw a circle
around a background area that does not contain any cells
(orange circle).

EVOS™ M7000 Imaging System User Guide 93

 Note: You can select up to 5 targets and 5 background areas. Increasing the
number of targets and background areas improves the accuracy and
reproducibility of the confluence measurements.

6. The software automatically calculates the confluence of your culture and

displays the results as a percentage of confluence.

7. To view the areas of the image counted as Target, select Show Mask. The areas
counted as Target are highlighted in the selected color.

8. Refine the sensitivity of the Confluence measurement using the Sensitivity

slider.

Note: As you adjust sensitivity, observe the image with the Show Mask
option on. Ensure that the target areas are selected with minimal coverage of
the background areas. Note that decreasing the sensitivity reduces the
confluence value.

9. When you complete the confluence measurement, Transfection Efficiency tool

becomes available. To calculate transfection efficiency, go to “Calculate
transfection efficiency”, page 95.
10. To save your Confluence results without calculating transfection efficiency,
click Save (see “Save analysis results”, page 98).
11. To save your confluence measurement settings for Batch Analysis, see “Save
current analysis settings”, page 100.

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 Calculate transfection efficiency
Transfection Transfection of a cell population typically results in a varying number of cells
Efficiency tool expressing the desired genes of interest. Transfection efficiency is the percentage of
cells that are transfected compared to the entire population.
The Transfection Efficiency tool calculates the fluorescence area (transfected cells
expressing the fluorescence marker) divided by the entire cell area in the image.

IMPORTANT! For analysis, only use 16-bit Raw image files, which contain the full
dynamic range and metadata needed for quantitative analysis.

Calculate 1. After you have completed the confluence measurement, click Transfection
transfection Efficiency to expand the controls for the Transfection Efficiency tool.
efficiency 2. Select the Fluorescence Channel for which you wish to measure the
transfection efficiency.
In this example, we want to calculate the percentage of cells that express GFP.
Therefore, the GFP channel is selected. The Fluorescence Channel and the
Transmitted Light Channel options are also checked, so that both channels are
displayed in the viewing area.

The software automatically calculates the Transfection Efficiency and displays

the results as % Confluence and % Transfection Efficiency.

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 Note: Transfection Efficiency calculation is based on the final measured
confluence (Step 8, page 94) and the fluorescence area that is above the set
fluorescence threshold value. To refine the Transfection Efficiency calculation,
adjust the Threshold such that only the cells that express at the desired level
are included in the calculation (see page 97).

3. To view the pure fluorescence signal and to observe the various levels of
fluorescence marker (GFP) expression, uncheck the Transmitted Light Channel.

4. Select Threshold Mask to highlight the fluorescence areas included in the

Transfection Efficiency calculation.

96 EVOS™ M7000 Imaging System User Guide

 5. To refine the Transfection Efficiency calculation, adjust the Threshold slider
until all the cells that express at the desired level are included in the calculation.

Note: Viewing the image only in the Fluorescence Channel and toggling the
Threshold Mask on and off will help you determine the best Threshold value
for your experiment.

As you adjust the Threshold, the software updates the calculation and displays
the new Transfection Efficiency value.

6. To save your Confluence and Transfection Efficiency results, go to “Save

analysis results”, page 98.

EVOS™ M7000 Imaging System User Guide 97

 Save analysis results
Save 1. When finished with the Auto count, Manual count, or Cell Culture analysis
(Confluence and Transfection Efficiency), click Save to open the Save Composite
Image dialog, then navigate to the destination folder to save your count results.

2. Enter File name and select File type.

3. If desired, select Save screenshot to preserve a detailed
account of your count results as a screenshot of the software user interface.
When Save screenshot is not selected, the software only saves the image as
displayed in the Viewing area. The saved image includes analysis results as
well counted objects, selected target and background areas, tags, in the selected
channels with or without mask as applicable to the analysis tool used.
Table 1 (page 99) lists the various options available for saving analysis results.
4. If you have performed an Auto count, select Save data to save a
separate CSV file of the count results with individual object
brightness, area, and circularity data.
This option is not available with Manual count, Confluence measurement, or
Transfection Efficiency calculation.
5. Click Save to save your analysis results.
6. To save your Transfection Efficiency calculation settings for Batch Analysis, see
“Save current analysis settings”, page 100.

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 Table 1. Save options for analysis tools.
Save Save as screenshot Save data
Auto Count Image and object count; Image (same as Save) and Separate image (same as
counted objects and user Auto Count tab with object Save) and CSV files; CSV file
selected target and count, histogram, and count contains brightness, area,
background areas identified options as displayed in the and circularity data
Auto Count tool
Manual Count Image and total object count; Image (same as Save) and N/A
object labels as tagged by Manual Count tab with labels,
user during count object counts, percentages,
and total count as displayed
in the Manual Count tool
Confluence Image in transmitted light Image (same as Save) and N/A
channel (with or without Cell Culture Confluence tab
mask) and % confluence; with % confluence and
user selected target and sensitivity as displayed in the
background areas identified Confluence tool
Transfection Image in selected channels Image (same as Save) and N/A
Efficiency (with or without Cell Culture Transfection
mask), % confluence, Efficiency tab
and % transfection efficiency with % confluence, % transfe
in the fluorescence channel ction efficiency, threshold,
and other options as
displayed in the Transfection
Efficiency tool
N/A: Not applicable.

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 Batch Analysis
Batch Analysis Batch analysis allows you to save and apply the analysis parameters set in Auto
function Count, Confluence, and Transfection Efficiency tools to other images that you have
collected and saved in an image folder.
Batch Analysis function is available as a Save Settings-Run Analysis split-button on
the Auto Count and Cell Culture tabs of the EVOS™ Analysis application. It is not
available for Manual Count.

IMPORTANT! Images to be batch analyzed should all be of the same cell type and
have the same magnification and illumination settings. For consistent
measurements, do not mix different cell types, magnifications, or illumination
settings in the same folder when performing batch analysis.

IMPORTANT! For batch analysis, only use 16-bit Raw image files, which contain the
full dynamic range and metadata needed for quantitative analysis.

Save current 1. When finished with the Auto count, Confluence measurement, or Transfection
analysis settings Efficiency calculation, click Batch, then select Save Settings.

2. Enter the name for the settings, then click Save. The current analysis settings
are saved for reuse, which you can apply to other images that you have
collected and saved an image folder.

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 Run Batch Analysis 1. Navigate to the folder that contains the images you wish to analyze using the
Batch Analysis tool, then click on a representative image to open it.
2. Perform Auto count, Confluence measurement, or Transfection Efficiency
calculation on the open image as described previously.
3. To analyze the remaining images in the same image folder, click Batch, then
select Run Analysis.

Note: You can also directly run Batch Analysis for images in an image folder
using previously saved Batch Analysis settings without first performing Auto
count, Confluence measurement, or Transfection Efficiency calculation (see
Step 4).

4. Select the Settings to use for Batch analysis.

• To use the current analysis settings (i.e., analysis parameters that you have
used in Step 2), select Current Settings.
• To use previously saved analysis settings, select the desired option from
the Select Settings list. You can sort the list by fluorescence channel, date
created, or last date used.
In this example, the previously saved U2OS Transfection Efficiency
setting is selected.

EVOS™ M7000 Imaging System User Guide 101

 5. To embed the measurements from the batch analysis in the images so that you
can compare them after the analysis, select Annotated Images.

Note: Summary Data is always selected. After the analysis, summary data is
included in the analysis folder as a separate CSV file.

6. To review the annotated images immediately after the analysis is complete,

select Review Annotated Images After Analysis.
7. Click Analyze to run batch analysis using the selected settings.
The software applies the analysis parameters used for the representative image
to all the images in the image folder.
When batch analysis is completed, the software saves the analysis results in a
separate folder in the same location as the analyzed images.
If Review Annotated Images After Analysis is selected, the software switches
to the Review mode and displays the list of analyzed images in the Review
panel.

Note: The analysis folder is named using the following format:

Batch <AnalysisDate><Unique Analysis ID>
For example, Batch 2021-01-19T114947
The images in the analysis folder retain their original name, but they are given the
prefix AN_.

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 7. Care and maintenance

General care
• When cleaning optical elements, use only optical-grade materials to avoid
scratching soft lens coatings.
• Use the appropriate cleaning solutions for each component, as indicated in the
Decontamination Procedures below.
• If liquid spills on the instrument, turn off the power immediately and wipe
dry.
• Do not exchange objectives between instruments unless you know that the
components have been approved and recommended by Thermo Fisher
Scientific.
• After using, cover the instrument with the supplied dust cover.

Note: Always use the correct power supply. The power adaptor specifications
appear on the serial number label (front of LCD hinge) and in the Specifications.
Damage due to an incompatible power adaptor is not covered by warranty.

CAUTION! Never disassemble or service the instrument yourself. Do not

remove any covers or parts that require the use of a tool to obtain access to
moving parts. Operators must be trained before being allowed to perform
the hazardous operation. Unauthorized repairs may damage the instrument
or alter its functionality, which may void your warranty. Contact your local
EVOS™ distributor to arrange for service.

IMPORTANT! If you have any doubt about the compatibility of decontamination or

cleaning agents with parts of the equipment or with material contained in it,
contact Technical Support (page 247) or your local EVOS™ distributor for
information.

Objective lens care

Clean each objective periodically or when necessary with an optical-grade swab
and a pre-moistened lens wipe (or lens paper moistened with lens cleaning
solution). To avoid scratching the soft lens coatings, use only optical-grade
cleaning materials and do not rub the lens.

Note: To protect all optical components of the instrument, use the dust cover
when the instrument is not in use.

EVOS™ M7000 Imaging System User Guide 103

 Stage care
• Clean the X-Y stage as needed with paper towels or Kimwipes™ tissues
dampened with 70% ethanol.
• Before moving the EVOS™ M7000 Imaging System to another location, use the
Shipping Restraints to lock the X-Y stage to prevent the stage from sliding
(page 112).

Decontamination procedures
In case hazardous material is spilled onto or into the components of the EVOS™
M7000 Imaging System, follow the decontamination procedure as described
below.
1. Turn power OFF.
2. Clean the LCD display.
a. Use a soft, dry, lint-free cloth to wipe off any dust from the screen.
b. Clean the LCD display with a non-alcohol-based cleaner made for flat-
panel displays.

IMPORTANT! Do not spray cleaning fluid directly onto the screen, as it may
drip into the display.

3. Lightly wipe working surfaces of the EVOS™ M7000 Imaging System (stage
top, objective turret, housing, etc.) with paper towels or Kimwipes™ tissues
dampened with 70% ethanol or 4,000 ppm hydrogen peroxide (H2O2).

IMPORTANT! Do not allow decontamination solution to get into lubricated

areas, such as the stage roller bearings, or any points of rotation such as stage
motors, condenser wheel, etc.
Do not soak any surface in decontamination solution.
NEVER spray liquid anywhere on the EVOS™ M7000 Imaging System.
Always wipe surfaces with dampened paper towels instead.

104 EVOS™ M7000 Imaging System User Guide

 Calibrate the stage
Stage Calibration establishes internal coordinates for the EVOS™ M7000 software to
properly execute its functions. The procedure requires the EVOS™ Calibration Slide
supplied with the instrument (also available separately; Cat. No. AMEP4720).

Calibrate the stage 1. Go to Settings tab, then click Stage Calibration.

2. Mount the vessel holder and the calibration slide as shown on the
screen, then click Calibrate XY.
As the instrument proceeds with the automated calibration procedure, the X-Y
stage moves between pre-set coordinates and the progress of the calibration is
shown on the screen.

EVOS™ M7000 Imaging System User Guide 105

 3. When the X-Y Stage Calibration is complete, click Next to proceed with Z Stage
Calibration.

4. Adjust the Brightness and Focus sliders to bring the image of the crosshairs into
focus, then click Done.

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 Calibrate vessel
Calibrate Vessel function establishes the internal coordinates of the selected vessel,
which allow the EVOS™ M7000 software to scan subsequent vessels of the same type
accurately.

Calibrate vessel The following protocol describes the Vessel Calibration process for a generic
96-well plate as an example. The Vessel Calibration process follows a similar
workflow for the calibration of other vessel types and the software provides on-
screen instructions at each step.
1. Mount the vessel holder and the sample vessel on the X-Y stage.
2. Go to the Settings  Vessels tab, then click the Vessel button to
open the Vessel Selection dialog.
3. Select the Vessel holder and Vessel type that corresponds to your
sample vessel, then click Done.

4. Click Calibrate Vessel to open the

Vessel Calibration dialog.
The first screen of the dialog provides an overview of the Vessel Calibration
process as well as options to cancel the calibration workflow or to restore the
default calibration values.

Note: If you need to restore the calibration values of a vessel to the default
settings, you can go to the first screen of the Vessel Calibration dialog and click
Restore Defaults, which replaces any existing calibration settings with the factory
default settings.

EVOS™ M7000 Imaging System User Guide 107

 5. Click Next to start the Vessel Calibration workflow.

6. Adjust lighting and focus.

7. Drag the image or use the Jog control to bring the Top edge of the A1 well into
view. If necessary, use the arrow buttons on the edges of the image to jump to
an adhacent field.

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 8. Drag the green line on the screen to align it with the top edge of the A1 well,
as illustrated on the screen.
If necessary, use the Zoom slider to zoom in and get a better view of the edge.

9. When you are satisfied with the alignment of the green line with the top edge
of the A1 well, click Next.
10. Following on-screen instructions, repeat the alignment procedure for the left,
bottom, and right edges of the A1 well, clicking Next after each alignment.
11. Following on-screen instructions, repeat the alignment procedure for the right
and bottom edges of the H12 well, clicking Next after each alignment.

EVOS™ M7000 Imaging System User Guide 109

 12. After you are done with the alignments, bring the sample in focus, then click
Next.

13. Review the images that show the alignment of the green line with the edges of
the A1 and H12 wells.

14. If satisfied with the results, click Save to update the existing vessel with the
new calibration values.
Alternatively, click Save As to save the vessel with the new calibration values
as new vessel with a name of your choice.

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 Change EVOS™ light cubes
You can add and remove EVOS™ light cubes to customize the EVOS™ M7000
Imaging System for your specific research needs. For a complete list of available
light cubes and to inquire about custom light cubes, go to thermofisher.com/evos
or contact Technical Support (page 247).

Change light cube 1. Remove the vessel holder from the X-Y stage.
2. On the Settings  Filter Cubes tab, select the Position of the light cube you
want to change, then click Move to filter cube change position.

3. Use the light cube tool to loosen the

two slotted screws (white arrows)
that are flush with the ridges on the
light cube.

4. Screw the threaded end of the light

cube tool into the hole in the center
of the light cube (as shown).

5. Use the light cube tool to tilt the light cube slightly toward you, then lift it out
gently. Unscrew and remove the light cube tool from the light cube.
6. Attach the tool to the new light cube, then lower the light cube into position.
Ensure that the electronic connections align properly (connections on the cube
facing the back of the microscope) and the light cube sits squarely in place.
7. Use the light cube tool to tighten the two slotted screws so that the screw
heads sit flush with the ridges on the light cube. Do not overtighten.

IMPORTANT! If the screws are not flush with the top of the light cube, they can
catch on the stage while moving and damage the system.

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 Change the objectives
You can add and remove objectives to customize the EVOS™ M7000 Imaging
System for your specific research needs.

Change objective 1. Remove the objective you want to replace from the objective turret. You may
need to move the stage so that the objectives are accessible. Note the indicated
position (1–5) of the removed objective on the turret (red arrow).

2. Screw the new objective into the open position in the objective turret. Note the
part number of the objective and the turret position. In the following example,
a new objective is installed into the turret position 5.

3. Go to the Settings  Objectives tab, and find objective in the Profiles list on
the left that matches the newly installed objective.

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 4. Click and drag the objective profile for the new objective to the appropriate
turret position on the right. In the following example, EVOS™ 40X objective
with 2.8-mm working distance is selected and dragged to the turret position 5.

5. Optional: If desired, type in a label in the Display Name text box. This creates
a label that is displayed on the Objective button in the Capture tab.
In the following example, “LWD” is entered as the Display Name.

The Objective button in the Capture tab displays the label “LWD” below the
objective magnification.

6. When finished, click Done to return to the Settings tab.

Note: For best results, calibrate the newly installed objectives before using it in
your experiments (page 114).

EVOS™ M7000 Imaging System User Guide 113

 Calibrate the objectives
Calibrate Objective is used to calibrate the field of view, parfocality, and
parcentration parameters of the selected objective.
Parfocality ensures that the sample stays in focus when the objective is changed,
and parcentration ensures that an object in the center of the field of view will stay
in the center of the field no matter which objective is being used.

Note: The pre-installed objectives supplied with the EVOS™ M7000 Imaging
System have been pre-calibrated. You do not need to calibrate them again unless
they are reinstalled after removal from the instrument.

Calibrate objective Calibration procedure involves two distinct steps and requires the use of the
EVOS™ M7000 calibration slide supplied with the EVOS™ M7000 Imaging System
(also available separately; Cat. No. AMEP4720).
First, the calibration crosshair on the screen is matched to the crosshair on the
calibration slide; then, the diameters of the reference circles on the calibration
slide are measured. Total time required objective calibration is about 5 minutes.
Note: For best parfocality and parcentration, calibrate the installed objectives one
after the other without removing the calibration slide.

1. Go to the Settings  Objectives tab, then click Calibrate to

launch the Objective Calibration tool.

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 2. Mount the EVOS™ Calibration Slide in the vessel holder as shown on the
screen, select the objective you want to calibrate, then click Calibrate
Objective.

The stage moves to the calibration target and the instrument automatically
finds the calibration crosshairs on the calibration slide (page 116).

Note: Depending on the type of objective being calibrated (e.g. Long Working
Distance vs. Coverslip Corrected objective), the calibration slide needs to be
mounted face up or face down. When mounting the slide, make sure to match the
orientation of the slide to the graphic shown on the calibration screen.

EVOS™ M7000 Imaging System User Guide 115

 3. Adjust the Brightness and Focus, then use the Jog Control button or click-drag
the screen to bring the black crosshairs on the calibration slide into view.

4. Manually align the green crosshairs over the black crosshairs or click Find
Crosshairs to automatically align once the black crosshairs are in view.

116 EVOS™ M7000 Imaging System User Guide

 5. Click Next to proceed to the second part of the calibration procedure,
measuring the diameter of the reference circle.
The stage automatically moves to the calibration circle and the software
automatically selects the appropriate reference circle on the slide for the
specific objective you are calibrating.

Note: If the circle on the screen is too large or too small, click Smaller Circle or
Bigger Circle to move to an appropriately sized circle. For best results, use the
largest circle possible.

6. Adjust the lighting and focus with the Brightness and Focus controls, then
click Find Circle to move the green calibration lines to the edges of the reference
circle. You can also manually move the lines by clicking and dragging them into
position.
7. When finished, click Next to complete the calibration.
8. Repeat the calibration process for each additional objective to be calibrated,
then click Done.

