Table of Contents
Related Manuals for Invitrogen EVOS M5000
Summary of Contents for Invitrogen EVOS M5000
- Page 1 EVOS M5000 Imaging System ™ USER GUIDE For Fluorescence and Transmitted Light Applications Catalog Number AMF5000 and AMF5000SV Publication Number MAN0017563 Revision F.0 For Research Use Only. Not for use in diagnostic procedures.
- Page 2 Life Technologies Corporation | 22025 20th Ave SE Ste. 100 | Bothell, Washington 98021 USA For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition. Revision history: MAN0017563 F.0 (English) Revision Date Description 16 November Add Stage View tool and update screens and content. 2023 14 December Update Connect account sign in, add information about Align Channels tool, update relevant screens.
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Page 3: Table Of Contents
Contents ■ CHAPTER 1 About this guide ............9 Audience . - Page 4 Contents Turn on the EVOS M5000 Imaging System ........22 ™...
- Page 5 Contents Count cells – manual count ............51 Perform manual count .
- Page 6 Contents Add measurements and annotations to saved images ......90 Analyze cell culture using saved images ......... . 90 Save analysis results .
- Page 7 Contents Change the objectives ............110 Procedure for objective change .
- Page 8 Contents ■ APPENDIX F Safety ............. . 147 Symbols on this instrument .
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Page 9: Chapter 1 About This Guide
About this guide Audience This user guide is for laboratory staff operating, maintaining, and analyzing data using the Invitrogen ™ EVOS M5000 Imaging System. ™ User attention words Two user attention words appear in this document. Each word implies a specific level of observation or action as described below. -
Page 10: Chapter 2 Product Information
EVOS M5000 Software ™ The EVOS M5000 Imaging System is controlled by the integrated Invitrogen EVOS M5000 Software ™ ™ ™ through a graphical user interface (GUI), which is accessed by the computer mouse and keyboard. The software is pre-installed and starts automatically when the instrument is powered on. -
Page 11: Product Use
Chapter 2 Product information Standard items included • Transfection Efficiency: Enables measuring percent transfection in a population of cells, as determined after measuring confluence. • Network and Connect connectivity: Allows Wi-Fi and Ethernet connectivity to the network and to your Connect account as part of the Connect-based platform to store and access your data files. Product use For Research Use Only. -
Page 12: Evos ™ M5000 Imaging System User Documentation
Chapter 2 Product information Standard items included • Condenser Slider, Block (Cat. No. AMEP4688) • Condenser Slider, 4X Pupil (Cat. No. AMEP4738) • Condenser Slider, Diffusion for Brightfield Applications (Cat. No. AMEPDFS1) • Universal power supply (12 V, 5 A) and power cord (type B, North America) •... -
Page 13: Instrument Exterior Components And Mechanical Controls
Chapter 2 Product information Instrument exterior components and mechanical controls Instrument exterior components and mechanical controls Front view LCD monitor Objective selection wheel Condenser Focusing knobs Condenser light shield USB-A 3.0 port Vessel holder and thumb screws Objective Mechanical X-Y stage Phase ring selector Stage X- and Y-axis positioning knobs EVOS M5000 Imaging System User Guide... -
Page 14: Rear View
Chapter 2 Product information Instrument exterior components and mechanical controls Rear view LCD monitor DisplayPort (video output) Mechanical X-Y stage Network port Focusing knobs USB-A 2.0 ports (2×) USB-A 3.0 port (2×) Stage X- and Y-axis positioning knobs Power switch Instrument passwords sticker Single-pin power input port (12 VDC, 5 A) EVOS M5000 Imaging System User Guide... -
Page 15: Graphical User Interface (Gui)
Chapter 2 Product information Graphical user interface (GUI) Graphical user interface (GUI) GUI layout The GUI of the system consists of the Viewing area on the left and Capture and Review tabs and the Settings and Virtual keyboard buttons on the right. Each tab and button opens the controls necessary to execute the selected function. -
Page 16: Chapter 3 Installation
Installation Operating environment and site requirements • The dimensions of the EVOS M5000 Imaging System are 18 × 23 × 18 in (46 × 59 × 46 cm) ™ (W × H × D). The system requires a benchtop of approximately 36 × 36 in (92 × 92 cm). • If the system includes the optional EVOS Onstage Incubator (Cat. -
Page 17: Prepare For Installation
Chapter 3 Installation Prepare for installation Prepare for installation Receive and inspect the shipment 1. Verify that the items shown on the shipping list are the same items that you ordered at the time of purchase. 2. Carefully inspect the shipping containers and report any damage to the Thermo Fisher Scientific service representative. -
Page 18: Remove Shipping Restraints
Chapter 3 Installation Install the instrument Note: Make sure to set aside packaging and foam for future transport and storage. Re-install the stage lock pin and the light cube shipping restraint before moving or transporting the instrument. Always ensure that the instrument is properly cushioned and braced to prevent damage. Remove shipping restraints The EVOS M5000 Imaging System is equipped with two shipping restraints (stage lock pin and light... - Page 19 Chapter 3 Installation Install the instrument Figure 2 After removing the stage lock pin and moving the stage to access the shipping restraint. 3. Unscrew and remove the light cube tool, which secures the shipping restraint block to the blank light cube. 4. Remove the shipping restraint block and store it in the accessories box. Removal of the restraint block provides access to the blank light cube in the light cube turret.
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Page 20: Install The Uv Light Shield
Chapter 3 Installation Install the instrument 5. Using the light cube tool, loosen the two screws that secure the blank light cube to the instrument. You do not need to remove the screws. 6. Screw the light cube tool in to the threaded hole in the blank light cube, then lift the blank light cube up and out of the microscope. - Page 21 Chapter 3 Installation Install the instrument 3. Secure the UV light shield mount to the top of the condenser light shield using the two screws and 2 mm hex key supplied with the UV shield assembly (not the protruding screws on the mount). 4.
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Page 22: Connect The Instrument
Chapter 3 Installation Turn on the EVOS M5000 Imaging System ™ Note: The UV light shield is provided as a safety feature and should be installed whenever the unit is in operation. The UV light shield is removable for access to the condenser sliders used in transmitted light mode. -
Page 23: Connect To The Thermo Fisher ™ Connect Platform
Chapter 3 Installation Connect to the Thermo Fisher Connect Platform ™ Connect to the Thermo Fisher Connect Platform ™ About the Thermo Fisher Connect Platform ™ The Thermo Fisher Connect Platform enables access to your EVOS M5000 instrument through ™ ™ Instrument Connect by way of a web browser or mobile device. Connecting to your Connect account allows you to save captured images in your unique user account in addition to local storage. -
Page 24: Link Instrument To Your Connect Account
Chapter 3 Installation Connect to the Thermo Fisher Connect Platform ™ Link instrument to your Connect account You can link the EVOS M5000 instrument to your Connect account using one of the following options: ™ 1. Mobile Device (QR code): Scan the QR code on your instrument using the Instrument Connect application on your mobile device (“Add instrument to your Connect account with QR code (Mobile Device)”... -
Page 25: Add Instrument To Your Connect Account With Qr Code (Mobile Device)
Chapter 3 Installation Connect to the Thermo Fisher Connect Platform ™ Add instrument to your Connect account with QR code (Mobile Device) 1. Click (Sign In) on the top left corner of the screen to open the Sign In dialog. 2. Click Link Account, then select Mobile Device. 3. -
Page 26: Set Up A New Administrator
Chapter 3 Installation Connect to the Thermo Fisher Connect Platform ™ 3. Enter your Connect PIN number. If you do not have a PIN number, set the PIN number in the Instrument Connect application (“Create a PIN number” page 23). 4. Click Sign In. When you connect to your Connect account, the Sign In button changes to display your user name (for example, 5. -
Page 27: Chapter 4 Capture Images
Capture images Overview Workflow Capture images Select the objective and light source page 28 Focus on the sample page 30 Find the region of interest page 31 Capture an image in a single channel page 32 Save page 62 Analyze and annotate the captured images page 36 EVOS M5000 Imaging System User Guide... -
Page 28: Capture Tab
Chapter 4 Capture images Capture images in a single channel Capture tab The basic functions of the EVOS M5000 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. -
Page 29: Adjust Brightness
Chapter 4 Capture images Capture images in a single channel Memory buffer thumbnail images Auto-capture channels (via the All button) Channel selection/light activation buttons Note: If the phase ring correctly matches the objective, the selected phase ring is depicted in blue. If the objective and the phase ring are mismatched, the phase ring (Condenser) is displayed in orange. -
Page 30: Focus On The Sample
Chapter 4 Capture images Capture images in a single channel Note: Optimize the brightness parameters as follows: · 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. -
Page 31: Optional: Adjust Display Settings
Chapter 4 Capture images Capture images in a single channel Note: Z-Offset specifies the focus position in the selected channel relative to the focus position in other channels. Setting the correct Z-Offset is especially important when the fluorescent markers in different channels are in different focal planes. Optional: Adjust display settings 1. -
Page 32: Capture Images For Each Channel
Chapter 4 Capture images Capture images in a single channel Capture images for each channel 1. Ensure that the Channel you want to capture is selected. When you select a Channel, the corresponding Capture channel checkbox above the button is automatically checked. Note: If you exit the Live mode, the current channel remains selected, as indicated by the highlighted color surrounding the channel button. - Page 33 Chapter 4 Capture images Capture images in a single channel 4. If needed, readjust the brightness and focus, then click Capture. The viewing area shows the image captured in the new channel superimposed on the image captured in the previous channel. A thumbnail of the image captured in the new channel is displayed along with the thumbnail of the image from the previously captured channel.
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Page 34: Capture Images In Multiple Channels
Chapter 4 Capture images Capture images in multiple channels Capture images in multiple channels Capture multiple channels automatically 1. To capture multiple channels automatically, select the desired channels by checking the corresponding boxes. 2. If needed, adjust brightness and focus for each of the selected channels as described. 3. - Page 35 Chapter 4 Capture images Capture images in multiple channels IMPORTANT! Captured images are stored in individual memory buffers with one for each channel. Memory buffers are for display and are not automatically saved. If unsaved, newly captured images overwrite the previously captured image in the selected channel. Images captured in other fields and channels are not affected.