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 Install the shipping restraints
Use the Shipping Restraints whenever you package the unit for shipment. If the
unit is being hand-carried and is not at risk of drops or excessive vibration there is
no need to install the restraint. If you need to remove light cubes or objectives,
remember to do so before installation of the Shipping Restraint.

Install procedure 1. Go to Settings  Service tab, then click

Move to Shipping Position.
2. Turn off the power to the instrument (but not to the computer), then proceed
to securing the restraints in the locations shown.

3. To install the X-Y Stage Shipping Restraint, attach to threaded holes in the
front right corner of stage, then tighten the screws in order from bottom to
top, 1-2-3.
If necessary, you can gently push on the side or front of the stage to move the
plates into alignment.

IMPORTANT! Do not over-tighten the screws. Once the lock washer

compresses, stop tightening.

118 EVOS™ M7000 Imaging System User Guide

 4. To install the Light Cube Shipping Restraint, place the white Light Cube
Shipping restraint into the Light Cube access hole, then insert the Light Cube
Tool into the hole in the shipping restraint. Secure the restraint by screwing
the Light Cube Tool into the top of the Light Cube.

IMPORTANT! Do not over-tighten. The Light Cube Tool is secure when it

moves with the Light Cube. The Light Cube should still be able to move
slightly side to side with respect to the X-Y Stage.

5. To install the Camera Shipping Restraint, insert the Camera Shipping

Restraint into the hole at the back of the X-Y Stage, then screw it into the
camera carriage until the red knob touches the top of the X-Y Stage. Do not
over-tighten.

6. After securing all restraints, click Exit Application, then turn off the power to
the computer.

IMPORTANT! Do not power the instrument back on until the Shipping Restraints
have been removed.

EVOS™ M7000 Imaging System User Guide 119

 Appendix A: Troubleshooting

Note: For additional technical support, contact your local EVOS™ distributor. If you do not have your
distributor information, visit thermofisher.com/evos or contact Technical Support (page 247).

Image quality issues

Problem Possible solutions
Image is too dim (at higher
Remove condenser slider, if one is in place.
magnifications)
Follow instructions under “Objective lens care” (page 103) to clean
Specks, dots, or blurs on image
objectives.
• Position sample, so that it lies flat on the stage; ensure that the
Uneven focus across screen sample’s thickness is even.
• Be sure vessel holder is mounted flat with respect to stage.
Place the slide so the coverslip is facing up (long working-distance
Difficulty focusing on coverslipped
objectives require a thick optical substrate, and image best
sample on standard slide
through 1.0–1.5 mm of glass or plastic).
• Click the Power button (onscreen).
Image display is black • Center sample over objective.
• Verify power supply is connected and power switch is on.
• Dim the illumination until the red highlights disappear to get
the maximum level of brightness without any overexposed
Image display is red, or red patches areas.
cover parts of the screen
• Disable the “Show saturated pixels” option in the Settings 
General tab.

120 EVOS™ M7000 Imaging System User Guide

 Software interface issues
Note: We recommend keeping the EVOS™ M7000 Imaging System up to date with the latest software.

Problem Possible solutions

Image does not respond to changes in
Click the Light button to return to live observation.
focus or stage position
The controls available on the EVOS™ M7000 Imaging System are
Some of the software controls are not
contextual; only the controls relevant for the chosen task will be
available
available.
Save button does not respond when Click capture first; it is only possible to save an image that is
clicked captured.
• Verify physical cable connections; confirm the Ethernet jack is
active.
Unable to connect to network
• Contact your network administrator to resolve any network
issues.

Mechanical issues
Problem Possible solutions
Automatic stage does not move Remove shipping restraint.
Filter Cube Axis does not move Remove shipping restraint.
Camera Axis does not move Remove shipping restraint.
Vessel does not sit securely on moving Use the correct vessel holder for the application (visit
stage thermofisher.com/evos).

EVOS™ M7000 Imaging System User Guide 121

 Appendix B: System overview

Technical specifications
Note: Technical specifications of the EVOS™ M7000 Imaging System are subject to change without notice.
For the latest product information, see the product page (thermofisher.com/evosm7000).

Physical Dimensions (L × W × H): 45.7 × 33.0 × 35.6 cm (18 × 14 × 13 inches)

characteristics Weight: 16 kg (35 lb)
Footprint: Approximately 92 cm × 92 cm (36 in × 36 in); entire system includes the
instrument, computer, and 23-inch touchscreen, high-resolution LCD monitor
Operating temperature: 4°–32°C (40°–90°F)
Operating humidity: <90%, non-condensing
Operating power: 100–240 VAC, 1.8 A
Frequency: 50–60 Hz
Electrical input: 24 VDC, 5 A

Hardware Optics: Infinity‐corrected optical system; Royal Microscopical Society (RMS)‐

threaded objectives with 45 mm parfocal distance
Imaging mode: Fluorescence, brightfield, color brightfield, and phase contrast
Imaging methods: Single color, multicolor, area scan with tiled or stitched images,
time lapse, z-stacking, movie capture
Illumination: Adjustable intensity LED (>50,000-hour life per light cube)
Light cubes (not included): 5 position chamber for 4 fluorescence light cubes plus
brightfield imaging; light cubes with integrated hard-coated filter set; broad
selection standard and specialty light cubes (page 124)
Contrast methods: Fluorescence and transmitted light (brightfield & phase contrast)
Objective turret: 5-position; front-mounted control
Objectives (not included): Wide selection of high‐quality long working distance
(LWD) and coverslip‐corrected objectives
Condenser: 60 mm LWD condenser; 4 position turret with a clear aperture and
3 phase annuli
Stage: Motorized X-Y scanning stage; 120 mm × 80 mm travel range with sub‐
micron resolution; drop‐in inserts to receive vessel holders and lockdown holders
to fix sample in place during long scans
Focus mechanism: Automated focus mechanism with sub‐micron resolution
LCD display: 23-inch high-resolution touchscreen color monitor (also fully
controllable via mouse); 1920 × 1080 pixel resolution
Cameras: High-sensitivity 3.2 MP (2048 ×1536 pixels) monochrome CMOS sensor
with 3.45 µm pixel resolution; high-sensitivity 3.2 MP (2048 ×1536 pixels) color
CMOS sensor with 3.45 µm pixel resolution
Captured images: 16‐bit Raw monochrome: TIFF, PNG; 8-bit per RGB channel:
TIFF, PNG, or JPG; movies and time‐lapse images: AVI or WMV

122 EVOS™ M7000 Imaging System User Guide

 Computer: External Dell™ PC with an Intel™ Core™ i7-8700 processor, 32 GB DDR4
RAM, 512 GB PCIe solid-state drive, NVIDIA™ Quadro™ P1000 graphics card with
4 GB memory, and Windows™ 10 software, designed to operate with touchscreen
monitor and microscope
Output ports: Instrument: USB 3.1 Type B, 4-pin power port;
Computer: 1 × USB 3.1 Gen 2 Type C; 5 × USB 3.1 Gen 1 Type A;
4 × USB 2.0 Type A; 1 serial; 2 × DisplayPort 1.2; 1 RJ45; 2 PS/2; 1 UAJ; 1 line-out
Networking capability: Connection through Windows/SMB network via an
Ethernet cable connection
Power supply: AC adapter with country‐specific power cords.

EVOS™ M7000 Imaging System User Guide 123

 Operation principles and technical overview
LED illumination The EVOS™ M7000 Imaging System utilizes an adjustable intensity LED light
source provided by the proprietary, user-interchangeable LED light cube (see
below). Because the LED light source is as close as possible to the objective turret,
the number of optical elements in the channel is minimized. High-intensity
illumination over a short channel increases the efficiency of fluorophore excitation,
providing better detection of weak fluorescent signals.
In contrast to traditional fluorescence microscopy light sources that use mercury, a
toxic carcinogen requiring special handling and disposal, the LED light source of
the EVOS™ M7000 Imaging System is more environmentally friendly, energy
efficient, and has a significantly longer life span (>50,000 hours versus 300 hours
for a typical mercury bulb and 1,500 hours for a metal halide bulb).

LED light cubes Each user-interchangable, auto-configured light cube contains an LED, collimating
optics, and filters. In addition to the channel dedicated to the transmitted light
from the condenser for brightfield contrast applications, the EVOS™ M7000
Imaging System can accommodate up to five fluorescent or specialty light cubes
for multiple-fluorescence research applications.
The table below lists some of the common fluorescent and specialty light cubes
available from Thermo Fisher Scientific. For a complete list of available light cubes
and to inquire about custom light cubes, go to thermofisher.com/evos or contact
Technical Support (page 247).

Light cube Dye

DAPI DAPI, Hoechst , BFP
™

TagBFP TagBFP
CFP ECFP, Lucifer Yellow, Evans Blue
GFP GFP, Alexa Fluor™ 488, SYBR™ Green, FITC
YFP EYFP, acridine orange + DNA
RFP, Alexa Fluor™ 546, Alexa Fluor™ 555, Alexa Fluor™ 568,
RFP
Cy™3, MitoTracker™ Orange, Rhodamine Red, DsRed
Texas Red™, Alexa Fluor™ 568, Alexa Fluor™ 594, MitoTracker™
Texas Red
Red, mCherry, Cy™3.5
Cy5 Cy™5, Alexa Fluor™ 647, Alexa Fluor™ 660, DRAQ5™
Cy5.5 Cy™5.5, Alexa Fluor™ 660, Alexa Fluor™ 680, Alexa Fluor™ 700
Cy7 Cy™7, IRDye 800CW
CFP-YFP em CFP/YFP (for FRET applications)
AO Acridine orange + RNA, simultaneous green/red with FL color
White Refracted light applications

124 EVOS™ M7000 Imaging System User Guide

 Image capture and save formats
Raw vs. Displayed Raw images contain minimally processed data from the image sensor. The purpose
images of raw image formats is to save, with minimum loss of information, data obtained
from the sensor, and the conditions surrounding the capturing of the image (the
metadata). As such, it is the recommended format for image analysis and
quantitation. EVOS™ M7000 Imaging System can save images captured in different
channels individually as 16-bit Raw images.
Available File format for viewing Raw image outputs in EVOS™ M7000 Imaging
System are TIFF, PNG, C01, and DIB. The changes made on a Raw image file to
produce the viewable output are non-destructive; that is, only the metadata that
controls the rendering is changed to make different output versions, leaving the
original data unchanged.
Displayed images are captured images that are saved in a format that can be
viewed in most image display applications. Displayed images give the best
(“prettiest”) results, especially when producing tiled and stitched images, but
result in loss of information when the data from the image sensor is rendered to
produce the viewable image.
We recommend saving captured images as both Raw and Displayed images to
preserve the option of using the Raw images in downstream image analysis and
quantitation, and the Displayed images for instances where “prettier” images are
required, such as publications, presentations, etc.

Tiled, Stitched, and If an object extends into multiple fields, a set of multiple images from the fields into
Merged images which the object extends may be required to show the object of interest in full.
Tiled images are images captured from multiple fields in an area and lined-up
close together into a tiled format, thus giving you a single image that includes
images from all the fields in the selected area.
When you use the Tiled image option without enabling Stitching, the images are
simply placed very close together with a barely noticeable seam to create an image
of all fields in that area. Tiling is quicker than stitching and is often sufficient.
If you notice that images from adjacent fields are not lining up correctly, you will
want to ensure that your instrument is fully calibrated and the camera is aligned.
Stitched images are images captured from multiple fields in an area and lined up
with a slight overlap into a tiled format. Stitching uses an algorithm to apply the
overlap, then removes the seam so you can visualize objects that cross fields.
Stitching option is available under Scan Area of Automate tab.
If you are analyzing images that cross fields (such as images of neurites), and the
images are slightly misaligned, you might want to consider Stitching the images.
Merged image is an image generated by combining the images captured in
different channels into a multicolor overlay. It is also referred to as a composite
image.

EVOS™ M7000 Imaging System User Guide 125

 Appendix C: Graphical user interface (GUI)

The EVOS™ M7000 Imaging System is controlled by the integrated Invitrogen™

EVOS™ M7000 Software through a graphical user interface (GUI), which is accessed
by the touchscreen monitor or the computer mouse and keyboard.

GUI layout The GUI of the EVOS™ M7000 Imaging System consists of the Viewing area on the left
and a series of tabs representing the main functions of the software (Capture,
Automate, Review, and Settings) on the right. Each tab contains the controls
necessary to execute the selected function. The Locations button, Area View/Field
View toggle, and the Zoom slider are located above the Viewing area, which can
contain additional controls depending on the view mode and the tab selected.

Locations: Opens the Location tools, which allow you to select locations or
designate capture fields for automated scan protocols (page 134).
Area View/Field View toggle: Switches between Area View and Field View.
Zoom slider: Zooms in and out of the Viewing area.
Capture tab: Contains the controls for the manual capture of images (page 139).
Automate tab: Used to create and run automated scan protocols (page 157).
Review tab: Allows you to review captured images (page 192).
Settings tab: Contains controls to select and adjust basic and advanced system
options and instrument functions (page 196).
Viewing area: Displays the sample in Area View or Field View mode
(page 127).

Note: Click on the View Help Content icon to open the Help window, which
provides EVOS™ M7000 Imaging System Help.
Position your pointer over the Tooltip icon without clicking it to view a
small Position box with additional information.

126 EVOS™ M7000 Imaging System User Guide

 Viewing area
Overview Viewing area displays the sample in Area View or Field View. The Area View/
Field View toggle and the Zoom slider are located above the Viewing area.

Area View In Area View, the Viewing area displays a graphical representation (i.e., map) of
the selected target area in its entirety. Depending on the vessel in use, the target
area may represent a single well of a multiwell plate, a chamber of chamber slide,
or an entire culture dish.
Area View allows you to select individual fields of view for manual image capture.

Locations (page 134) Toggle locations display (page 129)

Area View/Field View toggle (page 128) Toggle field borders (page 129)
Zoom slider (page 128) Image display settings (page 129)
Area map (page 128) Center on selected field (page 130)
Field selection tool (page 128) Delete image options (page 130)
Selected field (page 129) Area and pixel information
(page 130)
Captured field (page 129)

EVOS™ M7000 Imaging System User Guide 127

 Area View controls Locations button: Opens the Locations tools, which allow you
to mark (“pin”) specific points or draw fields of interest in the
Viewing area, which can be revisited for further examination or captured
during automated scan protocols.
Note that the Location tools accessed from the Area View differ from those that
are accessed through the Field View.
For detailed description of the Locations function, see page 134.
Area View/Field View toggle: Switches between Area View and
Field View.
Zoom slider: Zooms in and out of the Viewing
area.
The minimum and maximum zoom for Area View are 1X and 2X, respectively.
Area map: Represents the current sample area selected for view or capture (i.e.,
target area). The target area is selected using the Vessel map (page 141).

Note: If using a single culture dish, flask, or slide, the scan area corresponds to
the entire dish, flask, or slide. As such, the scan area displayed in Area View
represents the entire dish or flask selected.
In a multi-well plate, multi-chamber slide, or multiple dishes on multi-dish
holders, each scan area corresponds to a single well, chamber, or dish.

The following examples show the area map of a single well of a 96-well plate
(left) and a sample slide on a double-slide holder (right). The orange square in
each examle correspond to a single field view at 4X magnification and illustrate
the relative size of a single well in a 96-well plate and a sample slide.

Area map – Single well of a 96-well plate Area map – Sample slide

Field selection tool: Selects the target field you want to capture.
• The selection tool consists of an orange crosshair within a
broken orange rectangle. The crosshair indicates the position
of the objective lens over the target and the rectangle represents
the actual imaging area.
• The relative size of the field selection tool depends on the zoom level and
the selected objective.

128 EVOS™ M7000 Imaging System User Guide

 Selected field: Field that has been selected. Selected field
corresponds to the field of view displayed in the Field View.
• Selected field, whether captured or not, is indicated by orange
borders around it, which cannot be turned off.
• Clicking the Capture button (page 148) captures an image of the selected
field and stores it in the memory buffer.
Captured field: Field that has been captured. A thumbnail of the
captured image is displayed within the field borders.
If multiple images are captured for the same field in different
channels, the captured images are displayed as an overlay.
Toggle locations display: Switches the display of locations (defined with the
Location tools) in Area View on and off.

Locations display is on Locations display is off

Toggle field borders: Switches the display of the borders around captured
fields in Area View on and off. This option is not available in Field View.

Field borders are on Field borders are off

• When field borders are turned on, all captured fields in Area View are
displayed with blue borders around them.
• Currently selected field, whether captured or not, is displayed with an
orange border, which cannot be hidden.
Image display settings: Opens the Image display settings window,
which allows you to adjust image display parameters (brightness,
constrast, gamma) for the Viewing Area. Clicking the button again
closes the window.

Note: Adjustments made to Image display settings only affect how the image
is displayed in the Viewing Area; they do not affect how the image is captured.

• Visible Channels: Visible channels are the channels selected for

display in the Viewing area. They are selected using the Toggle channel
display checkbox located on the top left corner of the corresponding Light
source button (page 143).

Note: The controls for image display settings are contextual; only the controls
for the visible channels will be available. In the example above, only the
controls for the GFP DAPI, and RFP channels are displayed.

• To remove a channel from display, uncheck the corresponding Visible

Channel checkbox. To display it again, recheck the checkbox.

EVOS™ M7000 Imaging System User Guide 129

 • Brightness , Contrast , and Gamma parameters for each of the
selected channels are adjusted using the corresponding sliders.
• Toggle image color display: Option to display images in pseudocolor
or in grayscale in the Viewing area. By default, color display is on.
When on, images are displayed in default pseudocolor corresponding to
the channel in which they were captured. You can change the default
pseudocolor using in the Settings  Filter Cubes tab (page 192).
When off, images are displayed in grayscale.

Color display is on Color display is off

Center on selected field: Centers the Viewing area on the selected field.
Delete image options: Opens the Delete image options window, which
contains the controls listed below. Clicking the button again closes the
window.

• Delete Selected Field: Deletes only the selected field, if the selected field
contains a captured image. If the selected field is empty, this option is not
available.
• Delete Area Fields: Deletes only the fields that were captured in the
current scan area. Images captured in other areas are not deleted.
• Delete Session Fields: Deletes all fields in all areas that were captured in
the current session.
Area and pixel information: Displays the coordinates of the pointer and pixel
intensity at the pointer location.

• Area XY Location (Microns): Location of your pointer in relation to the

center of the selected well along the x-y axis, expressed in µm.
• Pixel XY Offset: Location of your pointer in relation to the top left corner of
the captured field, expressed in pixels along the x-y axis.
• Z Locations: Location of the captured plane along the z-axis, in µm.
• Pixel Intensities: Pixel intensity at the pointer location as reported by the
camera. It represents the number of photons detected by the camera sensor.
• Area XY Location at the pointer location is displayed for the entire scan
area. Pixel XY Offsets, Z Locations, and Pixel Intensities are displayed only
for captured fields.

130 EVOS™ M7000 Imaging System User Guide

 Field View Field View displays the currently selected field in the Viewing area. It allows you
to preview the sample and to adjust imaging parameters (e.g., brightness, focus) in
the Live mode before capture.
• If the selected field contains a captured image and the display is turned on for
the channel in which the image was captured, Field View displays the captured
image, allowing you to preview it before saving.
• If the field was captured in multiple channels, Field View displays an overlay
of the captured images.

Locations (page 134) Toggle scale bar (page 132)

Area View/Field View toggle (page 132) Toggle sample grid (page 132)
Zoom slider (page 132) Image display settings (page 132)
Delete field (page 132) Area and pixel information
(page 133)
Navigation arrows (page 132) Selected field of view (page 133)

EVOS™ M7000 Imaging System User Guide 131

 Field View controls Locations button: Opens the Locations tool, which allows you
to mark (“pin”) specific points in Field View.
The pinned points can be revisited later or used in a automated scan protocol.
Note that the Location tools accessed from the Field View differ from those
that are available through the Area View.
For detailed description of the Locations function, see page 134.

Area View/Field View toggle: Switches between Area View and

Field View.
Zoom slider: Zooms in and out of the Viewing
area.
The minimum and maximum zoom for Field View are 1X and 4X, respectively.
Delete field: Deletes the image captured in the current field on view.
This button is available only if there is an image captured in the current
field on view.
Navigation arrows: Moves the stage exactly one field of view in the
corresponding direction (up, down, left, right).

Toggle scale bar: Switches the display of the scale bar in the Viewing area on
and off.
Scale bar display is on Scale bar display is off

Toggle sample grid: Switches the display of the sample grid in the Viewing
area on and off.
Sample grid display is on Sample grid display is off

Image display settings: Opens the Image display settings window,

which allows you to adjust image display parameters (brightness,
constrast, gamma) for the Viewing Area. Clicking the button again
closes the window.
For more information on Image display settings window, see page 129.

132 EVOS™ M7000 Imaging System User Guide

 Area and pixel information: Displays the coordinates of the pointer and pixel
intensity at the pointer location.

This information is displayed only if the Light is on for the selected channel, or
if the current field of view contains a captured image and image display is on
for the channel in which the image was captured.
• Area XY Location (Microns): Location of your pointer in relation to the
center of the selected well along the x-y axis, expressed in µm.
• Pixel XY Offset: Location of your pointer in relation to the top left corner of
the captured field, expressed in pixels along the x-y axis.
• Z Locations: Location of the captured plane along the z-axis, in µm.
• Pixel Intensities: Pixel intensity at the pointer location as reported by the
camera. This represents the number of photons detected by the camera
sensor.
Selected field of view: Displays the entire selected field of view.

• If a previously captured field is selected in Area View, Field View displays

the image captured in the selected field.
• If the selected field is captured in multiple channels, Field View displays
an overlay of the captured images.
• Switching the Field View to Live mode (i.e., Light button on) displays the
live image of the selected field in the current channel, whether the field
contains a captured image or not.
• Switching the Live mode off (i.e., Light button off) returns the Field View
to its previous state (blank or displaying the previously captured image).

EVOS™ M7000 Imaging System User Guide 133

 Locations Locations function allows you to mark (“pin”) specific points or draw fields of
interest in the Viewing area. These locations of interest can then be revisited for
further examination or captured during automated scan protocols.
Note that the Location tools are contextual; the tools available depend on whether
the instrument is in Field View or Area View.

Locations – Area View Locations – Field View

Location tools (page 135) Show location numbers (page 138)

Locations directory (page 137) Delete all (page 138)
Show locations (page 138) Done (page 138)

134 EVOS™ M7000 Imaging System User Guide

 Locations controls Location tools: Used to define points and fields of interest.

Location tools – Area View Location tools – Field View

• Select\Pan: Allows you to explore different regions of the

sample vessel in the Viewing area when the Locations controls
are active.
To navigate to a different region in the Viewing area, select Select\Pan,
then click-drag the Viewing area in the desired direction.
• Ellipse: Allows you to define a field group that best fits an
ellipse drawn in Area View.
To define the capture region, select Ellipse, then click-drag an
ellipse shape in Area View.

• Rectangle: Allows you to define a field group that best fits a

rectangle drawn in Area View.
To define the capture region,select Rectangle, then click-drag a
rectangle shape in Area View.

EVOS™ M7000 Imaging System User Guide 135

 • Freeform: Allows you to draw a freeform shape based on
straight-sided polygonal shapes in Area View that will
encompass the capture fields.
To define the freeform region, select Freeform, then click on the desired
location in Area View to add your first anchor point.
At the next location, click again to add your second anchor point. When
you release the mouse button, both points will be joined by a straight line.
Continue moving around the Area View, clicking to add new anchor
points and fastening the end of the line to each new point as you go.
To close your freeform shape, double-click on your initial anchor point.
Alternatively, double-click anywhere on Area View to close your freeform
shape with a straight line from this point to your initial starting point.

• Point: Allows you to mark (“pin”) specific points in Area and

Field Views as locations of interest.
To mark a point in Area or Field View, select Point, then click on
the desired location.

136 EVOS™ M7000 Imaging System User Guide

 Locations directory: Lists the locations (points or shapes) created with the
Location tools in a table format. You can select a location from the directory to
view the sample and/or set the z-focus at that location.
In the following example, the directory lists three locations created in the first
well (Area: A01) of a 96-well plate: an ellipse, a rectangle, and a point.

• To select a location, click the desired location from the list. The X-Y axis
stage moves to align the objective with the selected (“current”) location.
In the example below, location number 3 (“Point_3”) is selected in the
Locations directory and the objective is centered on that location (as
indicated by the orange rectangle showing the field of view).

EVOS™ M7000 Imaging System User Guide 137

 • To set the z-focus for a location, select the location from the list, then click
Set Z. The z-focus will be set at the current z-position for that location.

• To change the z-position at a selected location, exit the Locations tool and
adjust the focus using the focus sliders in the Capture tab, then Set Z for
that location as described.
• To remove a location from the list, select the location, then click the
Delete Location button.

Show locations: Toggles the display of existing locations

in the Viewing area.
Show location numberss: Toggles the display of
location numbers for the existing locations in the
Viewing area.

Delete all: Deletes all locations in the Locations directory and removes them
from the Viewing area..
Done: Finalizes the location creation procedure and returns to the Capture tab.

138 EVOS™ M7000 Imaging System User Guide

 Capture tab
Overview Capture tab contains the controls for the manual capture of images. You can access
the Automate, Review, and Settings at any time by selecting the corresponding
tab. All current selections and settings within the Capture tab are preserved if you
navigate away from the Capture tab.

Vessel (page 140) Advanced focus settings (page 146)

Vessel map (page 141) Capture (page 148)
Zoom (page 142) Brightness controls (page 149)
Objective (page 142) Focus sliders (page 150)
Thumbnail images (page 142) Record Video (page 150)
Light source (Channel) (page 143) Incubator (page 151)
Light (Live mode) (page 143) Capture Z Stack (page 152)
Brightness and camera settings (page 144) Capture Channels (page 154)
AutoFocus (page 145) Save (page 154)

EVOS™ M7000 Imaging System User Guide 139

 Capture tab Vessel: Opens the Vessel Selection dialog, which allows you to
controls specify the vessel and vessel holder in use. You MUST select a vessel
prior to imaging.

• Select the Vessel category that corresponds to your sample vessel to

display the holder and vessel type selections available for that category.
Available vessel categories are Well Plates, Flasks, Dishes, and Slides.

The following images are example screens from the Vessel Selection dialog
for each of the vessel categories.

Well Plates Flasks Dishes Slides

Note: The controls available in the Vessel Selection dialog are contextual; the
dropdown menus display only the options available for the selected vessel category.

140 EVOS™ M7000 Imaging System User Guide

 • Select the appropriate Holder and Vessel type from the dropdown menus
available for your vessel category.
The following images are example screens from the Vessel Selection dialog
for vessel holder and vessel type selection for each of the vessel categories.

Well Plates Dishes

Flasks Slides

Vessel map: Represents the vessel container (vessel + vessel holder) in use, as
determined by the selection made in the Vessel selection dropdown. The well
selected for imaging is indicated in blue on the Vessel map.
• To select a well to display in the Viewing area, click on the desired well,
flask, dish, or slide.
In the following examples, the first well of a 96-well plate and the first dish
on a 35-mm quad dish holder have been selected.

96-well plate 35-mm dishes on a quad dish holder

• When in the Live mode (page 143), the Vessel map displays crosshair that
aid in navigation around the vessel or well, as shown below.

96-well plate 35-mm dishes on a quad dish holder

• To move the stage to another location on the sample vessel to display a
different field, click and drag the crosshair to the corresponding location
on the virtual vessel.
• When the Light is off (i.e, not in the Live mode), click on a different well on
the Vessel map to move the crosshair to the same relative position as the
previous well.

EVOS™ M7000 Imaging System User Guide 141

 Zoom: Opens a larger view of the Vessel map.
• This view shows the same content as the smaller Vessel map and behaves
the same way.
• Clicking the button again closes the zoom window.

Objective: Used to select from the currently installed objectives.

• The magnification displayed on the objective button reflects the profile
specified in Objective setup on the Settings tab.
• You can select only one objective at a time. In the example below, the 10X
objective is selected.

Thumbnail images: Display the most recently captured image stored in the
memory buffer for the specific channel.
• If unsaved, newly captured images will overwrite the previously captured
image in the channel.
• If no image has been captured for the channel, the corresponding
thumbnail will be blank.
• If z-stack was captured in the channel, the z-stack icon on the top left
corner of the thumbnail image for that channel identifies the image as
a z-stack projection.
• You can delete an image captured in a channel by clicking the Delete
channel image icon on the top right corner of the thumbnail image
for that channel.
In the example below, GFP channel displays the thumbnail for a z-stack
projection while the RFP channel displays the thumbnail for a field captured at
single focal plane. The rest of the channels do not have any images captured.

142 EVOS™ M7000 Imaging System User Guide

 Light source: Selects the desired light source from the installed LED light cubes
(fluorescent channels) or transmitted light from the condenser (brightfield).
• Click a Light source button to select the corresponding channel for
capture. The selected channel is indicated by the blue arrow underneath
the corresponding light source button. You can select only one light source
at a time for capture.
• Check the Toggle channel display option located on the top left corner of
the Light source button to select the corresponding channel for display in
the Viewing area. You can select multiple channels for display.
In the example below, the GFP channel is selected for capture and both
GFP and RFP channels are selected for display.

Light (Live mode): Turns on the excitation light for the currently selected light
cube and enters the instrument in the Live mode. The Live mode allows rapid
visualization of a large sample area illuminated with the selected light source
before image capture.

Light OFF Light ON

• Clicking the Light button a second time or capturing an image turns the
light off and exists the instrument from the Live mode.
• In the Live mode, the Viewing area shows the sample illuminated under the
selected light source in pseudo-color with the default emission color.
In the example below, the sample is illuminated in the GFP channel.

• The Vessel map displays the navigation crosshair, and the Jog
Control button appears next to the Vessel map.
• Jog Control moves the stage at an intermediate pace, allowing the quick
scanning of the sample in different parts of the sample vessel.

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 Brightness and camera settings: Opens the Brightness and camera settings
window, which allows you to set Mode, Camera, and Phase options for the
selected light source. Clicking the button again closes the window.

• Mode: Toggles between Simple and Actual modes for brightness controls.
- Simple mode allows you to control light intensity as a single
Brightness parameter (page 149).
- Actual mode allows you to adjust Light (i.e., LED intensity), Exposure,
and Gain parameters individually.
• Camera: Toggles between the Mono (monochrome)and Color cameras.
- Mono is used for image capture in fluorescence and transmitted light
(brightfield) channels. It uses a high-sensitivity 3.2 MP CMOS
monochrome camera with 2048 × 1536 pixel resolution.
- Color is used for image capture in the brightfield channel only. It uses
a high-sensitivity 3.2 MP CMOS color camera with 2048 × 1536 pixel
resolution.
• Phase: Selects between Phase options and Brightfield in the transmitted
light channel only. Phase contrast options change the phase annuli being
used to match the objective with a specific magnification. Available
options are:
- Small Ring: Used for objectives with low magnification (i.e.,
Olympus™ 4× PH)
- Medium Ring: Used for objectives with medium magnification (i.e.,
EVOS™ 4×/10× PH)
- Large Ring: Used for objectives with high magnification (i.e., EVOS™
20×/40× PH)
- Brightfield (phase contrast off)

Note: Phase options are available only for the transmitted light (brightfield)
channel; they are not available for fluorescence channels. The phase contrast
option does not require phase contrast objectives. However, to obtain a phase
image, you must first install a phase contrast objective.

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 Autofocus: Runs the autofocus algorithm in the currently
selected channel to find the best focus position for the sample.
This button is available on both Capture and Automate tabs.
• The Autofocus window displays the image from the selected channel in
real time as the instrument searches for the optimal focus.

• To minimize the Autofocus window, click the minimize button.

Once minimized, the Autofocus window can be moved around the

Viewing area.
To expand the Autofocus window, click the expand button.
• Click Stop AutoFocus in the Autofocus window to abort
the autofocus procedure.

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 Advanced focus settings: Opens the Advanced focus settings window, which
contains the controls listed below. Clicking the button again closes the window.

• Autofocus method: Allows you to select the autofocus method best suited
for your experimental needs. Available options are:
- Fluorescence Optimized: The focal plane is derived from the highest
ratio between detailed, high-contrast objects against the background.
This option is recommended for fluorescence imaging.
- Transmitted Optimized: The optimal focal plane is derived through
statistics-based edge detection over 9 different regions to determine the
highest ratio of edge-to-background.
This option is recommended for transmitted-light imaging.
- Small Structure: This method computes the energy according to the
size of image features and can measure the presence or absence of small
image features.
Choose this method when capturing images of samples that have many
fine, hair-like structures (e.g., filaments or structural stains).
- Large Structure: This method is statistics-based and looks for large
changes in image content as parts of the image go in and out of focus.
Choose this method when your sample contains large structures (e.g.,
whole cell stains).
- Small Bright Objects: This method looks for changes in brightness at
the center of the cell and optimizes focus on cells that have a bright
center with a dark surround area.
Choose this method when capturing samples with localized staining
(e.g., nuclei).

Note: The effectiveness of the autofocus method depends on the plate type,
sample type, biomarker intensity, magnification, and channel. It may be
necessary to use different algorithms for different channels. This ensures the
best autofocus is used for your sample.

• Lock Z-Offsets: Allows you to lock the Z-Offsets, which specify the optimal
focus position in each channel relative to the focus position in other
channels.
- When locked, adjusting the Z-position in one channel changes the
Z-position in all channels, preserving the relative Z-positions of the
channels.
- When unlocked, adjusting the Z-position in one channel does not affect
the Z-position set for the other channels.
For an example, see “Note” on page 147.
For more information on configuring the autofocus settings for an
automated scan protocol, see page 176.

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 Note: Consider a scenario where the initial Z-positions of the DAPI and RFP
channels are 50 µm and 80 µm, respectively. The autofocus is run from the
DAPI channel and finds the best focal plane at a Z-position of 60 µm.
If the autofocus is selected to run from a single channel, then the Z-Offsets
will be locked, and the RFP channel will be focused to a Z-position of 90 µm.
If the autofocus is run from all channels, then the Z-Offsets will not be locked,
and the autofocus algorithm will find the best focal plane for the RFP channel
independently of the DAPI channel.

• Clear Z-Offsets: Clears the Z-Offsets.

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 Capture: Captures an image of the selected field using the current capture
settings and stores it in the image cache of the channel in which it was captured.
• Field View displays the captured image. If multiple images are captured in
the same field of view using different channels, the captured images are
displayed as an overlay.
• The captured image is also be displayed above the Light source button for
the specific channel in which the image is captured.
• A thumbnail of the captured image is displayed in Area View at the
location of its capture when the zoom level is greater than one field of view.
The examples below show the Field View (top) and the Area View (bottom)
after the same field has been captured in DAPI and GFP channels.

Note: Captured images are stored in the image cache for the selected channel.
If unsaved, newly captured images of the same field will overwrite the
previously captured image in that channel.

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 Brightness controls: Control the brightness settings for the selected channel.
You can adjust the brightness settings for each channel independently without
affecting the settings for other channels.
To adjust the settings for a given parameter, move the corresponding slider in
the desired direction or enter the desired value in the text box.
• In the Simple mode, light intensity is controlled as a single Brightness
parameter and expressed as a percentage between 0% and 100%.

Brightness slider (Simple mode)

• In the Actual mode, brightness is controlled as individual Light (LED

intensity), Exposure, and Gain parameters.

Light Exposure, and Gain sliders (Actual mode)

Brightness parameters in the Actual mode have the following ranges:
Light: 0−100%
Exposure: 0−4 seconds
Gain: 1−8

Note: For best results, optimize the brightness parameters as follows:

• When searching for sample: Increase Gain for a brighter signal and decrease
Exposure for faster frame rate during navigation around the vessel.
• When capturing image: Decrease Gain to reduce background noise and
increase Exposure to regain signal intensity, as needed.
• For brighter signal: Increase Light intensity for brighter illumination. If
needed, follow by increasing Gain.
• For time lapse imaging: Increase Gain and Exposure, and decrease Light
intensity to reduce photobleaching and phototoxicity.
For example, for overnight time lapse experiments, capture one image every
30 minutes or less, limit the use of autofocus, and use a channel other than
DAPI for autofocus.

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 Focus sliders: Used for the manual adjustment of focus for the current channel.
The focus position is expressed in µm along the z-axis.

• The coarse focus slider bar represents the full focal range of the instrument
while the fine focus slider bar represents only a small fraction of the full
focal range.
• Coarse focus slider handle can be placed at any point within the focal
range of the instrument.
• Fine focus slider handle can be dragged as far as either end of the slider
bar, but it will snap back to the center position upon release.
• You can adjust the focus by moving the focus sliders in the desired
direction.
Alternatively, you can double-click on the handle of the focus slider to
activate the text box and enter the desired value for the focus position.

• If the instrument is in the Live mode, the live image changes in effective
real-time to reflect the changes made to the focus.
Record Video: Opens the Record Video controls, which allow
you to record capture a series of live images and compile them
into a video.
• Record Video controls are available only in the Live mode.

• Click Record to start recording live images.

Duration and frames recorded are displayed under the Record Video
controls.

• Click Pause to pause recording live images.

• Click Stop to finish recording.
• Click Save to open the Save Video window.
The Save Video window follows the standard Windows™ file navigation
system and allows you to save the captured video in the desired location.
You can choose to record your video in AVI or WMV formats.

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 Incubator: Opens Incubator tab, which allows you to control the
EVOS™ Onstage Incubator and monitor its status during your
experiments.

• Use Incubator: Enables the use of the EVOS™ Onstage Incubator.

• Temperature: Sets the incubator temperature (ambient to 40°C).
• CO2: Sets the CO2 level (0% to 20%).
• Oxygen: Sets the oxygen level (0% to 20%).
• Use humidity: Enables the use of humidified atmosphere in the incubation
chamber.
• Shutdown: Allows you select between manual shutdown or automatic
shutdown after a specified time period.
• Incubator status: Displays the current temperature, humidity, CO2, and
oxygen values against the target values.