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Page 36: Chapter 5 Measure, Annotate, And Analyze Captured Images
Measure, annotate, and analyze captured images Display settings and analysis tools Display settings and analysis tools allow you to change image display settings for live and captured images in the Viewing area, correct pixel shifts that can occur at higher magnifications in multichannel fluorescent images, annotate captured images, and perform cell count, measure confluence, and calculate transfection efficiency. -
Page 37: Adjust Image Display Settings
Chapter 5 Measure, annotate, and analyze captured images Adjust image display settings • Analyze cell culture (“Analyze cell culture” page 44): – Perform Auto Count (“Perform auto count” page 45) – Perform Manual Count (“Perform manual count” page 51) – Measure confluence (“Measure confluence” page 54) –... -
Page 38: Display Grid
Chapter 5 Measure, annotate, and analyze captured images Display grid the left may help reduce undesired background fluorescence. For images with very faint fluorescence signals, moving the Gamma slider to the right may help the intensity of faint signals to stand out from the background. -
Page 39: Display Scale Bar
Chapter 5 Measure, annotate, and analyze captured images Display scale bar Display scale bar 1. Click (Scale Bar) to superimpose a scale bar over the Viewing area. 2. To change Scale Bar Settings, click (Scale Bar Settings) (arrow on the Scale Bar split button) to open the Grid Settings tool. -
Page 40: Align Channels
Chapter 5 Measure, annotate, and analyze captured images Align channels Align channels Align channels The Align Channels tool allows you to correct pixel shifts that can occasionally appear when performing multichannel fluorescence imaging at higher magnifications. You can select individual channels in a multichannel image, move them to the correct position relative to other channels, and then save the corrected image. -
Page 41: View Pixel Intensity Histogram
Chapter 5 Measure, annotate, and analyze captured images View pixel intensity histogram 5. If the multichannel image was captured in more than two channels, repeat the process for the other channels until all the channels are correctly aligned. 6. Click Save to save the corrected image, then click the Align Channels button to hide the channel alignment controls. -
Page 42: Add Measurements And Annotations
Chapter 5 Measure, annotate, and analyze captured images Add measurements and annotations 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 triangle at the lower right corner of the plot, then drag the plot to the desired size. - Page 43 Chapter 5 Measure, annotate, and analyze captured images Add measurements and annotations 4. If desired, choose 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.
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Page 44: Analyze Cell Culture
Chapter 5 Measure, annotate, and analyze captured images Analyze cell culture Analyze cell culture Analysis tools If not already showing, hover the pointer over the Viewing area to reveal the Display Settings and Analysis Tools toolbar, then click (Show Cell Count) to display Auto Count, Manual Count, and Cell Culture options in the tabs area. -
Page 45: Count Cells - Auto Count
Chapter 5 Measure, annotate, and analyze captured images Count cells – auto count Count cells – auto count Perform auto count 1. Click (Show Cell Count), then select Auto Count. 2. If counting from the Capture tab, select the Channel in which to count objects. Available options depend on the channels used when the image was captured. - Page 46 Chapter 5 Measure, annotate, and analyze captured images Count cells – auto count 3. To identify targets, click Target, then click and drag to draw a circle (blue) around a representative target. 4. If needed, click Target again to identify other targets (for example, nuclei that might appear different) to improve the accuracy of your count.
- Page 47 Chapter 5 Measure, annotate, and analyze captured images Count cells – auto count 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.
- Page 48 Chapter 5 Measure, annotate, and analyze captured images Count cells – auto count The target objects that were counted are identified 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 representative targets and background that you have selected, the auto count algorithm can overcount or undercount the cells in the image.
- Page 49 Chapter 5 Measure, annotate, and analyze captured images Count cells – auto count 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. •...
- Page 50 Chapter 5 Measure, annotate, and analyze captured images Count cells – auto count 10. To change the color of the circles that identify the objects included in the count, select the desired color from the Count Color dropdown. 11. When finished with the count, save your count results (see “Save analysis results” page 92).
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Page 51: Count Cells - Manual Count
Chapter 5 Measure, annotate, and analyze captured images Count cells – manual count Count cells – manual count Perform manual count 1. Click (Show Cell Count), then select Manual Count. 2. Select the Channels to display in the Viewing area for manual count. You can select multiple channels that contain captured images. - Page 52 Chapter 5 Measure, annotate, and analyze captured images Count cells – manual count 3. Click in an Object Name field to enter a name for that label. You can use up to six labels to tag objects for the manual count. EVOS M5000 Imaging System User Guide ™...
- Page 53 Chapter 5 Measure, annotate, and analyze captured images Count cells – manual count 4. Click on the Label number to select a label, then left-click at each point on the Viewing area to tag the items in that label category. 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.
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Page 54: Measure Confluence
Chapter 5 Measure, annotate, and analyze captured images Measure confluence 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. - Page 55 Chapter 5 Measure, annotate, and analyze captured images Measure confluence 2. If not visible, hover the pointer over the Viewing area to reveal Display Settings and Analysis Tools, then click the Show Cell Count (123) button. 3. In the tabs area, select Cell Culture to display the Confluence tool. 4.
- Page 56 Chapter 5 Measure, annotate, and analyze captured images Measure confluence 5. Click Background, then click and drag on the image to draw a circle around a background area that does not contain any cells (orange circle). 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.
- Page 57 Chapter 5 Measure, annotate, and analyze captured images Measure 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. Increased the sensitivity results in higher confluence value.
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Page 58: Calculate Transfection Efficiency
Chapter 5 Measure, annotate, and analyze captured images Measure confluence Calculate transfection efficiency Transfection Efficiency tool Transfection of a cell population typically results in a varying number of cells expressing the desired genes of interest. Transfection efficiency is the percentage of cells that are transfected compared to the entire population. - Page 59 Chapter 5 Measure, annotate, and analyze captured images Measure confluence The software automatically calculates the Transfection Efficiency and displays the results as % Confluence and % Transfection Efficiency. Note: The Transfection Efficiency calculation is based on the final measured confluence (Step 8 page 57) 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 step 5 page 61).
- Page 60 Chapter 5 Measure, annotate, and analyze captured images Measure confluence 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.
- Page 61 Chapter 5 Measure, annotate, and analyze captured images Measure confluence 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.
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Page 62: Chapter 6 Save Captured Images
Save captured images Save Save images manually 1. After capturing an image in a single channel or a set of images in multiple channels, click Save to open the Save dialog, then navigate to the destination folder to save your captured images. 2. -
Page 63: Quick Save Images
Chapter 6 Save captured images Quick Save images 4. Select Save individual channels to save images captured in different channels individually. Saving as type TIFF or PNG are the recommended formats for image analysis. Leave the Save individual channels option unselected to save only the captured image displayed in the viewing area. - Page 64 Chapter 6 Save captured images Quick Save images 4. Enter a Base Filename. The Starting number automatically changes to 1 when the Base Filename is changed. Note: The default base filename is Image. Base Filename is appended by a numerical suffix and channel name.