Note: The EVOS™ Onstage Incubator (Cat. No. AMC1000) is an optional accessory
that enables the incubation of cells directly on the automatic X-Y stage, allowing
the capture of images from the same sample over long periods of time and
recording of time lapse movies.

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 Capture Z Stack: Opens Z-Stack Settings window, which
allows you to capture multiple images along the z-axis based
on your specifications.

• Light: Turns on the light source in the selected channel to illuminate the
sample as you locate the top and bottom boundaries of the z-stack using
the focus sliders. Click the button again turns off the light.
• Set Top Position: Sets the current focal plane reached using the focus
sliders as the top position of the z-stack.
• Set Default Focus: Specifies the new default focus position for the z-stack.
• Set Bottom Position: Sets the current focal plane reached using the focus
sliders as the bottom position of the z-stack.
• Step Size: Describes the z-distance in µm between the successive focal
planes that are captured for the z-stack. Available methods to specify the
step size are:
- Multiple of Depth of Field: Enter the step size as multiple of depth of
field.
- Step Size: Enter the step size in µm in the corresponding text box.
• Number of planes: Specifies the number of focal planes that are captured
to generate the z-stack. Each captured image along z-axis represents a
single “optical section” of the sample.

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 • Illustration: Graphically demonstrates the z-stack parameters. As you
enter the z-stack parameters, Illustration is automatically updated to
reflect the new settings.

- The Z-Stack Midpoint is represented by a blue line. Half of the images

captured for the z-stack are above this position and half are below it.
- The default focus position is represented by a white line.
- The orange rectangle represents the z-stack depth. It is the product of
step size and number of planes.
- The positions of the bottom and the top focus positions and the
position of the Z-Stack Focus are indicated in µm next to the orange
rectangle representing the z-stack depth.
• Projection Method: Determines the mathematical algorithm that will be
used to extract the most in-focus pixels from the images captured at
different focal planes to generate the z-stack projection.
- Maximum: For fluorescent images that are bright on dark background.
- Standard deviation (StDev): For unstained transmitted images where
the objects are identified by the contrast instead of the intensity.
- Average: For fluorescent or histochemically-stained transmitted
images when assessing concentration of the stain/marker.
• Capture Z Stack: Captures the z-stack images based on your specifications.
• Save: Saves the z-stack settings for future use in the current or another
selected channel.
Clicking Save closes the Z-Stack Settings window. To capture z-stack
images, click Capture Z-Stack to re-open the Z-Stack Settings window.
• Cancel: Closes the Z-Stack Settings window without capturing z-stack
images or saving z-stack settings.

Note: You can capture z-stack images in only one channel at a time; however,
you can save z-stack images of a field captured in different channels as a
single merged video.

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 Capture Channels: Captures a selected field in multiple
channels simultaneously.
Before capture, each channel to be acquired must be selected by clicking on the
small circular checkbox located on the upper left corner of the corresponding
light source button.
In the example below, DAPI, RFP, and transmitted light (brightfield) channels
have been selected for simultaneous capture.

Save: Opens the Save options window, which allows you to

select a save location and to set save options.

• Select fields to save: Allows you to select the captured fields you want to
save. You can choose from the following options:
- Currently selected field: Saves images only from the current field.
- All newly captured fields: Saves images that have been captured and
stored in the image cache, but not yet saved. This option is available
only if you have previously saved images from the same session.
- All captured fields: Saves images from all captured fields that are held
in the image cache. This is typically all of the images that you have
captured during an imaging session.
• Location: Describes the location of the current folder or file.

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 • File name prefix: Allows you assign a
prefix to your saved images.
The default prefix for images captured in the Capture tab is “image”.
• Browse: Opens the Select Folder window, which allows you
to assign a destination folder for your saved images.

• Select file types to save: Allows you to set I mage save options. You can
select one or more of the following options:
• Raw images: Saves images from
individual channels as 16-bit raw
images. This is the recommended
format for image analysis and
quantitation (see “Raw vs. Displayed
images”, page 125).
Available save options for raw images are:
- Single field, individual channels: Saves images captured in each
field and each channel individually.
- Z-stack planes, individual channels: Saves individual z-stack images
as well as the z-stack projection for each field and channel.
Available file formats for Raw images are:
- TIFF: No or low image compression with no loss in image quality.
- PNG: High image compression with no loss in image quality.
- C01: Thermo Fisher™ proprietary format. Only images captured by
monochrome cameras may be saved in this format.
- DIB: Color corrects across devices for true viewing. Only images
captured by monochrome cameras may be saved in this format.

Note: When viewing 16-bit images on most standard image viewers, they will
appear very dark or almost totally black, especially if a low exposure setting
was used. These images either need to be converted for display or opened in a
software application that allows viewing of 16-bit images such as the Thermo
Scientific™ Celleste™ Image Analysis Software (Cat. No. AMEP4816).

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 • Displayed images: Allows you to save
images in a format that can be viewed in
most image display applications. This is
the recommended format for “prettiest”
images (see “Raw vs. Displayed images”,
page 125).
Displayed images from individual
channels can be saved separately or as a
merged or tiled image, using either 24-bit
pseudocolor (8-bit per RGB channel) or
grayscale (16-bit).

Note: While pseudocolors help differentiate the channels used in multi-channel

overlays, grayscale images usually show more detail.

Note: 24-bit images (8-bit per RGB channel) are NOT recommended for image
analysis as not all channels will display in many image analysis applications.

Available save options for raw images are:

• Single field, individual channels: Saves images captured in each field and
each channel individually. You can select Grayscale (16-bit) or Pseudocolor
(24-bit RGB; 8-bit per RGB channel).
• Merged image: Combines the images captured in different channels into a
multicolor overlay and saves the merged image in 24-bit Pseudocolor (8-bit
per RGB channel). It is also referred to as a composite image.
• Tiled image: Merges the images captured in each channel and places them
very close together into a tiled format without applying an algorithm. The
resulting image is not seamless, but the process is quicker than stitching
and is often sufficient for most applications.
You can create tiled images with the following dimensions:
- Small (2000 × 2000 pixels)
- Medium (4000 × 4000 pixels)
- Large (10,000 × 10,000 pixels)
- Maximum (26,000 × 26,000 pixels)

Note: For more information about when to use tiled or stitched images, see
“Tiled, Stitched, and Merged images”, page 125.

Available file formats for raw images are:

• TIFF: No image compression with either no loss in image quality (16-bit
Raw) or reduced (8-bit) dynamic range (Microsoft-compatible TIFF).
• PNG: Medium image compression with no loss in image quality.
• JPEG: High image compression with little loss in image quality; good for
sharing.
• BMP: No image compression with no loss in image quality.

Note: If you plan to analyze your images, save them in 16-bit TIFF Raw format.
The 16-bit dynamic range is required for analysis and there is additional
information in the file headers, including OME data, that can be useful.

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 Automate tab
Automate tab Automate tab consists of a series of panels, which are organized by functionality
overview and contain the controls necessary to create and recall automated scan protocols.

Hardware: Allows you to configure hardware options (such as sample vessel,

objective, light source etc.) for the scan protocol (page 158).
Scan Area: Allows you to specify the scan areas and fields to capture for the
scan protocol (page 159).
AutoFocus and Z Stacks: Allows you to configure AutoFocus options and
Z-Stack settings for the scan protocol (page 172).
Time Lapse and Incubator: Allows you to specify time lapse options (duration,
capture frequency etc.) and incubator settings (temperature, oxygen etc.) for the
scan protocol (page 180).
Image Save Settings: Allows you to select a save location for captured images
and to set image save options (page 186).
Current Protocol: Displays the name of the currently selected scan protocol and
provides additional information (total number of images, estimated scan file
size, estimated temporary file size, and drive space available).
Save: Saves the automated scan protocol for future experiments.
Load: Opens the Load dialog, which allows you to recall a previously saved scan
protocol to run with new samples.
Run: Runs the automated scan protocol (newly created or recalled).

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 Hardware Hardware panel allows you to select and edit hardware options (such as sample
vessel, objective, light source etc.) and adjust settings for the current scan protocol.

Selected Vessel
Selected Camera
Select Objective
Select Channels
Phase Ring
Adjust Settings

Hardware controls Selected Vessel: Displays the selected vessel type. For more information, see
“Vessel” in “Capture tab controls”, page 140.
Selected Camera: Displays the selected camera. For more information, see
“Brightness and camera settings” in “Capture tab controls”, page 144.
Select Objective: Allows you select the objective for the scan protocol from the
currently installed objectives.
You can select only one objective at a time. In the example below, the 10X
objective is selected.

Select Channels: Allows you to select the channels you want to capture in the
scan protocol. The brightness value set in the Capture tab for each channel is
displayed below the corresponding checkbox.
You can select multiple channels by checking the corresponding channel boxes.
In the example below, the DAPI and GFP channels have been selected.

Phase Ring: Displays the phase option selected for the transmitted light
(brightfield) channel. For more information, see “Phase” in “Capture tab
controls”, page 144.
Adjust settings: Allows you to modify channel settings for the
scan protocol in the Capture tab (page 134).
In the capture tab, you can change Brightness and camera settings (page 144),
adjust Brightness parameters (page 149), and select a different Vessel
(page 140) and Objective (page 142).

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 Scan Area Scan Area panel allows you to define the areas and fields you want to scan in the
automated scan protocol and specify the order in which they are captured.

Area View (page 160) Vessel map (page 169)

Capture region (page 161) Area acquisition order (page 169)
Zoom slider (page 161) Field acquisition order (page 169)
Scan Locations (page 161) Stitching (page 170)
Scan Pattern (page 166) Toggle locations display (page 170)
Create Locations (page 167) Toggle field borders (page 170)
Edit scan area (page 168) Image display settings (page 171)

Note: If using a single culture dish, flask, or slide, the scan area corresponds to the
entire dish, flask, or slide. As such, the scan area displayed in Area View represents
the entire dish or flask selected.
In a multi-well plate, multi-chamber slide, or multiple dishes on multi-dish holders,
each scan area corresponds to a single well, chamber, or dish.
You can specify multiple areas to scan, but the Area View displays only the current
(i.e., active) scan area.

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 Scan Area controls Area View: Represents the target scan area from which specific fields are
selected for the scan protocol.
• In a single culture dish or flask, the scan area corresponds to the entire dish
or flask. As such, the scan area displayed in Area View represents the
entire dish or flask selected.
In the example below, the sample vessel is a single 60-mm culture plate,
and the Area View corresponds to the entire plate.

• In a multi-well plate or a multi-chamber slide, each scan area corresponds

to a single well or chamber. You can specify multiple areas to scan, but the
Area View displays only the current (i.e., active) scan area.
In the example below, the sample vessel is a 6-well plate. Although the first
three wells have been selected for capture, the Area View displays only the
active first well.

• When using the Scan Locations tool (page 161), the scan areas are selected
using the Assign scan area button (page 164).
When using the Scan Pattern tool (page 166), the scan areas are selected
from the Vessel map using the Edit scan area button (page 168).
• When multiple scan areas have been defined, you can specify the order in
which they are captured using the Area Acquisition Order (page 169).
• You can specify the order in which the selected fields are captured using
the Field Acquisition Order (page 169).

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 Capture region: Represent the regions to be captured in a scan protocol.
The regions to be captured consist of fields of view that are defined using the
Scan Locations or Scan Pattern tools. You can select multiple regions for
automated capture.
In the following example, the capture region was defined as 15 fields of view
(5 W × 3 H) using the Scan Pattern tool.

Zoom slider: Zooms in and out of the Viewing area.

Scan Locations: Opens the Locations tool, which allows you to

define the capture regions and assign scan areas for the scan
protocol.
• Locations are defined using the Locations tool (page 134).
• Locations that have already been defined with the Locations tool are listed
in the Available Locations table.
The Available Locations table also provides information about the name
and shape of each location, and the number of fields it contains.

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 • If there are no previously defined locations, the Available Locations table
displays the message “No locations are available for selection”.

• To create new locations for the scan protocol, click

Create Locations to open the Locations tool (page 134).
Using the Locations tool, define the locations as described (page 135), then
navigate back to the Automate tabScan Locations.
Newly created locations will be listed in the Available Locations table.
• To select a location listed in the Available Locations table, click the desired
location. The selected location will be highlighted in blue and the Viewing
area will display the selected location and shape (if the Toggle locations
display is on).

In the following example, the first location in the list (an ellipse) is selected.
The Viewing are displays the selected location and the field group that best
fits the shape of the location.

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 • To add a location to the scan protocol, click the desired location in the
Available Locations table to select, then click Add Selected.

• To add multiple locations to the scan protocol simultaneously, Shift-click

the desired locations in the Available Locations table to select them, then
click Add Selected.

• To add all of the locations listed in the Available Locations table, click
Add All.
• The locations added to the scan protocol are displayed in the Scan Locations
table and removed from the Available Locations table.
The Scan Locations table also provides information about the name and
shape of each location, and the number of fields it contains.
In the example below, two locations have been added to the Scan locations
list, and one location remains in the Available Locations table.

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 • To remove a location from the Scan Locations table, click the desired
location in the table to select, then click Remove Selected.

• To remove multiple locations from the Scan Locations table simultaneously,

Shift-click the desired locations in the table to select them, then click
Remove Selected.
• To remove all of the locations listed in the Scan Locations table, click
Remove All.
• The locations removed from the Scan Locations table appear in the
Available Locations table.
• To assign a scan area for the selected scan locations,
click Assign scan area to open the Vessel map, then
select the desired areas from the Vessel map to define the scan area
(page 165).
Depending on the vessel in use, scan areas can consist of wells in multi-well
plates, chambers in multi-chamber slide, or entire culture flasks or dishes.

• When assigning scan areas, each selected scan location (i.e., highlighted in
Scan Locations table) is assigned to each selected scan area.
If no location is selected in the Scan Locations table, all scan locations listed
in the table are assigned to each selected scan area.
If the Vessel map contains no selected scan areas, the scan protocol uses
the default scan area associated with each scan location.

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 • To define scan areas in the Vessel map:
- Click on a single area (well,
chamber, or dish) to select it.
Selected area will be
displayed in blue.

- Ctrl-click to select multiple

areas individually. Selected
areas will be displayed in
blue.

- Click-drag to select multiple

areas as a single block on the
Vessel map.

- Ctrl-click-drag to select
multiple blocks on the Vessel
map.

• To unselect scan areas:

- To unselect a single area, Ctrl-click the area you want to unselect.
- To unselect all areas, click on any region on the Vessel map that is not
selectable (i.e., not a well, chamber, or dish).
• Click Done to close the Vessel map and exit the area selection mode.

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 Scan Pattern: Opens the Scan Pattern tool, which allows you to
define the capture regions for the scan protocol as a pattern based on
defined parameters.
• You can define the scan pattern as a matrix (X fields wide × Y fields high)
or as a percentage of area covered by scan fields, counted either from the
center or from the edges.
In the following example, the capture region has been defined as a matrix
of 5 fields wide × 3 fields high.

• As you define the parameters, the Area View displays a preview of the
selected fields.

• To define the capture region as a matrix, select W × H fields of view, then

enter the desired dimensions into the corresponding text boxes. You can
only enter integers.
In this example, the capture region is
defined as a pattern of 5 fields wide
× 3 fields high.

166 EVOS™ M7000 Imaging System User Guide

 • To define the capture region as a percentage of area covered by scan fields,
select % of well area, enter the desired value (an integer between 1 and 100)
into the text box, then select From center or From edge.
In this example, the capture region is
defined as 50% of well area covered by
the scan fields, counted from the edge.

In this example, the capture region is

defined as 50% of well area, counted
from the center.

• When you are finished defining the parameters for the capture pattern,
click Create.
Create Locations: Open the Locations tool (page 134), which
allows you to create new locations for the scan protocol.
Using the Locations tool, define the locations as described (page 135), then
navigate back to the Automate tabScan Locations.
Newly created locations will be listed in the Available Locations table.

EVOS™ M7000 Imaging System User Guide 167

 Edit scan area: When using the Scan Pattern tool, it allows you to
specify the areas you want to capture by selecting the
corresponding areas on the Vessel map.
• Click Edit scan area to enable area selection on the Vessel map. Edit scan
area button changes to Done button and the Vessel map becomes active.

• To define scan areas in the Vessel map, follow the same procedure as
described for the Assign scan area (page 165).
• If you want to view a larger version of the Vessel map, click the
Zoom button. The larger map displays the same content as the
smaller vessel map and behaves the same way.

• When in the edit mode (i.e., Edit scan area button is clicked), clicking on
an area in the larger map selects the corresponding area in the smaller map
and marks it for the scan protocol.
• Clicking the zoom button again closes the zoom window.
• When finished defining the scan areas, click Done to exit the selection
mode.

168 EVOS™ M7000 Imaging System User Guide

 Vessel map: Represents the vessel container (vessel + vessel holder) in use.
In the Automate tab  Scan Area panel, the Vessel map is used to select
specific areas for the automated scan protocol.
• To select an area (well, chamber, or dish) for the scan protocol, click Edit
scan area, then click on the corresponding area on the Vessel map (page 168). A
blue fill indicates that the area has been selected for the scan protocol.
• The scan area shown in the Viewing area is indicated with a dark blue
circle on the Vessel map. Areas that are not active have white circles.
• In the example below, A1, A2, B1, and B2 wells have been selected for the
scan protocol. A1 is the active scan area (with dark blue flame), while the
inactive wells (A2, B1, B2) display white frames. The Area View shows the
active well A1.

Area Acquisition Order: Allows you

specify the order in which the selected
areas are captured in the scan protocol.
Available options are:
- Serpentine Horizontal
- Serpentine Vertical
- Random Selection

Field Acquisition Order: Allows you

specify the order in which the fields are
captured in each area selected for the scan
protocol. Available options are:
- Spiral Outward Counterclockwise
- Spiral inward Clockwise
- Serpentine Horizontal
- Serpentine Vertical
- Random Selection

EVOS™ M7000 Imaging System User Guide 169

 Stitching: Applies an overlap when assembling captured images
into a mosaic image of the scan area, which allows you to
visualize objects in the sample that cross fields.

Stitching off Stitching on

Toggle locations display: Switches the display of locations (defined with the
Location tool) in Area View on and off.

Field borders are on Field borders are off

Toggle field borders: Switches the borders around captured fields in Area
View on and off. This option is not available in Field View.

Field borders are on Field borders are off

• When field borders are turned on, all captured fields in Area View are
displayed with blue borders around them.
• Currently selected field, whether captured or not, is displayed with an
orange border, which cannot be hidden.

170 EVOS™ M7000 Imaging System User Guide

 Image display settings: Opens the Image display settings window,
which allows you to adjust image display parameters (brightness,
constrast, gamma) for the Viewing Area. Clicking the button again
closes the window.

Note: Adjustments made to Image display settings only affect how the image
is displayed in the Viewing Area; they do not change how the image is
captured.

• Visible Channels : Visible channels are the channels selected for

display in the Viewing area. They are selected using the Toggle channel
display checkbox located on the top left corner of the corresponding Light
source button (page 143).

Note: The controls for image display settings are contextual; only the controls
for the visible channels will be available. In the example above, only the
controls for the GFP, DAPI, and RFP channels are displayed.

• Brightness , Contrast , and Gamma parameters for each of the

selected channels are adjusted using the corresponding sliders.
• Reset button resets the Brightness, Contrast, and Gamma parameters to
their default values.
• Toggle image color display: Option to display images in pseudocolor
or in grayscale in the Viewing area. By default, color display is on.
When on, images are displayed in default pseudocolor corresponding to
the channel in which they were captured. You can change the default
pseudocolor in the Settings tab (page 192).
When off, images are displayed in grayscale.

Color display is on Color display is off

EVOS™ M7000 Imaging System User Guide 171

 AutoFocus and AutoFocus and Z Stacks panel allows you to configure the autofocus options and
Z Stacks determine the Z-Stack settings for the scan protocol.

Capture Z-Stacks
Use AutoFocus
Channel
Focus Position
Z-Offsets

Note: The options available on this panel are contextual; the controls that are
displayed depend on the selections made in this panel or elsewhere in the
Automate tab. Objects and controls that are not available as a function in a
particular context are not shown in the panel.

Note: Click on the View Help Content icon to open the Help window, which
provides EVOS™ M7000 Imaging System Help.

AutoFocus and Z Capture Z-Stacks: Allows you to capture a series of images at various focal
Stacks controls planes along the z-axis within the sample.
Checking this option reveals the controls for Z-Stack Settings (page 173).
Use AutoFocus: Allows you configure the autofocus method and set the
autofocus frequency to use during automated scan.
Checking this option reveals the controls for AutoFocus Settings (page 176).
Channel: Indicates the channels selected for capture in scan protocol, and
allows you set the default focus position.
Depending on the context, Channel displays channel selection buttons for the
Default Focus Position and the Z-Offsets.
Focus Position: Indicates the active z-stage position (i.e., the position of the
focal plane along the z-axis) for the selected channel (page 178).
Z-Offsets: Allows you to specify the focus position in each channel relative to
the focus position in other channels when the fluorescent markers in different
channels are in different focal planes (page 178).
This option is not available when the scan protocol is set to capture images in a
single channel.

172 EVOS™ M7000 Imaging System User Guide

 Z-Stack Settings Z-Stacking allows you to capture multiple images along the z-axis based on your
specifications during the automated scan protocol.
Images captured for the z-stack can be used to create a Z-Stack Projection. Z-Stack
Projection is a digital image processing technique that combines multiple images
taken at different focal planes to generate an image with a greater depth of field
than any of the individual source images.
The settings for the Z-Stack procedure are configured in the Z-Stack Settings
window in the Capture tab (Capture tab  Capture Z-Stack, page 152).

Capture Z-Stacks (page 174) Adjust Settings (page 174)

Focus Position (page 174) Illustration (page 175)
Z-Stack Summary (page 174)

Note: When the Capture Z-Stacks option is checked, the Z-Offsets option is not
available as one of the Focus Position controls and the Z-Offsets that have been
previously set are not preserved.

Note: Click on the View Help Content icon to open the Help window, which
provides EVOS™ M7000 Imaging System Help.

EVOS™ M7000 Imaging System User Guide 173

 Z-Stack Settings Capture Z-Stacks: Allows you to capture z-stack images
controls based on your specifications during your automated scan
protocol.
When the Capture Z-Stacks option is checked, the AutoFocus and Z Stacks
panel displays the Z-Stack Summary, the Adjust Settings button, and
Illustration.
Focus Position: Indicates the position of the current focal plane along the z-axis
for the selected channel, which is used as a reference point when defining the
different focal planes captured for the Z-Stacks.

Z-Stack Summary: Provides a summary of the Z-Stack Settings that were set up
in the Capture tab (Capture tab  Capture Z-Stack, page 152).
The summary includes information on depth of field, step size, number of
planes, total range of the z-stack, top and bottom z-positions, default focus
position of the selected channel, and the z-stack focus position.

Adjust Settings: Opens the Z-Stack Settings window in the

Capture tab, which allows you to modify the z-stack settings for
the scan protocol (see “Capture Z-Stack”, page 152).

174 EVOS™ M7000 Imaging System User Guide

 Illustration: Graphically demonstrates the Z-Stack Settings. Illustration is
available only if Capture Z-Stacks option is selected (page 172).

• The Z-Stack Midpoint is represented by a blue line. Half of the images

captured for the z-stack are above this position and half are below it.
• The default focus position is represented by a white line.
• The orange rectangle represents the z-stack depth. It is the product of step
size and number of planes.
• The positions of the bottom and the top focus positions and the position of
the Z-Stack Focus are indicated in µm next to the orange rectangle
representing the z-stack depth.

EVOS™ M7000 Imaging System User Guide 175

 AutoFocus Settings Autofocus performance depends on the z-information contained within a vessel
and vessel holder combination. When the system is properly calibrated for the
selected vessel, autofocus is able to use the dimensions used during the vessel
creation to find the optimal focal plane.
During automated scan, if the autofocus is unable to find the focal plane using a
small range, it extends the range within which it searches for focal plane.

Use AutoFocus (page 177) Start Focus Position (page 178)

Single Channel/All Channels (page 177) Z-Offsets (page 178)
Configure AutoFocus Method (page 177) AutoFocus Frequency (page 179)
Channel (page 178)

Note: Click on the View Help Content icon to open the Help window, which
provides EVOS™ M7000 Imaging System Help.

176 EVOS™ M7000 Imaging System User Guide

 AutoFocus Settings Use AutoFocus: Reveals the controls for setting autofocus
controls options for the scan protocol.
Single Channel/All Channels: Option to run the autofocus
in a single channel or in all channels selected for the scan.
• When Single Channel is selected, autofocus is run in a
single channel. This option preserves the Z-Offsets between the channels.
• By default, the last channel used for imaging or viewing a live image is
selected for single channel autofocus. You can change the autofocus
channel using the Channel radio buttons (page 178).
• When All Channels is selected, autofocus is run separately in all channels
that were selected in the Hardware panel (page 158). This option does not
preserve the Z-Offsets between channels.
Configure AutoFocus Method: Reveals the controls to configure the
autofocus method for each channel selected for the scan protocol.

Available options are:

• Fluorescence Optimized: The focal plane is derived from the highest ratio
between detailed, high-contrast objects against the background. This
option is recommended for fluorescence imaging.
• Transmitted Optimized: The optimal focal plane is derived through
statistics-based edge detection over 9 different regions to determine the
highest ratio of edge-to-background. This option is recommended for
transmitted-light imaging.
• Small Structure: This method computes the energy according to the size of
image features and can measure the presence/absence of small image
features. Choose this option when capturing samples that have many fine,
hair-like structures (e.g., filaments or structural stains).
• Large Structure: This method is statistics-based and looks for large
changes in image content as parts of the image go in and out of focus.
Choose this option when your sample contains large structures (e.g., whole
cell stains).
• Small Bright Objects: This method looks for changes in brightness at the
center of the cell and optimizes focus on cells that have a bright center with
a dark surround area. Choose this option to capture samples with localized
staining (e.g., nuclei).
After selecting the autofocus method, click Close to return to the AutoFocus
and Z Stacks panel.

Note: The effectiveness of the autofocus method depends on the plate type,
sample type, biomarker intensity, magnification, and channel. It may be
necessary to use different autofocus methods for different channels. This
ensures that the best autofocus method is used for the relevant biology.

EVOS™ M7000 Imaging System User Guide 177

 Channel: Allows you to select the channel in which to run the autofocus
during the automated scan when the Single Channel option is selected.
• Only the channels that have been selected for the automated scan protocol
in the Hardware panel will be available for selection.
• In the example below, DAPI channel has been selected for the autofocus
procedure during the scan protocol.

Start Focus Position: Indicates the currently active z-stage position (i.e.,
position of the current focal plane along the z-axis) for the selected channel.
• The Start Focus Position is used as starting point around which the
autofocus algorithm searches for optimal focus.
• You can only select a single channel for the Start Focus Position.
• When Z-Offsets option is checked, Start Focus Position is used as reference
point against which the focus position in other channels is set.
Z-Offsets: Allows you to specify the focus position in each channel relative to
the focus position in other channels. Setting the correct Z-Offsets is especially
important when the fluorescent markers in different channels are in different
focal planes.

• The Z-distance difference between channels is expressed as an offset from

the Start Focus Position in the reference channel.
• You can enter both positive and negative values in µm for the Z-Offsets.
In the example above, the reference start focus position is set to 384.44 µm
in the DAPI channel, while the Z-Offset for the GFP channel is 5.00 µm.
Therefore, the autofocus algorithm will use 389.44 µm as the default focus
position for the GFP channel.
• If the autofocus algorithm is configured to run on a single channel
(page 177), the current Z-distance differences between channels will be
preserved as if the Z-Offsets have been locked (page 146).
• If the autofocus algorithm is configured to run on all channels (page 177),
then the autofocus run in one channel will not affect the Z-distances of the
other channels as if the Z-Offsets have been unlocked (page 146).
• The Z-Offsets option is not available when the Capture Z-Stacks option
has been selected (page 173).

178 EVOS™ M7000 Imaging System User Guide

 Note: Consider a scenario where the initial Z-positions of the DAPI and GFP
channels are 50 µm and 80 µm, respectively. The autofocus is run from the
DAPI channel and finds the best focal plane at a Z-position of 60 µm.
If the autofocus is selected to run from a single channel, then the Z-Offsets will
be locked, and the GFP channel will be focused to a Z-position of 90 µm.
If the autofocus is run from all channels, then the Z-Offsets will not be locked,
and the autofocus algorithm will find the best focal plane for the GFP channel
independently of the DAPI channel.

AutoFocus Frequency: Allows you to specify which fields in a scan area are
used for autofocus during the automated scan protocol. Specifying fewer fields
per scan area for autofocus reduces the overall scan times.

Available options for AutoFocus Frequency are:

• First field only each area: Performs the autofocus operation only on the
first field of each scan area. No further autofocus is performed on
subsequent fields in the scan area. The focus position obtained on the first
field is used for the remaining fields in the scan area.
• First field only each location: Performs the autofocus operation only on
the first field of each location in every scan area. No further autofocus is
performed on subsequent fields..
• Every field: Performs the autofocus operation on every field of the scan
area.
• Every <X> fields: Allows you to specify the distance between fields in
each scan area before the autofocus is triggered.
The distance between fields is expressed as “unit fields” (equivalent to a
field of view as determined by the objective used); it is not determined by
the order in which the fields are collected.
For example, if you want to autofocus every 3 fields, autofocus will only
occur when the stage is three fields away from another autofocused field.
If the stage is less than three fields away, autofocus will not occur.

EVOS™ M7000 Imaging System User Guide 179

 Time Lapse and Time Lapse and Incubator panel gives you the option of running your automated
Incubator scan protocol over a time period, with or without the use of the EVOS™ Onstage
Incubator, where the selected fields are captured at given intervals based on your
specifications. The images captured in a time lapse scan protocol can then be
compiled into a video sequence.

Use Time Lapse (page 181) Image capture frequency (page 183)
Use Incubator (page 181) Incubator (page 184)
Run (page 182) Add run (page 184)
Duration (page 182) Autofocus Settings (page 185)
Delay Start (page 182)

Note: The options on this panel are contextual. Objects and controls that are not
available as a function in a specific context are not shown in the panel.

Note: Click on the View Help Content icon to open the Help window, which
provides EVOS™ M7000 Imaging System Help.

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 Time Lapse and Use Time Lapse: Allows you to create time lapse routines to
Incubator controls capture individual images at given intervals over a time
period based on your specifications.
• Checking the Use Time Lapse box enables all other controls in this panel,
allowing you to set the specific options for your time lapse routine (e.g.,
imaging frequency, use of the incubator etc.).

• A time lapse routine can be divided into individual “runs”, which allow
for different imaging frequency, duration, and incubator settings (see
“Run”, below).
• To add additional runs to your time lapse routine, click the Add run
button (page 184).
• The fields and locations to be captured during a time lapse experiment are
selected in the Scan Area panel (page 159).
• The autofocus options for the time lapse routine are set in the AutoFocus
and Z Stacks panel (page 172).
• The captured images are stored with time lapse information in their
headers and can be stitched together into a video.
Use Incubator: Enables the use the EVOS™ Onstage Incubator your time lapse
experiments. This option is available only when the EVOS™ Onstage Incubator
is connected to the EVOS™ M7000 Imaging System.
When checked, Use Incubator option reveals the controls for configuring the
incubator settings (see “Incubator”, page 184).

Note: The EVOS™ Onstage Incubator (Cat. No. AMC1000) is an optional

accessory that enables the incubation of cells on the automatic X-Y stage,
allowing the capture of images from the same sample over long periods of
time and the recording of time lapse movies.

EVOS™ M7000 Imaging System User Guide 181

 Run: Contains the controls for the corresponding run.

• The options available in this area are contextual and depend on the
selections made for Use Time Lapse and Use Incubator.
• Click the collapse/expand button to hide the controls for the run.
Click the button again to expand the controls.
• To add additional runs to your time lapse routine, click the Add run
button (page 184).
• If there is more than one run in the time lapse routine, each run has its own
set of controls that are similarly collapsible/expandable.
• Click the Delete Run button to delete a run and remove it from the
panel.
Duration: Allows you to specify the duration of the corresponding run in the
time lapse routine.

• Enter the desired values for the duration of the run into the corresponding
Hours, Minutes, and Seconds fields.

Delay Start: Allows you to postpone image capture for the corresponding run
of the time lapse routine and specify the duration of the delay.

• Enter the desired values for the delay into the corresponding Hours,
Minutes, and Seconds fields.
• When the Delay Start option is not selected, image capture starts
immediately after the time lapse experiment is initiated.
• By default the Delay Start option is deselected, with 0 hours entered for the
duration. After this feature has been used once, the most recent delay
period entered is used as the default value.

182 EVOS™ M7000 Imaging System User Guide

 Image capture frequency: Sets the frequency with which the image sets are
captured for the corresponding run in the time lapse routine.
The fields and scan areas are captured in the order specified in the Scan Area
panel (page 169).

Available options for image capture frequency are:

• Frequency: Determines the time period that must elapse before a new set
of images are captured. Note that the initial set of images is captured at
time point 0.
For example, if your run duration is 1 hour and you have entered
12 minutes for image capture frequency, the instrument captures one set of
images every 12 minutes starting at time point 0 for a total of 6 image sets
(60 minutes/12 minutes per image set plus the initial image set at time
point 0).
• Intervals: Determines the total number of time intervals between captured
image sets for a given run duration. Note that the initial set of images is
captured at time point 0.
For example, if your run duration is 1 hour and you have specified
5 Intervals, the instrument captures a total of 6 image sets 12 minutes apart
(60 minutes/5 intervals plus the initial image set at time point 0).
• As fast as possible: Captures a new set of images immediately after
completing the capture of the previous set without any delay between the
image sets.
Note that the speed with which the images are captured depends on your
specifications for the scan protocol such as the autofocus frequency and
exposure settings.
When this option is selected, the instrument captures all selected fields in
each scan area in the order specified, and then repeats the image capture
process without any delay, starting with the first field in the first area for
the duration of the run.

EVOS™ M7000 Imaging System User Guide 183

 Incubator: Determines the incubation parameters (i.e., culture conditions) and
the shutdown method for the EVOS™ Onstage Incubator.

• The incubation parameters are set separately for each run in the time lapse
routine.
• Temperature: Sets the incubator temperature (ambient to 40°C).
• CO2: Sets the CO2 level (0% to 20%).
• Oxygen: Sets the oxygen level (0% to 20%).
• Use humidity: Enables the use of humidified atmosphere in the incubation
chamber.
• Shutdown: Allows you to select the shutdown method for the EVOS™
Onstage Incubator at the end of your time lapse routine.
- Turn off manually: The incubator remains on until the Use Incubator
option is manually deselected.
- Turn off at the end of experiment: Heat, humidity, and the flow of gas
are automatically turned off at the end of the experiment.
- Turn off after: Enter the time period in hours and minutes that must
elapse before the heat, humidity, and the flow of gas are automatically
turned off.
Add run: Adds a new run to the time lapse routine.
• Clicking the Add run button adds a new set of run controls
to the panel, which are identical to the existing run controls.
This allows you set the parameters for each run (delay start, duration,
capture frequency, and incubator settings) independently for each run.
• The runs are performed in chronological order, beginning with Run 1 and
ending with Run N.
• You can remove a run from the time lapse routine by clicking the
corresponding Delete Run button .

184 EVOS™ M7000 Imaging System User Guide

 Autofocus Settings: Allows you to set the autofocus frequency and autofocus
fail options for the time lapse routine only.

• First time point only: Performs the autofocus procedure only once at the
first time point of the run before proceeding with image capture.
• Every time point: Performs the autofocus procedure at every time point of
the run, ensuring that the sample is in focus for each image set captured.
• Autofocus fail options: Selecting Yes causes the instrument to skip the
autofocus procedure at locations where no optimal focal plane was found
at subsequent time points.
Selecting No ensures that the autofocus is run at each selected autofocus
location every time regardless of previous autofocus failure.

Note: The Autofocus Settings options available in this panel apply only to the
time lapse routine. Autofocus locations and method are determined
separately in the AutoFocus and Z Stacks panel (page 176).

EVOS™ M7000 Imaging System User Guide 185

 Image Save Image Save Settings panel allows you to assign a destination directory for your
Settings saved images and to specify the file types to save.

Location (page 187) Raw images (page 188)

Browse (page 187) Displayed images (page 189)
File name prefix (page 187) More options (page 190)

Note: Click on the View Help Content icon to open the Help window, which
provides EVOS™ M7000 Imaging System Help.
Position your pointer over the Tooltip icon without clicking it for additional
information.

186 EVOS™ M7000 Imaging System User Guide

 Image Save Location: Describes the location of the current folder or file.
Settings controls

Browse: Opens the Select Folder window to assign a destination

directory for your saved images.
The Select Folder window follows the standard Windows file navigation
system.

• Selected folder: Displays the location of the current folder.

• New: Creates a new folder in the current location.
• Select: Selects the current folder as the destination directory for your saved
files. If the destination directory contains other files that could get
overwritten, a warning message is displayed in the Select Folder window.

Note: We recommend that you save your captured images to an external

hard drive.

File name prefix: Allows you to assign a

prefix to the automatically generated file
names when saving captured images.
The default prefix for images captured in an automated scan is “scan”.

Note: For more information on the file naming convention when saving, see
“File naming convention”, page 191.

EVOS™ M7000 Imaging System User Guide 187

 Raw images: Allows you to save images and image sets captured in individual
channels as 16-bit Raw images. This is the recommended format for
performing image analysis and quantitation (see “Raw vs. Displayed images”,
page 125).