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Page 65: Chapter 7 Capture Time Lapse Images
Capture time lapse images Time lapse tool With the Time Lapse tool, you can set up your cells and program the EVOS M5000 Imaging System ™ to capture images (including Z-Stack images) at given intervals over a time period based on your specifications. - Page 66 Chapter 7 Capture time lapse images Run a time lapse routine 5. (Optional) If the EVOS Onstage Incubator is installed and used for a time-lapse routine, click Use ™ Incubator. Note: This option is only available if you have installed and connected the EVOS Onstage ™...
- Page 67 Chapter 7 Capture time lapse images Run a time lapse routine 10. Define the Z-Stack parameters to determine the number of “optical sections” captured in the Z-Stack image set: • Automatically compute Z-Stack parameters: Select to calculate the number of images and their Z-positions automatically based on the Z-Stack boundaries and the default Step Size.
- Page 68 Chapter 7 Capture time lapse images Run a time lapse routine 14. Set the Image capture options for Run 1. Available options are Frequency, Intervals, and As fast as possible. • Frequency: Enter the time in Hours, Minutes, and Seconds that must elapse before a new set of images are captured.
- Page 69 Chapter 7 Capture time lapse images Run a time lapse routine 22. Select the Frame Rate for image capture: 1, 3, or 5 images/second. Note: Frame Rate specifies how many images are captured per second during the time lapse recording. You can view the saved time lapse recording at different playback speeds ranging from 1 fps (frames per second) to 120 fps (step 3 page 72).
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Page 70: Run A Saved Time Lapse Routine
Chapter 7 Capture time lapse images Run a time lapse routine Run a saved time lapse routine Each time you run a Time Lapse routine, the system saves the specifics of the routine, which you can then recall and run with a new sample. 1. -
Page 71: Review Images And Video Captured In A Time Lapse Routine
Chapter 7 Capture time lapse images Run a time lapse routine Review images and video captured in a time lapse routine 1. To review the results from your completed time lapse routine, click (Results). The viewing area displays the first image captured in the time lapse routine and the Review tab shows the Image Properties, and Time Lapse and Auto Focus Settings. - Page 72 Chapter 7 Capture time lapse images Run a time lapse routine 3. To play the time-lapse movie, select the (Time-lapse Video) file, then click (Play) on the Time Lapse Video Player. 4. To change the playback speed, select the desired speed from the Playback Speed menu. Note: Playback Speed is independent of the Frame Rate, which specifies how many images are captured per second during the time-lapse recording (step 19 page 68).
- Page 73 Chapter 7 Capture time lapse images Run a time lapse routine 5. To review individual images captured during the time-lapse routine, select the desired image from the Review tab. The viewing area displays the selected image and the Review tab shows the Image Properties, and Time Lapse and Auto Focus Settings.
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Page 74: Chapter 8 Capture Z-Stack
Capture Z-Stack Z-Stack tool The Z-Stack tool 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. - Page 75 Chapter 8 Capture Z-Stack Capture Z-stack images 5. Move the focus sliders up from the Current Z position to the desired Top position (upper boundary of the Z-Stack), then click Set for Top. 6. Move the focus sliders down from the Current Z position to the desired Bottom position (lower boundary of the Z-Stack), then click Set for Bottom.
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Page 76: Chapter 9 Stage View
Stage View Overview ™ Note: Stage View requires an updated stage manufactured in early 2024 for all new EVOS M5000 ™ systems. Prior EVOS M5000 systems using a stage with separate X- and Y-axis control knobs will not have access to the Stage View feature but can be updated to have all of the other software features described in the this user guide. -
Page 77: Workflow
Chapter 9 Stage View Overview Workflow Stage View Set the Stage Origin page 78 Mark the pin locations page 81 Find the pins using the Find Target screen page 81 Save the pin maps page 81 Load the pin map and find the pins using the Find Target screen page 84 EVOS M5000 Imaging System User Guide... -
Page 78: Set Stage Origin
Chapter 9 Stage View Set Stage Origin Set Stage Origin 1. Select the Stage View tab. 2. Switch to a low-power objective (2x, 4x, or 10x). This allows for easier locating of the calibration pinhole and ensures enough space for the objective beneath the stage for movement. 3. - Page 79 Chapter 9 Stage View Set Stage Origin 7. Click Set Origin. The stage is now initialized for tracking its position. 8. Click Vessel to select a vessel type: • Well Plates • Slide or Dish • Blank Map • Load Pins 9. Click the plate size or vessel size/type or load a pin map from the Load Pin Map window: •...
- Page 80 Chapter 9 Stage View Set Stage Origin • Load Pins: Loads a previous map with pins and the appropriate vessel. Click Load Pins..Click the desired Map Name row. Click Load. The Find Target window will open. Click Done to return to the main screen. Note: When selecting a well plate, any captured images will automatically add the well lD to the individual saved filename.
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Page 81: Mark Pin Locations
Chapter 9 Stage View Mark pin locations Mark pin locations 1. Find a point of interest by moving the stage using the knobs. 2. Once on a desired spot, click (Add pin). This will be Pin 1 or can be custom named by clicking in the field next to the icon. - Page 82 Chapter 9 Stage View Find pins using the Find Target screen 1. Select a target by clicking on the desired pin or the pin name in the list. The pin will change color from blue to amber and Target will update with the name of the selected pin. 2.
- Page 83 Chapter 9 Stage View Find pins using the Find Target screen 5. (Optional) Each pin name in the Find Target window can be modified or deleted by hovering the mouse over the pin name, then clicking (Edit) or (Delete). Click to complete the edit. All pins can be deleted by clicking Clear all above the pin list.
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Page 84: Load Pin Map
Chapter 9 Stage View Load Pin Map Load Pin Map • To use a previous map, select a row, then click Load. – This window also allows for searching and editing saved maps. Saved maps can be selected from the current list by using the Search function or sorting by vessel type. –... -
Page 85: Chapter 10 Review And Analyze Saved Images
Review and analyze saved images The Review tab allows you to review, annotate, and analyze still images, including those captured in Time Lapse routines and Z-Stacks. You can also re-save or delete saved files. • Review saved images (“Review images” page 86) •... -
Page 86: Review Images
Chapter 10 Review and analyze saved images Review images Note: You can also view and analyze captured images that are saved to your Connect account with the EVOS Image Analysis application. ™ For more information on the EVOS Image Analysis application, see Appendix C, “System overview”. ™... - Page 87 Chapter 10 Review and analyze saved images Review images 6. Click the image to select. The selected image is indicated with a box around it and the viewing area displays the selected image. The Image Properties panel shows the metadata associated with the selected image. EVOS M5000 Imaging System User Guide ™...
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Page 88: Configure Display Settings
Chapter 10 Review and analyze saved images Configure display settings Configure display settings Adjust display settings for saved images Display settings and analysis tools allow you to change image display settings, and to analyze and annotate captured images. Hover the pointer over the Viewing area to reveal the buttons for Display Settings and Analysis Tools. Note: Display Settings are available in both the Capture and the Review tabs and function the same way. -
Page 89: Display Grid
Chapter 10 Review and analyze saved images View pixel intensity histogram Display grid • Click (Grid) to superimpose a grid over the Viewing area. For instructions on how to change the Grid settings, see “Display grid” page 38. Display scale bar • Click (Scale Bar) to superimpose a scale bar over the Viewing area. -
Page 90: Add Measurements And Annotations To Saved Images
Chapter 10 Review and analyze saved images Add measurements and annotations to saved images Note: The Histogram tool is available in both the Capture and the Review tabs and functions the same way. Add measurements and annotations to saved images 1. Hover the pointer over the Viewing area to reveal the buttons for Display Settings and Analysis Tools at the bottom of the screen. - Page 91 Chapter 10 Review and analyze saved images Analyze cell culture using saved images 2. Click to select from the following analysis options: • Auto Count: Automatically counts the objects displayed in the Viewing area based on your specifications. With Auto Count, you can only count objects in a single fluorescence channel (typically a nuclear stain channel).
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Page 92: Save Analysis Results
Chapter 10 Review and analyze saved images Save analysis results 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. - Page 93 Chapter 10 Review and analyze saved images Save analysis results 3. Select Save as screenshot to preserve a detailed account of your count results as an image. When Save as screenshot is not selected, the software only saves the image as displayed in the Viewing area.
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Page 94: Batch Analysis
Chapter 10 Review and analyze saved images Batch analysis Batch analysis Batch Analysis function 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. - Page 95 Chapter 10 Review and analyze saved images Batch analysis 4. Select the Settings to use for Batch Analysis. • To use the current analysis settings (that is, analysis parameters that you have used in Step 2 page 94), select Current Settings. • To use previously saved analysis settings, select the desired option from the Select Settings list.
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Page 96: Chapter 11 Configure Instrument Settings
Configure instrument settings Overview Settings tab The Settings tab allows you to assign objectives to the objective turret, to calibrate objective magnification, to set image and general instrument options, add and remove light cubes, and to connect to a Wi-Fi network and to map network drives. •... - Page 97 Chapter 11 Configure instrument settings Overview Objectives: Set up and calibrate objectives and assign objective profiles (“Adjust objective settings” page 98). Display: Use White Balance Calibration and Hot Pixel Correction to adjust the white balance for the color camera (“Calibrate white balance” page 100) and set saturated pixel options (“Set saturated pixel options” page 101).
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Page 98: Adjust Objective Settings
Chapter 11 Configure instrument settings Adjust objective settings Adjust objective settings Assign objectives After adding a new objective to the objective turret or replacing an older objective, assign the new objective to the appropriate turret position. For instructions on how to remove an objective, see “Change the objectives”... -
Page 99: Calibrate Objective Magnification
Chapter 11 Configure instrument settings Adjust objective settings Calibrate objective magnification 1. Go to the Settings4Objectives, click (Actions) next to the newly installed objective, then select Calibrate to launch the Objective Calibration tool. 2. Mount the EVOS Calibration Slide in the 2-slide vessel holder (Cat. No. AMEP-VH001) and select ™... -
Page 100: Calibrate White Balance
Chapter 11 Configure instrument settings Calibrate white balance Calibrate white balance Calibrate white balance Calibrate White Balance calibrates color channel lighting. 1. Go to Settings4Display tab, then click White Balance Calibration. 2. Follow the screen instructions. Select a 20X or higher objective, then set the phase ring to Brightfield using the Phase ring selector (“Rear view”... -
Page 101: Set Saturated Pixel Options
Chapter 11 Configure instrument settings Set saturated pixel options Set saturated pixel options Highlight saturated pixels Highlight saturated pixels function displays overexposed pixels on an image with the user-defined color, which provides a visual aid for optimal illumination when adjusting the brightness settings. 1. -
Page 102: Define Display Settings Toolbar Behavior
Chapter 11 Configure instrument settings Configure network settings 1. To automatically unselect channel checkboxes after multichannel image capture, click Uncheck Capture-All channels after Capture in the (Settings)4General tab. 2. Click Done. Define Display Settings toolbar behavior By default, the Display Settings and Analysis Tools toolbar is displayed automatically after image capture and remains visible. -
Page 103: Connect To A Wi-Fi Network
Chapter 11 Configure instrument settings Configure network settings Connect to a Wi-Fi network 1. Click (Settings). 2. Click Network. 3. Click Show Wi-Fi Networks, then select the network you want to join. 4. Click Close. Map network drive 1. Go to Settings4Network, then click Map Network Drive. EVOS M5000 Imaging System User Guide ™... - Page 104 Chapter 11 Configure instrument settings Configure network settings 2. Select the drive you want to map from the Drive menu. 3. Click Browse to open the Browse for Folder dialog. 4. Navigate to the folder you want to map, then OK. 5. If you want to reconnect to the selected drive and folder when you turn on the instrument, click Reconnect at logon.