Available save options for Raw images are:

• Single field, individual channels: Saves images captured in each field and
each channel individually.
• Z-planes, individual channels: Saves individual images captured along
the z-axis as well as the z-stack projection for each field and channel.
Available file formats for Raw images are:
• TIFF: No image compression with either no loss in image quality (16-bit
Raw) or reduced (8-bit) dynamic range (Microsoft-compatible TIFF).
• PNG: Medium image compression with no loss in image quality.
• C01: Thermo Fisher™ proprietary format. Only images captured by
monochrome cameras may be saved in this format.
• DIB: Color corrects across devices for true viewing. Only images captured
by monochrome cameras may be saved in this format.

Note: If you plan to analyze your images, save them in 16-bit TIFF Raw format.
The 16-bit dynamic range is required for analysis, and there is additional
information in the file headers, including OME data, that can be useful.

Note: When viewed on most standard image viewers, 16-bit images will appear
very dark or almost totally black, especially if a low exposure setting was used.
These images will either need to be converted for display or opened in a
software application that allows viewing of 16-bit images.

188 EVOS™ M7000 Imaging System User Guide

 Displayed images: Allows you to
save images in a format that can be
viewed in most image display
applications. This is the
recommended format for
“prettiest” images (see “Raw vs.
Displayed images”, page 125).
You can save displayed images in
24-bit pseudocolor (8-bit per
RGB channel) or 16-bit grayscale,
either individually from each
channel or as a merged image.

Note: While pseudocolors help differentiate the channels used in multi-channel

overlays, grayscale images usually show more detail.
24-bit images (8-bit per RGB channel) are NOT recommended for image
analysis as not all channels will display in many image analysis applications.

Available save options for displayed images are:

• Single field, individual channels: Saves images captured in each field and
each channel individually in Grayscale (16-bit) or Pseudocolor (24-bit RGB;
8-bit per RGB channel).
• Merged image: Combines images of a field captured in different channels
into a multicolor overlay and saves the image in 24-bit Pseudocolor (8-bit
per RGB channel).
• Tiled image, merged channels: Merges the images captured in each
channel and aligns them close together into a tiled format without
applying a stitching algorithm. The resulting tiled image is not seamless,
but this method is quicker than stitching and is often sufficient for most
applications. You can create tiled images with the following dimensions:
- Small (2000 × 2000 pixels)
- Medium (4000 × 4000 pixels)
- Large (10,000 × 10,000 pixels)
- Maximum (26,000 × 26,000 pixels).

Note: If you are analyzing images that cross fields and the images are slightly
misaligned, you might want to consider stitching the images (page 170).
Stitching applies an algorithm to apply an overlap, and then removes the seam
so you can visualize objects that cross fields. For more information about tiled
and stitched images, see “Tiled, Stitched, and Merged images”, page 125.

Available file formats for raw images are:

• TIFF: No image compression with either no loss in image quality (16-bit
Raw) or reduced (8-bit) dynamic range (Microsoft-compatible TIFF).
• PNG: Medium image compression with no loss in image quality.
• JPEG: High image compression with little loss in image quality; good for
sharing.
Include Grid option superimposes a grid
on the displayed images with the following
options for grid size (in pixels):
Auto, 10 × 10, 50 × 50, 100 × 100, 200 × 200, and 500 × 500.

EVOS™ M7000 Imaging System User Guide 189

 More options: Displays the full menu of save options in a
convenient table format, allowing you to save your images in
various formats simultaneously.

Note: We recommend saving captured images as both Raw and Displayed

images to preserve the option of using the Raw images in downstream image
analysis and quantitation, and the Displayed images for instances where
“prettier” images are required. For more information on Raw and Displayed
images, see “Raw vs. Displayed images”, page 125.

Note: For more information about when to use tiled or stitched images, see
“Tiled, Stitched, and Merged images”, page 125.

190 EVOS™ M7000 Imaging System User Guide

 File naming Files are saved using the following file naming convention:
convention <Prefix>_Image format_Time point_Z-plane_Grid_Row_Column_Field_Channel_
File type
where:
• <Prefix>: User defined prefix that is assigned to the automatically generated
default file name.
By default, the prefix is “image” for images captured manually in the Capture
tab, and “scan” for images captured automatically in the Automate tab.
• Image format: Refers to the format in which the images was saved (R = Raw,
D = Displayed, M = Merged).
• Time point: Describes the time point at which the image was captured and
has the format p0, p1, p2... etc.
For manually captured images, the time point is set to “p0”.
• Z-plane: Describes the plane in the z-axis where the image was captured and
has the format z0, z1, z2…etc.
• Grid: Refers to vessels containing multiple grid patterns on them, such as
slides with groupings of microarrays. It has the format 0, 1, 2… etc.
• Row: Refers to the row number in the selected culture vessel.
• Column: Refers to the column number in the selected culture vessel.
• Field: Refers to the field of view that was captured and has the form f01, f02,
f03… etc. Fields are numbered based on the order in which they were
captured.
• Channel: Refers to the channel in which the image was captured and has the
form d0, d1, d2… etc. A single-channel assay will be labeled with d0 in the file
name.
For example, the image with the following name was captured at time point 2
through channel 1 in a time lapse z-stack scan. It shows the field 1 in the z-plane 1
in grid 0, row B, and column 3.

EVOS™ M7000 Imaging System User Guide 191

 Review tab
Overview Review tab allows you to review saved images and associated metadata, and to
re-save or delete saved files.

Viewing area (page 192) Preview size (page 195)

Folders (page 194) Thumbnail view/List view (page 195)
Images/Metadata (page 194) Image preview/Image list (page 195)
View (page 195) Display options (page 195)

Viewing area: Displays the selected image.

• To display an image in the Viewing area, select the image in the Image
preview/Image list area, then click Load Scan.
• Alternatively, double-click the image you have selected in the Image
preview/Image list area to display it in the Viewing area.

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 • When you open a folder containing images, the Scan Metadata panel
shows metadata about the scan and the Field Metadata panel shows the
metadata concerning the imaged field displayed.

• The Vessel map shows the location of the Field that is displayed in the
Viewing area.

Note: You can also position your pointer over a scan or plate in the Browse panel or
over an image in the Image preview/Image list panel without clicking to display
the metadata associated with that scan, plate, or image.

EVOS™ M7000 Imaging System User Guide 193

 Folders: Allows you to navigate to the folder
containing your saved images, and to select
saved images display in the Image preview
area.
The folder containing saved images can be in
an external storage device (USB flash drive),
on the computer workstation running the
EVOS™ M7000 Imaging System, or on the local
network (if connected).

Images/Metadata: Lists the images or scans saved within the selected folder (if an
image folder is selected in the Folders panel) or the scan metadata for the selected
scan (if a scan is selected in the Folders panel).

Image/Scans list (Recent Scans folder is selected in Folders panel)

Scan metadata (A scans file is selected in Folders panel)

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 View: Allows you to filter the image files displayed
in the preview area.
Available options are All, Raw,Displayed, Tiled, Merged, and Other.
Preview size buttons: Allow you to increase or decrease the display
size of the image thumbnails in the Preview area.
Thumbnail view/List view toggle: Allows you to display the saved
images in the Preview area as thumbnails or as a list.
Image preview/Image list: Allows you to select the image to display in the
Viewing area. The selected image is indicated with a blue box around it.
Double-click the selected image to display it in the Viewing area.

Thumbnail view List view

Display options: Allows you to set image display options in the
Viewing area.
The following display options are available in the Review tab:
1. Toggle field borders (page 129)
2. Toggle scale bar (page 132)
3. Toggle sample grid (page 132)
4. Image display settings (page 132)
5. Center on selected field (page 130)

Note: Display options in the Review tab are visible only when an image is
displayed in the Viewing area.

EVOS™ M7000 Imaging System User Guide 195

 Settings
Settings tab Settings tab is used for selecting basic and advanced system options and for
overview performing calibration and maintenance procedures.
To access the Settings tab, click the Settings tab anytime from the Capture,
Automate, and Review tabs.
To close the Settings tab and return to the previous tab, click Done.

General settings General: Allows you to set the Saturated Pixel, Jog Control, Scale Bar, Grid
controls Settings, and AutoFocus options (page 197).
Cameras: Allows you to perform Hot Pixel Correction for the monochrome
camera and to adjust White Balance for the color camera (page 199)
Objectives: Allows you to set up and calibrate objectives and assign objective
profiles (page 201).
Stage Calibration: Opens the Stage Calibration tool, which allows you to
calibrate the automatic X-Y axis stage for accurate instrument function
(page 204).
Vessels: Allows you to select sample vessels, calibrate the instrument for the
selected vessel, to import and export vessel information, and to import stage
insert information (page 205)
Incubator: Allows you to configure the gas inputs, calibrate the oxygen sensor,
and set the temperature offsets for the EVOS™ Onstage Incubator (page 208).
The Incubator panel is only visible when an EVOS™ Onstage Incubator is
connected to your EVOS™ M7000 Imaging System.
Service: Provides version information about instrument software and firmware
(page 210).
Camera Rotation: Allows you to calibrate the camera rotation for accurate
instrument function (page 212).
Filter Cubes: Allows you to add or remove EVOS™ LED light cubes and to
assign pseudocolors for installed light cubes (page 213).

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 General General panel in the Settings tab allows you to set the Saturated Pixel, Jog Control,
Scale Bar, and Grid Settings options. To expand or collapse the controls for a setting,
click the corresponding arrow.

Saturated Pixels (page 197) Grid Settings (page 198)

Jog Control (page 197) AutoFocus (page 198)
Scale Bar (page 197)

General settings Saturated Pixels: Displays saturated pixels on an

controls image with the user-defined color, which provides a
visual aid for optimal illumination when adjusting
the brightness settings in Live mode.
Available colors for saturated pixels are
Blue, Red, and Green.
Jog Control: Allows you to invert the direction of the
stage movement as input by the Jog Control button
(page 143).
Available options are Invert Horizontally and Invert
Vertically.
Scale Bar: Allows you to set the display options for
the scale bar.
• Select Move Freely to be able to drag the scale bar
anywhere in the Viewing area or select Dock
Location to dock it in the bottom or top left or
right corners of the Viewing area.
When set to move freely, click Reset to Default to
place the scale bar in the bottom left corner.
• Other scale bar display options include:
- Display End Bars: Adds end bars to the scale
bar.
- Display Text: Displays the size of the scale
bar. You can select to display the scale
information using small, medium, or large
sized fonts.
- Select Scale Bar Width: Allows you select the
width of the scale bar (narrow, medium,
wide).
- Select Color: Allows you to select the color of
the scale bar (black or white).

EVOS™ M7000 Imaging System User Guide 197

 Grid Settings: Allows you to set the display
options for the sample grid that is displayed
in the Viewing area.
• Select Color: Sets the color of the grid.
Available options are white, light grey,
dark grey, and black.
• Grid Alignment: Determines how the
grid is aligned to the Viewing area.
Available options are:
- Cross-hair at center
- Grid Square at the center
• Grid Size: Allow you select the grid size (i.e., the unit size of the squares
that form the grid), which is expressed in µm.
You have the following options for the grid size (in pixels):
Auto, 10 × 10, 50 × 50, 100 × 100, 200 × 200, and 500 × 500.
When the Auto Size option is selected, the software automatically adjusts
the grid spacing if the chosen value results in more than 50 grid lines per
image.
• Show grid size label: Displays the unit size of the squares that form the
grid.

AutoFocus: Allows you set AutoFocus

options.
• Quick AutoFocus: If checked, the
software runs coarse focus until it
identifies the first high-contrast object
that can be found, then runs fine autofocus.
If unchecked, the software runs through the entire autofocus range, then
runs fine autofocus around the highest-contrast object that can be detected.

Note: For samples that contain air bubbles or debris, we recommend that the
Quick AutoFocus option is disabled.

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 Cameras Cameras panel in the Settings tab allows you to perform Hot Pixel Correction for
the monochrome camera and to adjust White Balance for the color camera.

Hot Pixel Correction (page 199) White Balance (page 200)

Cameras controls Hot Pixel Correction: Opens the Hot Pixel Correction
dialog, which allows you to search for hot pixels in the
monochrome camera and reset the brightness value of
all hot pixels to a base pixel intensity level.

Hot pixels are caused by electrical charges that leak into the sensor wells and
they appear brighter than the other pixels in the camera. Hot Pixel Correction
algorithm of the EVOS™ M7000 software stores the location and value of the
hot pixels in its memory for subsequent automatic hot pixel correction.
• Search for Hot Pixels: Prompts the software to search for hot pixels in the
image.
Click OK to save the location of hot pixels for subsequent correction.
• Reset Hot Pixels: Resets the hot pixel locations to the factory default.

EVOS™ M7000 Imaging System User Guide 199

 White Balance: Opens the White Balance dialog, which
allows you to adjust the white balance on the color
camera manually or automatically.

• To automatically adjust the white balance, find a white area on the image,
click and drag to select that area on the field of view, then click Auto
Correct.
• To manually adjust the white balance, find a white area on the image, then
use the Red and Blue sliders.

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 Objectives Objectives panel in the Settings tab allows you to assign and unassign objectives on
the objective turret, and to calibrate objective magnification.

Profiles (page 202) Display name (page 203)

Active (page 202) Details (page 203)
Calibrate (page 203)

Note: For specific instructions on how to change and calibrate objectives, see
“Change the objectives” (page 112) and “Calibrate the objectives” (page 114).

EVOS™ M7000 Imaging System User Guide 201

 Objectives controls Profiles: Displays the objective profiles that are available to be assigned to the
active objectives in the turret.
The profiles are grouped by magnification and include manufacturer, part
numbers, and working distance information.

• To add a new profile, click Add.

• To copy a profile, click Copy.
• To edit a profile, click Edit.
Active: Lists the objectives installed in the objective turret and allows you to
assign and unassign objectives from the Profiles list.

• The active objective list provides information about magnification,

manufacturer, working distance, and part number for the objectives
assigned to each turret position.
• If there is no objective assigned to a turret position, he list displays
“empty”.
• Currently selected (active) objective in the turret is highlighted in blue. To
select another objective, click the desired objective position.
• To assign a profile to a newly installed objective, click and drag the profile
from the Profiles list to the appropriate Turret position in the Active list
(see “Change the objectives”, page 112).

202 EVOS™ M7000 Imaging System User Guide

 Calibrate: Opens the Objective Calibration tool, which allows
you to calibrate the field of view, parfocality, and parcentration
parameters of the selected objective (see “Calibrate the objectives”, page 114).

Display name: Allows you assign a label for the

selected objective, which is displayed on the
Objective button in the Capture tab.
In the following example, “NoPH” is entered as the Display Name. The
Objective button in the Capture tab displays the label “NoPH” below the
objective magnification.

Details: Displays the detailed information about the active objective in the
turret.

EVOS™ M7000 Imaging System User Guide 203

 Stage calibration Stage Calibration panel allows you to calibrate the position of the automatic
X-Y stage for accurate instrument function.
Note that the stage calibration procedure requires the EVOS™ Calibration Slide
supplied with the instrument (also available separately; Cat. No. AMEP4720).

Note: For specific instructions on how to calibrate the stage, see “Calibrate the
stage” (page 105).

204 EVOS™ M7000 Imaging System User Guide

 Vessels Vessels panel allows you to select sample vessels, calibrate the instrument for the
selected vessel, to import and export vessel information, and to import stage insert
information.

Vessel (page 206) Export Vessel (page 207)

Vessel map (page 206) Import Vessel (page 207)
Calibrate Vessel (page 207) Import Stage Insert (page 207)

Note: For specific instructions on how to calibrate the instrument for the selected
vessel, see “Calibrate vessel” (page 107).

EVOS™ M7000 Imaging System User Guide 205

 Vessels controls Vessel: Opens the Vessel Selection dialog, which allows you to
select the vessel you want to calibrate or export.

• Select the Vessel category that corresponds to your sample vessel to

display the holder and vessel type selections available for that category.
Available vessel categories are Well Plates, Flasks, Dishes, and Slides.

• Select the appropriate Holder and Vessel type from the dropdown menus
available for your vessel category.

• Click Export to open the Save As dialog, which allows you to save the
vessel information as a *.vessel file at the desired destination directory.
Vessel map: Represents the vessel container (vessel + vessel holder) in use, as
determined by the selection made in the Vessel selection dropdown.

206 EVOS™ M7000 Imaging System User Guide

 Calibrate Vessel: Opens the Vessel Calibration dialog, which allows you to
update the selected vessel with new calibration values or to save them as a new
vessel. The calibration values are vessel-specific coordinates that allow the
EVOS™ M7000 Imaging System to correctly scan the sample vessel.

Note: For specific instructions on how to calibrate the instrument for the selected
vessel, see “Calibrate vessel” (page 107).

Export Vessel: Allows you to export the

vessel information and associated calibration
values as a *.vessel file in the desired
destination directory.
Import Vessel: Allows you to import *.vessel
files that contain vessel information and
associated calibration values.
Import Stage Insert: Allows you to import
*.stageinsert files that contain stage insert
information and associated calibration values.

EVOS™ M7000 Imaging System User Guide 207

 Incubator Incubator panel allows you to configure the gas inputs, calibrate the oxygen sensor,
and set the temperature offsets for the EVOS™ Onstage Incubator (page 208).

Note: The Incubator panel is only visible when an EVOS™ Onstage Incubator is
connected to your EVOS™ M7000 Imaging System.

Reset Safety Shutoff (page 209) Gas Inputs (page 209)

Temperature (page 209) Oxygen Sensor (page 209)

Note: For specific instructions on how to set up the EVOS™ Onstage Incubator and
use it with the EVOS™ M7000 Imaging System, see “Appendix D: EVOS™ Onstage
Incubator” (page 224).

208 EVOS™ M7000 Imaging System User Guide

 Incubator controls Reset Safety Shutoff: Resets the EVOS™ Onstage
Incubator after an automatic safety shutoff event.
Temperature: Used to set the Static Offset, which
allows you to adjust temperature readings based on
the ambient room temperature. This should remain at
0 by default.
Gas Inputs: Allows you to configure Gas Inputs that reflect your set-up for the
EVOS™ Onstage Incubator.

• For Port 1, select Air or Premix.

If you select Premix, manually enter the percentage of the CO2 and O2 to
reflect the specifics of your set-up.
• Port 2 is reserved for Nitrogen only.
• Port 3 is reserved for CO2 only.
For instructions on how to configure the gas inputs to the EVOS™ Onstage
Incubator, see page 234.
Oxygen Sensor: Allows you to calibrate the oxygen sensor when using separate
tanks for gases.

For instructions on how to calibrate the EVOS™ Onstage Incubator oxygen

sensor, see page 235.

EVOS™ M7000 Imaging System User Guide 209

 Service Service panel display the EVOS™ M7000 software and firmware versions, and
allows you to calibrate the objective turret, copy log files to a USB drive, move the
X-Y stage to the shipping position, and change the cache location.

Software version (page 210) Copy Log Files to USB (page 211)
Firmware version (page 210) Move to Shipping Position (page 211)
Objective Turret (page 211) Cache (page 211)

Service controls Software version: Displays the installed EVOS™ M7000 software information.