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Page 105: Service
Chapter 11 Configure instrument settings Service Service The Service tab displays the current software and firmware versions of the system and allows you to copy error logs, set date and time, and update software and firmware from the cloud or the USB flash drive. -
Page 106: Update From Cloud
Chapter 11 Configure instrument settings Service Note: You cannot set the time more than 24 hours in the past. Update from cloud 1. Restart your EVOS M5000 instrument before an update to optimize the update process. ™ 2. To update the instrument software and firmware from the Cloud, sign in to your Connect account (“Connect to the Thermo Fisher Connect Platform”... -
Page 107: Chapter 12 Instrument Care And Maintenance
Instrument 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. •... -
Page 108: Objective Lens Care
Chapter 12 Instrument care and maintenance Objective lens care 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. -
Page 109: Change Evos ™ Led Light Cubes
Chapter 12 Instrument care and maintenance Change EVOS LED light cubes ™ Change EVOS LED light cubes ™ To customize your EVOS M5000 Imaging System, you can add and remove LED light cubes to fit the ™ instrument’s functionality to your own specific research needs. Each LED light cube is coded to allow the imaging system to automatically recognize it in any position. -
Page 110: Change The Objectives
Chapter 12 Instrument care and maintenance Change the objectives 6. Attach the tool to the new light cube and lower the cube into position so that the electronic connection aligns properly (facing the back of the microscope) and the cube sits squarely in place with the label facing toward the front. - Page 111 Chapter 12 Instrument care and maintenance Change the objectives 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 this example, the new objective is installed into the turret position 5.
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Page 112: Calibrate The Objectives
Chapter 12 Instrument care and maintenance Calibrate the objectives Calibrate the objectives Calibrate objective magnification allows the calibration of the field of view, parfocality, and parcentration parameters of the selected objective. When calibrated, parfocality ensures that the sample stays in focus when the objective is changed. Note: The pre-installed objectives supplied with the EVOS M5000 Imaging System have been pre- ™... - Page 113 Chapter 12 Instrument care and maintenance Calibrate the objectives 5. Focus on the calibration circle using the focus controls. 6. Click and drag the green calibration lines to align them along the outer borders of the calibration circle. 7. When finished, click Save to complete the calibration. Repeat the calibration process for each additional objective to be calibrated.
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Page 114: Appendix A Troubleshooting
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. Image quality issues Observation Recommended action Transmitted light image is too Remove the condenser slider, if one is in place. dim (at higher magnifications) Specks, dots, or blurs on image Follow the instructions under “Objective lens care”... -
Page 115: Stage View Issues
Appendix A Troubleshooting Stage view issues Stage view issues Observation Recommended actions Cannot locate pinhole for Use a low-power objective (10x or less). aligning the stage Note the orientation of your slide or vessel in the vessel holder when creating pins. Use the same orientation when re-locating pins. Use a low-power objective (10x or less). -
Page 116: Mechanical Issues
Appendix A Troubleshooting Mechanical issues (continued) Observations Recommended actions • Verify physical cable connections; confirm that the Ethernet jack is Unable to connect to network active (for Ethernet connection). • Verify that the USB Wi-Fi adaptor for wireless network connection is installed into one of the USB ports in the back (for wireless connection). -
Page 117: Appendix B Graphical User Interface (Gui)
Graphical user interface (GUI) Capture tab 1. Sign In/User: Allows you to sign in to and sign out of your Connect account. 2. Viewing area: Displays live or captured images of the sample. 3. Zoom slider: Zooms in and out of the Viewing area. The zoom range is 100% to 1000%. 4. - Page 118 Appendix B Graphical user interface (GUI) Capture tab 7. Channel: Selects the light source from the installed LED light cubes (fluorescent channels) or from the condenser (transmitted light). RGB Trans option outputs a color image from interlaced RGB images in the Live mode. •...
- Page 119 Appendix B Graphical user interface (GUI) Capture tab Time Lapse: Opens the Time Lapse tool, which allows you to create and run time lapse routines to capture images at given intervals over a time period based on your specifications. Z-Stack: Opens the Z-Stack tool, which allows you to capture multiple images along the Z-axis based on your specifications.
- Page 120 Appendix B Graphical user interface (GUI) Capture tab Incubator: Opens the Incubator Control, which allows you to control the optional EVOS ™ Onstage Incubator and monitor its status during your experiments. Note: The Incubator Control window only appears if an EVOS Onstage Incubator is connected to ™...
- Page 121 Appendix B Graphical user interface (GUI) Capture tab 19. Focus controls: • Autofocus: Runs autofocus in the selected channel. Note: Do NOT use autofocus with oil-immersion objectives, as this can create air bubbles in the oil or cause the objective to collide with the sample. •...
- Page 122 Appendix B Graphical user interface (GUI) Capture tab (Display Scale Bar/Scale Bar Settings): Display Scale Bar button switches 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.
- Page 123 Appendix B Graphical user interface (GUI) Capture tab (Show Cell Count): Allows you to perform cell counts using the Auto Count or Manual Count tools and to measure the confluence and transfection efficiency of your culture from the captured image using the Cell Culture tools. •...
- Page 124 Appendix B Graphical user interface (GUI) Capture tab • Manual Count: Allows you to manually mark items onscreen using up to 6 separate labels and keep a running tally of the counts with percentages for each label. See “Manual count controls” on page 129 for Manual Count controls.
- Page 125 Appendix B Graphical user interface (GUI) Capture tab • Cell Culture: Allows you to measure the confluence of your culture and calculate the transfection efficiency. See “Cell culture – confluence controls” on page 130 for Confluence controls and “Cell culture – transfection efficiency controls” on page 131 for Transfection Efficiency controls.
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Page 126: Review Tab
Appendix B Graphical user interface (GUI) Review tab Review tab 1. Sign In/User: Allows you to sign in to and sign out of your Connect account. 2. Image file name: File name of the image displayed in the Viewing area. 3. Zoom slider: Zooms in and out of the image. The zoom range is 100% to 1000%. 4. - Page 127 Appendix B Graphical user interface (GUI) Review tab 12. Image preview/Image list: Displays the preview or list of the image files and subfolders in the current folder. 13. Image Properties: Displays the metadata for the selected image file. 14. Export: Allows you to export the currently selected folder to a storage device. 15.
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Page 128: Auto Count Controls
Appendix B Graphical user interface (GUI) Auto count controls Auto count controls 1. Analysis tools: Allows you to toggle between Auto Count, Manual Count, or Cell Culture (Confluence and Transfection Efficiency) tools for image analysis. 2. Select Target and Background: Allows you to select representative target objects and background areas for Auto Count. -
Page 129: Manual Count Controls
Appendix B Graphical user interface (GUI) Manual count controls 10. Exit: Exits the Auto Count tool and displays the Review tab. 11. Save: Saves the analysis results as an image in the selected file format (see “Save analysis results” on page 92 for more information). Manual count controls 1. -
Page 130: Cell Culture - Confluence Controls
Appendix B Graphical user interface (GUI) Cell culture – confluence controls 8. 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 “Batch analysis” on page 94 for more information). -
Page 131: Cell Culture - Transfection Efficiency Controls
Appendix B Graphical user interface (GUI) Cell culture – transfection efficiency controls 7. Transfection Efficiency: Expands or hides the controls for the Transfection Efficiency tool. The Transfection Efficiency tool is inactive until the confluence measurement is completed. 8. % Confluence: Displays the percentage of the area covered by cells in the image, based on the selected target and background areas and sensitivity. -
Page 132: Settings
Appendix B Graphical user interface (GUI) Settings 5. Fluorescence Channel: Toggles the display of the fluorescence channel. 6. Transmitted Light Channel: Toggles the display of the transmitted light. 7. Threshold: Adjusts the fluorescence threshold value. Only the cells that express above the set threshold are used in the tranfection efficiency calculation. - Page 133 Appendix B Graphical user interface (GUI) Settings 1. Objectives: Allows you to assign and unassign objectives on the objective turret, and to calibrate objective magnification. 2. Display: Allows you to calibrate color channel lighting (White Balance Calibration) and to assign saturated pixel colors in the Fluorescence, Transmitted, and RGB Transmitted channels. 3.
- Page 134 Appendix B Graphical user interface (GUI) Settings 5. Network: Allows you to connect to a Wi-Fi network and to map network drives. 6. Service: Displays the EVOS M5000 hardware and software information, allows you to update ™ the EVOS M5000 firmware and software from Connect, Connect-based platform, or using a USB ™...
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Page 135: Appendix C System Overview
System overview Technical specifications Note: Technical specifications of the EVOS M5000 Imaging System are subject to change without ™ notice. For the latest product information, see the product page (thermofisher.com/evos). Physical characteristics Dimensions (W × H × D): 46 × 59 × 46 cm (18 × 23 × 18 in) Weight: 16 kg (35 lbs.) Footprint: Approximately 92 cm ×... -
Page 136: Operation Principles And Technical Overview
Appendix C System overview Operation principles and technical overview Captured images: 16‑bit monochrome (12‑bit dynamic range) TIFF or PNG; 8‑bit per RGB channel TIFF, PNG, or JPG. Output ports: 4‑pin power input port (12 VDC, 5 A), 2 USB 3.0, 2 USB 2.0 Networking capability: Connection through Windows /SMB network via an Ethernet cable connection ™... - Page 137 Appendix C System overview Operation principles and technical overview (continued) Light cube 5, Alexa Fluor 647, Alexa Fluor 660, DRAQ5 ™ ™ ™ ™ ™ 5.5, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor ™ ™ ™ ™ ™ 7, IRDye 800CW ™...