Firmware version: Displays the installed EVOS™ M7000 firmware information.

210 EVOS™ M7000 Imaging System User Guide

 Objective Turret: Allows you to calibrate the objective turret.

• Begin Turret Calibration: Initiates the turret calibration process.

The progress of the calibration is updated on the calibration progress bar.
• Reset to Factory Default: Resets the objective calibration values to factory
default values.
Copy Log Files to USB Drive: Saves the EVOS™
M7000 log files from the instrument cache to an
external USB drive.
Move to Shipping Position: Moves the X-Y stage
to the shipping position and provides instructions
for the installation of shipping restraints.

For instructions on how to install the shipping restraints to prevent damage to

the EVOS™ M7000 Imaging System, see “Install shipping restraints”, page 118.
Cache: Displays the location and size of the instrument memory cache and
allows you to change the location where the cache is stored.

EVOS™ M7000 Imaging System User Guide 211

 Camera rotation Camera Rotation panel allows you to calibrate the camera rotation to maintain high
image tiling quality.
Note that the camera rotation calibration requires the EVOS™ Calibration Slide
supplied with the instrument (also available separately; Cat. No. AMEP4720).

• To calibrate the camera rotation, insert the EVOS™ Calibration Slide into the
instrument, adjust the Brightness, then Focus on the crosshairs of the
calibration slide.
When you have focused on the crosshairs, click Next to begin the calibration of
camera rotation.

212 EVOS™ M7000 Imaging System User Guide

 Filter Cubes Filter Cubes panel allows you to add or remove EVOS™ LED light cubes from the
instrument and to assign pseudocolors to installed light cubes.

Move to filter cube change Color spectrum (page 214)

position (page 214) Color slider (page 214)
Position (page 214) Wavelength (page 214)
Edit Pseudocolors (page 214) Apply changes (page 214)
Filter cube tabs (page 214) Restore Default (page 214)
Current pseudocolor (page 214)

EVOS™ M7000 Imaging System User Guide 213

 Filter Cubes Move to filter cube change position: Moves the cube carriage into position so
controls that the light cube at the selected position can be changed.

Position: Selects the position of the light cube to be changed.

Note: To insert or remove a light cube, first select the Position of the light cube you
want to change, then click Move to filter cube change position. For detailed
instructions, see “Change EVOS™ light cubes” (page 107).

Edit Pseudocolors: Contains the controls to edit the pseudocolor for installed
light cubes.
To select a new pseudocolor, move the Color slider to the desired position on
the spectrum or enter the corresponding wavelength into the Pseudocolor
wavelength text box.

Note: You can assign pseudocolors only to non-transmitted light (fluorescence)

light cubes. The assigned pseudocolor is only relevant for the monochrome camera.

Filter cube tabs: Each tab represents an installed light cube and contains the
controls to change the psudocolor assigned to that cube.
Current pseudocolor: Displays the pseudocolor assigned to the light cube.
Color spectrum: Represents the color spectrum available to choose as a
pseudocolor for the light cube.
Color slider: Allows you to select the desired pseudocolor.
Wavelength: Allows you to enter the wavelength that corresponds to the
pseudocolor you want to assign to the light cube.
Apply changes: Applies the selected pseudocolor to the light cube. To save the
changes, click Done after applying the changes.
Restore Default: Restores the factory default pseudocolor to the light cube.

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 EVOS™ Analysis Application
Overview The EVOS™ Analysis application is an image analysis and annotation tool that
allows you to analyze and annotate saved images, perform auto or manual cell
count, determine the confluence of the cell culture, and calculate the transfection
efficiency.
When the EVOS™ Analysis application is launched (page 75), it opens to the
Review screen, but does not display an image. To view an image, you must first
select it from an image folder using the Review tab controls (page 216).

For detailed descriptions of specific EVOS™ Analysis application screens, refer to

the following:
• Review tab (page 216)
• Display settings and analysis tools (page 217)
• Auto Count controls (page 219)
• Manual Count controls (page 220)
• Cell Culture tools – Confluence (page 221)
• Cell Culture tools – Transfection Efficiency (page 222)
• Settings tab (page 223)

EVOS™ M7000 Imaging System User Guide 215

 Review tab

Review tab controls Viewing area: Displays the image selected from the Image preview/Image list.
Image file name: File name of the image displayed in the Viewing area.
Zoom slider: Zooms in and out of the image. The zoom range is 100% to 1000%.
Folder: Displays the location of the current folder or image.
Search: Allows you to search by file name in the selected folder.
Layout: Allows you to toggle between grid or list view, increase or decrease the
display size of the folders or image files, and sort by name, file type, or date
created in ascending or descending order.
Image preview/Image list: Displays the preview or list of the files and
subfolders in the current folder.
Image properties: Displays the metadata for the selected image file.
Display and analysis tools: Allow you to change image display settings in the
Viewing area, annotate and analyze the captured images, and perform cell
count and cell culture analysis (confluence and transfection efficiency). See
page 217 for more information.
Settings: Opens the Settings tab, which allows you to select image format
options for saving TIFF files, view EVOS™ Analysis software version, and to
copy error logs to a storage device.
Refresh: Refreshes the list or grid of images in the current folder.
Display settings: Displays or hides the Layout controls.
Export: Allows you to export the currently selected folder or image to a storage
device.
Save: Saves the currently opened image.

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 Display settings
and analysis tools

Image Display Settings: Opens the Image display settings tool, which allows
you to adjust image display parameters (Brightness , Contrast , Gamma
Correction ) for the selected channels.

Display Grid/Grid Settings: Display Grid

button toggles the grid display in the
Viewing area on and off. Grid Settings
allows you to set the grid size.
Display Scale Bar/Scale Bar Settings:
Display Scale Bar button toggles the display
of the scale bar in the Viewing area on and
off. Scale Bar Settings allows you to select
scale bar color and to display or hide end
bars.

Pixel Intensity: Opens the

Pixel Intensity window,
which displays the Pixel
count vs. Intensity
histogram, where Intensity
is a value based on the
number of photons detected
by the camera sensor.

Measurements and
Annotations: Allows you to
draw regions of interest
(rectangle, ellipse, polygon,
line, or free-form) on the
captured image and
measure dimensions, area,
or perimeter of the drawn region.

EVOS™ M7000 Imaging System User Guide 217

 Show Cell Count: Allows you to perform cell counts and cell culture analysis.
• Auto Count: Allows you define auto count parameters by selecting
representative target objects and background areas, then count the objects
by intensity, area, and circularity (page 219). Auto Count is not supported
for the transmitted light channel; it requires the image to be collected in a
fluorescence nuclear stain channel.
• Manual Count: Allows you to manually mark items onscreen using up to
six separate labels and keep a running tally of the counts with percentages
for each label (page 220).
• Cell Culture: Allows you to select up to 5 reference objects each for target
(i.e., cells) and background in your image to automatically measure the
confluence of your culture, then calculate the transfection efficiency
(fluorescence area divided by the entire cell area in the image) (page 221).

Auto Count Manual Count Cell Culture

Toggle pseudocolor: Allows you to display images in pseudocolor or in

grayscale in the Viewing area. By default, color display is on.

218 EVOS™ M7000 Imaging System User Guide

 Auto Count controls

Analysis tools: Allows you to toggle between Auto Count, Manual Count, or Cell
Culture (Confluence and Transfection Efficiency) tools for image analysis.
Channel: Selects the nuclear stain channel for Auto Count. You can select only a
single channel.
Select Target and Background: Allows you to select representative target objects
and background areas for Auto Count.
Split Cells: Allows you to split multiple objects that have been counted as one into
individual objects based on shape or pixel intensity to increase the count accuracy.
Refine: Selects intensity, area, or circularity by which to refine Auto Count results
using the Count Histogram.
Count Histogram: Allows you to set pixel intensity, area, or circularity thresholds to
refine the Auto Count results.
Object Count: Displays the object count based on the Auto Count parameters.
Object color: Selects the color by which the counted objects are identified.
Reset: Resets the count to 0 and clears the selected targets and background areas.
Batch Analysis: Allows you to save and apply the analysis parameters to other
images that you have collected and saved in an image folder (see page 100 for more
information).
Exit: Exists the Auto Count tool and displays the Review tab.
Save: Saves the analysis results as an image in the selected file format (see page 98
for more information).

EVOS™ M7000 Imaging System User Guide 219

 Manual Count
controls

Analysis tools: Allows you to toggle between Auto Count, Manual Count, or Cell
Culture (Confluence and Transfection Efficiency) tools for image analysis.
Channel: Selects the channels for Manual Count. You can select multiple channels.
Object #: Allows you to select the label (Object #) with which to tag objects in the
Viewing area. Left-click on the objects in the Viewing area to tag them with the
selected label; right-click to delete a tag. In this example, Object #2 is selected. You
can switch labels as desired. See page 90 for more information.
Object Name: Allows you assign a name for the Object #.
% and Count: Displays the label count and its percentage of the total object count
(total count of all labels).
Delete: Selects a label for deletion.
Total Count: Displays the total count of objects tagged with all labels.
Trash: Deletes the tags for the label and resets the label count to 0.
Reset: Resets all label counts and the total count to 0 and clears the Viewing area of
all tags.
Batch Analysis: Allows you to save and apply the analysis parameters to other
images that you have collected and saved in an image folder (see page 100 for more
information).
Exit: Exists the Manual Count tool and displays the Review tab.
Save: Saves the analysis results as an image in the selected file format (see page 98
for more information).

220 EVOS™ M7000 Imaging System User Guide

 Cell Culture –
Confluence controls

Analysis tools: Allows you to toggle between Auto Count, Manual Count, or Cell
Culture (Confluence and Transfection Efficiency) tools for image analysis.
Confluence: Expands or hides the controls for the Confluence tool.
Select Target and Background: Allows you to select representative cell and
background areas for the confluence measurement.
Sensitivity: Adjusts the algorithm sensitivity to pixel intensity (higher intensity =
more pixels included). Decreasing the sensitivity reduces the confluence value.
Show Mask: Indicates the areas included in the confluence measurement.
Mask Color: Selects the mask color.
Transfection Efficiency: Expands or hides the controls for the Transfection
Efficiency tool. The Transfection Efficiency tool is inactive until the confluence
measurement is completed.
% Confluence: Displays the percentage of the area covered by cells in the image,
based on the selected target and background areas and sensitivity.
Reset: Resets the Confluence and Transfection Efficiency measurements to 0 and
clears the selected targets and background areas.
Batch Analysis: Allows you to save and apply the cell culture analysis parameters to
other images that you have collected and saved in an image folder (see page 100).
Exit: Exists the Cell Culture tool and displays the Review tab.
Save: Saves the analysis results as an image in the selected file format (see page 98).

EVOS™ M7000 Imaging System User Guide 221

 Cell Culture –
Transfection
Efficiency controls

Confluence: Expands or hides the controls for the Confluence tool.

Transfection Efficiency: Transfection Efficiency: Expands or hides the controls for
the Transfection Efficiency tool.
Fluorescence Channel selection: Selects the fluorescence channel for transfection
efficiency calculation.
Fluorescence Channel: Toggles the display of the fluorescence channel.
Transmitted Light Channel: Toggles the display of the transmitted light.
Threshold: Adjusts the fluorescence threshold value. Only the cells that express
above the set threshold are used in the tranfection efficiency calculation.
Threshold Mask: Indicates the areas above the set threshold value and are included
in the transfection efficiency calculation.
Mask Color: Selects the threshold mask color.
% Confluence and %Transfection Efficiency: Displays the calculated confluence
and transfection efficiency values.
Reset: Resets the Confluence and Transfection Efficiency measurements to 0 and
clears the selected targets and background areas.
Batch Analysis: Allows you to save and apply the cell culture analysis parameters to
other images that you have collected and saved in an image folder (see page 100).
Exit: Exists the Cell Culture tool and displays the Review tab.
Save: Saves the analysis results as an image in the selected file format (see page 98).

222 EVOS™ M7000 Imaging System User Guide

 Settings tab

General: Allows you to configure TIFF file saving options. You can select to
save TIFF files in a Microsoft-compatible format (8-bit with reduced dynamic
range), uncompressed (16-bit Raw format with no loss in image quality), or
both.
Service: Display the EVOS™ Analysis Software version.
Copy Error Logs: Allows you to copy error logs to an external storage device
(USB drive).
Done: Saves your changes and returns to the Review tab.

EVOS™ M7000 Imaging System User Guide 223

 Appendix D: EVOS™ Onstage Incubator

EVOS™ Onstage The EVOS™ Onstage Incubator (Cat. No. AMC1000) is an optional accessory for the
Incubator EVOS™ FL Auto Imaging System that enables the incubation of cells on the
automatic X-Y stage, allowing the capture of images from the same sample over
long periods of time and recording of time lapse movies.
The EVOS™ Onstage Incubator consists of a Stagetop Environmental Chamber that
is placed on the automatic X-Y stage of the imaging system and a separate Control
Unit that supplies the power and gas (air or air-CO2 premix, CO2-only, and
nitrogen-only). The onstage incubator is controlled by the same software and user
interface that controls the EVOS™ M7000 Imaging System.

Standard items • Stagetop Environmental Chamber

included • Control unit
• Master Stage Plate (also available separately as EVOS™ Onstage Master Plate,
Cat. No. AMEPVH035)
• Vessel holder for multi-well plates (also available separately as EVOS™
Onstage Vessel Holder, Multiwell Plates, Cat. No. AMEPVH028)
• Cable with 6-pin connector
• Cable, USB A-to-B, 180 cm/6 ft
• Heated hose with temperature control, 180 cm/6 ft (also available separately as
EVOS™ Onstage Incubator Hose, Cat. No. AMEP4728)
• Gas line, 1/8 in ID, 1/4 in OD (also available separately as EVOS™ Onstage
Incubator Gas Line, Cat. No. AMEP4732)
• Push-to-connect gas line adaptor (3 each)
• Standard-head open-end wrench
• Hex screw driver
• Power Cord, Type A (North America)

Note: A country-specific power cord must be ordered separately in regions not

using the Type A power plug.

224 EVOS™ M7000 Imaging System User Guide

 Technical specifications
Note: Specifications of the Onstage Incubator are subject to change without notice.
Refer to the EVOS™ product page at www.thermofisher.com/evos for the latest
product information.

Physical Stagetop
characteristics Environmental Chamber Control Unit
Height: 25 cm (9.7 in) 37 cm (15 in)
Depth: 19 cm (7.6 in) 16 cm (6.3 in)
Width: 3.7 cm (1.5 in) 20 cm (7.9 in)
Weight: 1.5 kg (3.3 lb) 10 kg (22 lb)
Temperature range: Ambient to 40°C (± 0.1°C)
Humidity: >80% relative humidity (RH) at 37–40°C
CO2 range: 0% to 20%
O2 range: 0% to ambient
Operating power: 100–240 VAC, 1.8 A
Frequency: 50–60 Hz
Electrical input: 24 VDC, 5 A

Hardware Compatible vessels: Multi-well plates, 35-mm Petri dishes, T-25 flasks
Gas input ports: Air or air-CO2 premix, CO2-only, and N2-only (max. 50 psi input)
Stagetop environmental chamber accessories: Master Stage Plate, Vessel holder
for multi-well plates

EVOS™ M7000 Imaging System User Guide 225

 EVOS™ Onstage Incubator components
Control unit and EVOS™ Onstage Incubator consists of an environmental chamber and a separate
environmental control unit that supplies the power and gas (air or air-CO2 premix, CO2, and N2 for
O2 displacement in hypoxia experiments), and controls the humidity and
chamber
temperature.

Control unit
Hose heater connector
Heated hose
Sensor data cable with
6-pin connector
Environmental chamber

Control unit rear Sensor data cable jack

view USB control cable jack
Power input jack
Port 1: Air
Port 2: N2
Port 3: CO2
Power switch

226 EVOS™ M7000 Imaging System User Guide

 Environmental The environmental chamber of the EVOS™ Onstage Incubator consists of the
chamber incubator chamber, the vessel holder/adaptor, the heated glass lid, the light
shield, and the light shield cover.
The environmental chamber sits on the onstage incubator master plate attached to
the X-Y stage of the EVOS™ M7000 Imaging System (see “Assemble the
environmental chamber”, page 229).

Light shield cover

Light shield
Heated glass lid
Vessel holder/adaptor
Incubator chamber
Master plate
Assembled environmental chamber

EVOS™ M7000 Imaging System User Guide 227

 Set up the EVOS™ Onstage Incubator
Install the Onstage 1. Remove the X-Y stage base plate from the X-Y stage by unscrewing the four
Incubator Master 3.0-mm screws (indicated by red arrows) on the base plate . If necessary,
unscrew and remove the vessel holder/adaptor before removing the base
Plate
plate.

2. Secure the onstage incubator master plate to the X-Y stage using the four
thumb screws (indicated by red arrows).

228 EVOS™ M7000 Imaging System User Guide

 Assemble the 1. Place the incubator chamber on the onstage incubator master plate and secure
environmental it in place using the four 2.0-mm hex screws (indicated by red arrows).
chamber

2. Attach the vessel holder/adaptor to the incubator chamber using the four
thumb screws (indicated by red arrows).

Note: Place an empty “dummy” culture plate into the vessel holder/adaptor for the
initial warm up and equilibration to prevent build-up of condensation on the optical
components and the inside of the EVOS™ M7000 Imaging System.

EVOS™ M7000 Imaging System User Guide 229

 3. Place the heated glass lid with the no-fog glass window on the incubator
chamber. The heated glass lid is guided and held secure in its place by the two
magnets on its rim.

4. Place the light shield with tinted plastic window on top of the heated glass lid.
Use of the light shield is required for fluorescence imaging applications.
5. If desired, place the light shield cover on the light shield for fluorescence
imaging applications. The light shield cover completely blocks any ambient
light from entering the environment chamber and improves image quality in
fluorescence imaging applications.

230 EVOS™ M7000 Imaging System User Guide

 Set up for operation Follow the procedure below to set up the EVOS Onstage Incubator for operation.
™

For the locations of the various input jacks and gas ports, refer to “Control unit
rear view”, page 226.

IMPORTANT! Do not position the control unit so that it is difficult to turn off the main
power switch. In case of an instrument malfunction, turn the main power switch to
the OFF position and disconnect the power cord from the wall outlet.

1. Plug power cord into the power input jack on the control unit and the wall
outlet.
2. Plug USB cable into the USB control cable jack on the control unit and the USB
port on the computer.
3. Connect each gas line to the appropriate gas tank via the PTC (push-to-click)
connectors threaded into the regulator. To do this, push the tubing into the
PTC connector until it clicks into place. Pull on tubing slightly to ensure a tight
connection; the tubing should not come out.

4. Attach the gas lines to the control unit via the PTC connectors for the
appropriate gas intake port.
• If using pre-mixed air, attach to Port 1: Air In
• If using compressed air and CO2, attach to Port 1: Air In and Port 3: CO2 In
• For oxygen displacement, attach to Port 1: Air In and Port 2: Nitrogen In
5. Plug the 6-pin sensor data cable to the environmental chamber and the
appropriate input jack on the control unit.

EVOS™ M7000 Imaging System User Guide 231

 6. Assemble the water reservoir and add warm water (approximately 50°C) to
the max fill line through the fill hole (see image below). Do not overfill the
water reservoir.

7. Place the water reservoir into the control unit with the fill holes to the front
and close the lid.

8. Attach the heated hose between the environmental chamber and the control unit
9. Plug the hose heater cable to the connector on the heated hose.

232 EVOS™ M7000 Imaging System User Guide

 Use the EVOS™ Onstage Incubator
Turn ON the EVOS™ 1. Turn ON the EVOS™ M7000 Imaging System as described on page 18.
Onstage Incubator 2. Turn the computer and monitor ON.
3. Turn ON the power switch to the control unit (page 226).
4. Restart the M7000 software.
5. On the Capture tab, click the Incubator button to display the
Incubator control panel.
6. Select Use Incubator, then enter the target values for Temperature, CO2, and
Oxygen. If desired, select Use humidity.
7. Select the desired Shutdown option:
• Turn off manually: The incubator will remain on until the Use Incubator
option is manually deselected and the Apply button is clicked.
• Turn off after: Enter the time period in hours and minutes that must
elapse before the incubator is shut down automatically.
8. Click Apply. Incubator status will change to “Running” and the control panel
will display the Current, Target, and Actual values for Temperature,
Humidity, CO2, and Oxygen.

9. Click Done to return to the Capture tab.

Note: Place an empty “dummy” culture plate into the vessel holder/adaptor for the
initial warm up and equilibration to prevent build-up of condensation on the optical
components and the inside of the EVOS™ M7000 Imaging System.

EVOS™ M7000 Imaging System User Guide 233

 Configure gas 1. In the Settings tab (page 192), click Incubator to open the incubator
inputs configuration panel.

2. Select the appropriate options for the Gas Inputs that reflects your set-up for
the EVOS™ Onstage Incubator.
• For Port 1, you may select Air or Premix.
If you select Premix, manually enter the percentage of the CO2 and O2 to
reflect the specifics of your set-up.
• Port 2 is reserved for Nitrogen only.
• Port 3 is reserved for CO2 only.
3. Click Done once you have configured the gas connections for each port.
4. Turn on the regulators on the gas tanks. The meters on the regulators show
the tank fill on the right and gas flow on the left.
5. Set the flow on the regulators as follows. Do not exceed 50 psi of pressure.
• Air: 40–50 psi
• CO2: 40–50 psi
• Nitrogen: 40–50 psi

234 EVOS™ M7000 Imaging System User Guide

 Calibrate oxygen Calibrating the oxygen sensor ensures that the atmosphere in the environmental
sensor chamber is replenished with the appropriate gasses in the correct proportion.
1. In the Settings tab (page 192), click Incubator to open the incubator
configuration panel, then expand the Calibrate panel under Oxygen Sensor.

2. Verify that your gas connections have been correctly configured.

3. Verify that the Oxygen % has been set correctly. If not, enter the correct value
for oxygen content.
4. Select the purge gas source for your calibration (Nitrogen or CO2).
5. Click Begin Calibration. The EVOS™ M7000 Imaging System automatically
calibrates the oxygen sensor for the proper functioning of the EVOS™ Onstage
Incubator. The entire calibration process takes approximately three minutes.

EVOS™ M7000 Imaging System User Guide 235

 Appendix E: Safety

Safety conventions used in this document

Safety alert words Three safety alert words appear in this document at points where you need to be
aware of relevant hazards. Each alert word—CAUTION, WARNING, DANGER—
implies a particular level of observation or action, as defined below:

CAUTION! – Indicates a potentially hazardous situation that, if not avoided,

may result in minor or moderate injury. It may also be used to alert against
unsafe practices.

WARNING! – Indicates a potentially hazardous situation that, if not

avoided, could result in death or serious injury.

DANGER! – Indicates an imminently hazardous situation that, if not

avoided, will result in death or serious injury. This signal word is to be
limited to the most extreme situations.

236 EVOS™ M7000 Imaging System User Guide

 Symbols on instruments
Electrical symbols The following table describes the electrical symbols that may be displayed on
on instruments Thermo Fisher Scientific instruments.

Symbol Description
Indicates the On position of the main power switch.

Indicates the Off position of the main power switch.

Indicates a standby switch by which the instrument is switched on to

the Standby condition. Hazardous voltage may be present if this
switch is on standby.

Indicates the On/Off position of a push-push main power switch.

Indicates a terminal that may be connected to the signal ground

reference of another instrument. This is not a protected ground
terminal.
Indicates a protective grounding terminal that must be connected to
earth ground before any other electrical connections are made to the
instrument.
Indicates a terminal that can receive or supply alternating current or
voltage.
Indicates a terminal that can receive or supply alternating or direct
current or voltage.

EVOS™ M7000 Imaging System User Guide 237

 Safety symbols The following table describes the safety symbols that may be displayed on
Thermo Fisher Scientific instruments. Each symbol may appear by itself or in
combination with text that explains the relevant hazard (see “Safety labels on
instruments”). These safety symbols may also appear next to DANGERS,
WARNINGS, and CAUTIONS that occur in the text of this and other product-
support documents.

Symbol Description
Indicates that you should consult the manual for further information
and to proceed with appropriate caution.
Indicates the presence of an electrical shock hazard and to proceed
with appropriate caution.
Indicates the presence of a hot surface or other high-temperature
hazard and to proceed with appropriate caution.
Indicates the presence of a laser inside the instrument and to proceed
with appropriate caution.
Indicates the presence of moving parts and to proceed with
appropriate caution.
Indicates the presence of a biological hazard and to proceed with
appropriate caution.
Indicates the presence of an ultraviolet light and to proceed with
appropriate caution.

Environmental The following symbol applies to all Thermo Fisher Scientific electrical and
symbols on electronic products placed on the European market after August 13, 2005.
instruments
Symbol Description
Do not dispose of this product as unsorted municipal waste. Follow
local municipal waste ordinances for proper disposal provisions to
reduce the environmental impact of waste electrical and electronic
equipment (WEEE).
European Union customers:
Call your Customer Service representative for equipment pick-up and
recycling. See www.thermofisher.com for a list of customer service
offices in the European Union.

238 EVOS™ M7000 Imaging System User Guide

 Safety labels on instruments
The following CAUTION, WARNING, and DANGER statements may be
displayed on Thermo Fisher Scientific instruments in combination with the safety
symbols described in the preceding section.

Hazard
English Français
Symbol
CAUTION! Hazardous chemicals. Read the ATTENTION! Produits chimiques dangereux.
Safety Data Sheets (SDSs) before handling. Lire les fiches techniques de sûreté de
matériels avant toute manipulation de
produits.
CAUTION! HAZARDOUS WASTE. Refer to ATTENTION! Déchets dangereux. Lire les
SDS(s) and local regulations for handling and fiches techniques de sûreté de matériels et la
disposal. régulation locale associées à la manipulation
et l’élimination des déchets.
DANGER! High voltage. DANGER! Haute tension.
WARNING! To reduce the chance of electrical AVERTISSEMENT! Pour éviter les risques
shock, do not remove covers that require tool d’électrocution, ne pas retirer les capots dont
access. No user-serviceable parts are inside. l’ouverture nécessite l’utilisation d’outils.
Refer servicing to Thermo Fisher Scientific L’instrument ne contient aucune pièce
qualified service personnel. réparable par l’utilisateur. Toute intervention
doit être effectuée par le personnel de service
qualifié venant de chez Thermo Fisher
Scientific.
DANGER! Class 3B visible and/or invisible DANGER! Rayonnement visible ou invisible
laser radiation present when open. Avoid d’un faisceau laser de Classe 3B en cas
exposure to beam. d’ouverture. Evitez toute exposition au
faisceau.
CAUTION! Moving parts. Crush/pinch ATTENTION! Pièces en mouvement, risque de
hazard. pincement et/ou d’écrasement.

EVOS™ M7000 Imaging System User Guide 239

 General instrument safety
WARNING! PHYSICAL INJURY HAZARD. Use this product only as specified
in this document. Using this instrument in a manner not specified by
Thermo Fisher Scientific may result in personal injury or damage to the
instrument.

Moving and lifting CAUTION! PHYSICAL INJURY HAZARD. The instrument is to be moved and
the instrument positioned only by the personnel or vendor specified in the applicable site
preparation guide. If you decide to lift or move the instrument after it has
been installed, do not attempt to lift or move the instrument without the
assistance of others, the use of appropriate moving equipment, and proper
lifting techniques. Improper lifting can cause painful and permanent back
injury. Depending on the weight, moving or lifting an instrument may
require two or more persons.

Moving and lifting WARNING! Do not attempt to lift or move the computer or the monitor
stand-alone without the assistance of others. Depending on the weight of the computer
computers and and/or the monitor, moving them may require two or more people.
monitors Things to consider before lifting the computer and/or the monitor:
• Make sure that you have a secure, comfortable grip on the computer or the
monitor when lifting.
• Make sure that the path from where the object is to where it is being moved is
clear of obstructions.
• Do not lift an object and twist your torso at the same time.
• Keep your spine in a good neutral position while lifting with your legs.
• Participants should coordinate lift and move intentions with each other before
lifting and carrying.
• Instead of lifting the object from the packing box, carefully tilt the box on its
side and hold it stationary while someone slides the contents out of the box.

Operating the Ensure that anyone who operates the instrument has:
instrument • Received instructions in both general safety practices for laboratories and
specific safety practices for the instrument.
• Read and understood all applicable Safety Data Sheets (SDSs). See “Safety
Data Sheets (SDS)”.

Cleaning or CAUTION! Using cleaning or decontamination methods other than those

decontaminating recommended by the manufacturer may compromise the safety or quality
the instrument of the instrument.

Removing covers or CAUTION! PHYSICAL INJURY HAZARD. The instrument is to be serviced

parts of the only by trained personnel or vendor specified in the user guide. Do not
instrument remove any covers or parts that require the use of a tool to obtain access to
moving parts. Operators must be trained before being allowed to perform
the hazardous operation.

240 EVOS™ M7000 Imaging System User Guide

 Safety requirements for EVOS™ Onstage Incubator
WARNING! Thermo Fisher Scientific recommends the use of nitrogen,
oxygen, and carbon dioxide gas with the Onstage Incubator. The use of
alternative gasses is currently not supported and may adversely affect
system performance

Gas cylinders
You must supply the required nitrogen, oxygen, and carbon dioxide gas cylinders
and accessories for the installation. This instrument requires pressurized house
lines, or one size 1-A gas cylinder that holds approximately 7.2 m3 (257 ft3) of gas
when full for each gas. Use only pre-purified gasses of 99.9% or greater purity.

CAUTION! Damage to the instrument and its products can result from
using impure gas, gases other than specified, or an inadequate amount of
gas.

WARNING! EXPLOSION HAZARD. Pressurized gas cylinders are potentially

explosive. Always cap the gas cylinder when it is not in use, and attach it
firmly to the wall or gas cylinder cart with approved brackets or chains.

WARNING! Gas cylinders are heavy and may topple over, potentially
causing personal injury and tank damage. Cylinders should be firmly
secured to a wall or work surface. Please contact your environmental
health and safety coordinator for guidance on the proper installation of a
gas cylinder.

Pressure regulator
You must supply a two-gauge regulator with a Compressed Gas Association
(CGA) 580-cylinder adaptor on the inlet side and a Swagelok™-type end-fitting
that accepts 6.35-mm (0.25-in.) o.d. tubing. The primary gauge (0 to 3000 psi;
0 to 25,000 kPa recommended) measures tank pressure, and the secondary gauge
(0 to 200 psi; 0 to 2000 kPa recommended) measures regulated pressure. The
secondary gauge must allow regulation to 50 psi. Compressed Gas Association
(CGA) 580-cylinder adaptor with a needle-type shutoff valve on the exit side. The
needle valves should have Swagelok™-type end-fittings ready for connection to
6.35-mm (0.25-in.) o.d. tubing.
Attaching the cylinder
Attach the pressurized gas cylinder firmly to a wall or gas cylinder cart by means
of approved straps or chains.

Ventilation WARNING! The Onstage Incubator should be installed and operated in a

requirements well-ventilated environment as defined as having a minimum airflow of
6–10 air changes per hour. Please contact your environmental health and
safety coordinator to confirm that the Ion instruments will be installed and
operated in an environment with sufficient ventilation.

Ventilation requirements
Allow at least 50 cm (20 in) of clearance around the Instrument for ventilation.

EVOS™ M7000 Imaging System User Guide 241

 Chemical safety
Chemical hazard WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to
warning the Safety Data Sheet (SDS) provided by the manufacturer, and observe all
relevant precautions.

WARNING! CHEMICAL HAZARD. All chemicals in the instrument, including

liquid in the lines, are potentially hazardous. Always determine what
chemicals have been used in the instrument before changing reagents or
instrument components. Wear appropriate eyewear, protective clothing,
and gloves when working on the instrument.

WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a

glass container because of the risk of breaking or shattering. Reagent and
waste bottles can crack and leak. Each waste bottle should be secured in a
low-density polyethylene safety container with the cover fastened and the
handles locked in the upright position. Wear appropriate eyewear,
clothing, and gloves when handling reagent and waste bottles.

General safety To minimize the hazards of chemicals:

guidelines • Read and understand the Safety Data Sheets (SDSs) provided by the chemical
manufacturer before you store, handle, or work with any chemicals or
hazardous materials. (See “Safety Data Sheets (SDS)”)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood). For additional
safety guidelines, consult the SDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer’s cleanup procedures as recommended in the SDS.
• Comply with all local, state/provincial, or national laws and regulations
related to chemical storage, handling, and disposal.

242 EVOS™ M7000 Imaging System User Guide

 Chemical waste safety
Chemical waste CAUTION! HAZARDOUS WASTE. Refer to Safety Data Sheets (SDSs) and
hazard local regulations for handling and disposal.

Chemical waste To minimize the hazards of chemical waste:

safety guidelines • Read and understand the Safety Data Sheets (SDSs) provided by the
manufacturers of the chemicals in the waste container before you store,
handle, or dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks
from the primary container. Both containers must be compatible with the
waste material and meet federal, state, and local requirements for container
storage.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood). For additional
safety guidelines, consult the SDS.
• Handle chemical wastes in a fume hood.
• After emptying the waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with
good laboratory practices and local, state/provincial, or national
environmental and health regulations.

Waste disposal If potentially hazardous waste is generated when you operate the instrument, you
must:
• Characterize (by analysis, if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and
disposed of according to all local, state/provincial, and/or national
regulations.

IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

EVOS™ M7000 Imaging System User Guide 243

 Electrical safety
DANGER! ELECTRICAL SHOCK HAZARD. Severe electrical shock can result
from operating the EVOS™ M7000 Imaging System without its instrument
panels in place. Do not remove instrument panels. High‐voltage contacts
are exposed when instrument panels are removed from the instrument.

Fuses WARNING! FIRE HAZARD. For continued protection against the risk of fire,
replace fuses only with fuses of the type and rating specified for the
instrument.

Power DANGER! ELECTRICAL HAZARD. Grounding circuit continuity is vital for

the safe operation of equipment. Never operate equipment with the
grounding conductor disconnected.

DANGER! ELECTRICAL HAZARD. Use properly configured and approved

line cords for the voltage supply in your facility.

DANGER! ELECTRICAL HAZARD. Plug the system into a properly

grounded receptacle with adequate current capacity.

Overvoltage rating The EVOS™ M7000 Imaging System has an installation (overvoltage) category of
II, and is classified as portable equipment.

244 EVOS™ M7000 Imaging System User Guide

 Physical hazard safety
Moving parts WARNING! PHYSICAL INJURY HAZARD. Moving parts can crush and cut.
Keep hands clear of moving parts while operating the instrument.
Disconnect power before servicing the instrument.

Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,
and blood of humans and other animals have the potential to transmit
infectious diseases. Follow all applicable local, state/provincial, and/or
national regulations. Wear appropriate protective eyewear, clothing, and
gloves. Read and follow the guidelines in these publications.

ATTENTION! BIOHAZARD. Les échantillons biologiques tels que les tissus,

les fluides corporels et le sang des humains et d’autres animaux ont la
possibilité de transmettre des maladies infectieuses. Suivre tous les
règlements municipaux, provinciaux/provincial et / ou nationales en
vigueur. Porter des lunettes de protection approprié, des vêtements et des
gants.

In the U.S.:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories
(stock no. 017-040-00547-4;
www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4toc.htm)
• Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030;
www.access.gpo.gov/nara/cfr/waisidx_01/29cfr1910a_01.html)
• Your company’s/institution’s Biosafety Program protocols for working
with/handling potentially infectious materials.
• Additional information about biohazard guidelines is available at:
www.cdc.gov

In the EU:
• Check your local guidelines and legislation on biohazard and biosafety
precaution, and the best practices published in the World Health Organisation
(WHO) Laboratory Biosafety Manual, third edition
www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_
2004_11/en/

EVOS™ M7000 Imaging System User Guide 245

 Safety and electromagnetic compatibility (EMC) standards
This section provides information on:
• U.S. and Canadian safety standards
• European safety and EMC standards
• Australian EMC standards

U.S. and Canadian The CSA C/US Mark signifies that the product meets applicable U.S. and
safety standards Canadian standards, including those from CSA, CSA America, ANSI, ASME,
ASSE, ASTM, NSF and UL.

European safety The CE Mark symbolizes that the product conforms to all applicable European
and EMC standards Community provisions for which this marking is required. Operation of the
instrument is subject to the conditions described in this manual.
The protection provided by the instrument may be impaired if the instrument is
used in a manner not specified by Thermo Fisher Scientific.

Australian EMC The C-Tick Mark indicates conformity with Australian and New Zealand
standards standards for electromagnetic compatibility.

246 EVOS™ M7000 Imaging System User Guide

 Documentation and support

Obtaining support
Technical support For the latest services and support information for all locations, visit
www.thermofisher.com.
At the website, you can:
• Access worldwide telephone and fax numbers to contact Technical Support
and Sales facilities
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support (thermofisher.com/contact)
• Search for user documents, SDSs, vector maps and sequences, application
notes, formulations, handbooks, certificates of analysis, citations, and other
product support documents
• Obtain information about customer training
• Download software updates and patches

Limited product Life Technologies Corporation and/or its affiliate(s) warrant their products as set
warranty forth in the Life Technologies’ General Terms and Conditions of Sale found on
Life Technologies’ website at www.thermofisher.com/us/en/home/global/terms-
and-conditions.html. If you have any questions, please contact Life Technologies
at www.thermofisher.com/support.

IMPORTANT! Wiping the computer supplied with the EVOS™ M7000

Imaging System (i.e., erasing the hard drive to remove all programs, files,
and the operating system) voids the product warranty.

EVOS™ M7000 Imaging System User Guide 247

For support visit thermofisher.com/support or email [email protected]
16 February 2021