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Page 138: Appendix D Evos ™ Image Analysis
EVOS image analysis ™ EVOS image analysis ™ EVOS Image Analysis is an easy to use online application for storing, viewing, and analyzing images ™ from your EVOS M5000 Imaging System. ™ After you have signed in to your Connect account from the EVOS M5000 Imaging System, you can ™... -
Page 139: Gallery
Appendix D EVOS image analysis ™ Gallery Gallery The EVOS Image Analysis Gallery contains the images saved from your EVOS M5000 Imaging ™ ™ System to your Connect account. You can view the images and image metadata in this tab and select individual images for further image adjustment and analysis. -
Page 140: Adjust Tab
Appendix D EVOS image analysis ™ Edit image Adjust tab The Adjust tab allows you to zoom in to your selected image, adjust brightness and contrast values, and add line measurements and annotations. The measurement and annotation functions of the EVOS ™ Image Analysis application are identical to the Analysis tools of the EVOS M5000 Imaging System ™... -
Page 141: Analyze Tab
Appendix D EVOS image analysis ™ Edit image Analyze tab The Analyze tab allows you to use Auto Count and Manual Count. The Auto Count and Manual Count functions of the EVOS Image Analysis application are identical to the count tools of the EVOS ™... -
Page 142: Appendix E Glossary
Glossary Software controls and functions • Autofocus: The Autofocus button triggers the microscope to move through a Z-axis range (defined by the triangle sliders in the Coarse focus control) and search for the optimal focal plane. The display shows a live view as the objective sweeps through the defined Z-axis range. Autofocus uses an image-based focusing algorithm to achieve the desired focus. -
Page 143: Image Files
Appendix E Glossary Image files • Sync Z: The Sync Z feature is designed to enable the user to focus on a single channel while the other channels are adjusted automatically. This is accomplished by first adjusting the focus of each channel individually with the Sync Z feature unchecked. This will establish unique offsets for each channel. - Page 144 Appendix E Glossary Image files • Pseudocolor image: A pseudocolor image is a modified version of a grayscale image that uses a single color (for a fluorescence channel) or multiple distinct colors (for a fluorescence overlay image) to represent the image as it would be seen through a microscope's oculars. The chosen color(s) typically correspond to the emission wavelength of the fluorescence channel used to capture the image.
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Page 145: Microscope Controls And Optics
Appendix E Glossary Microscope controls and optics Microscope controls and optics • Light cube: A light cube is a proprietary optical module that can be easily installed by the user in an EVOS fluorescence microscope or fluorescence-capable Countess cell counter. Each ™ ™... -
Page 146: Stage View Feature
Appendix E Glossary Stage View feature Stage View feature • Crosshairs: The crosshairs are a visual aid that provides real-time information about the position of the objective lens relative to the stage. The crosshairs can be seen on both the small vessel map in the Stage View tab and the larger vessel map in the Find Target window. -
Page 147: Appendix F Safety
Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. - Page 148 Appendix F Safety Symbols on this instrument (continued) Symbol and description CAUTION! Potential biohazard. CAUTION! Ultraviolet light. Symbole et description MISE EN GARDE ! Risque de danger. Consulter le manuel pour d’autres renseignements de sécurité. MISE EN GARDE ! Risque de choc électrique. ...
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Page 149: Location Of Labels
Appendix F Safety Symbols on this instrument Location of labels Labels and location Figure 5 Instrument information labels (back panel) Instrument information label Technical support information Instrument passwords label Control and connection symbols Symbols and descriptions On (Power) Off (Power) Protective conductor terminal (main ground) Alternating current Conformity symbols Conformity mark... -
Page 150: Safety Information For Instruments Not Manufactured By Thermo Fisher Scientific
Appendix F Safety Safety information for instruments not manufactured by Thermo Fisher Scientific (continued) Conformity mark Description Indicates conformity with European Union requirements. Indicates conformity with Australian standards for electromagnetic compatibility. Indicates conformity with the WEEE Directive 2012/19/EU. CAUTION! To minimize negative environmental impact from disposal of electronic waste, do not dispose of electronic waste in unsorted municipal waste. -
Page 151: Physical Injury
Appendix F Safety Instrument safety Physical injury CAUTION! Moving and Lifting Injury. Improper lifting can cause painful and permanent back injury. Things to consider before lifting or moving the instrument or accessories: · Depending on the weight, moving or lifting may require two or more persons. ·... -
Page 152: Electrical Safety
Appendix F Safety Instrument safety Electrical safety WARNING! Ensure appropriate electrical supply. For safe operation of the instrument: · Plug the system into a properly grounded receptacle with adequate current capacity. · Ensure the electrical supply is of suitable voltage. · Never operate the instrument with the ground disconnected. Grounding continuity is required for safe operation of the instrument. -
Page 153: Cleaning And Decontamination
Appendix F Safety Safety and electromagnetic compatibility (EMC) standards Cleaning and decontamination CAUTION! Cleaning and Decontamination. Use only the cleaning and decontamination methods that are specified in the manufacturer user documentation. It is the responsibility of the operator (or other responsible person) to ensure that the following requirements are met: ·... -
Page 154: Safety Standards
Appendix F Safety Safety and electromagnetic compatibility (EMC) standards Safety standards Reference Description EU Directive 2014/35/EU European Union “Low Voltage Directive” EN 61010-1 Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1: General requirements UL 61010-1 CAN/CSA C22.2 No. 61010-1 EN 61010-2-081 Safety requirements for electrical equipment for measurement, control and laboratory use –... -
Page 155: Environmental Design Standards
Appendix F Safety Safety and electromagnetic compatibility (EMC) standards Environmental design standards Reference Description Directive 2012/19/EU European Union “WEEE Directive”—Waste electrical and electronic equipment Directive 2011/65/EU and European Union “RoHS Directive”—Restriction of hazardous substances in electrical Commission Delegated and electronic equipment Directive 2015/863 SJ/T 11364-2014 “China RoHS”... -
Page 156: Chemical Safety
Appendix F Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions: · 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. -
Page 157: Biological Hazard Safety
Appendix F Safety Biological hazard safety · Veiller à utiliser des récipients à déchets primaire et secondaire. (Le récipient primaire contient les déchets immédiats, le récipient secondaire contient les fuites et les écoulements du récipient pri maire. Les deux récipients doivent être compatibles avec les matériaux mis au rebut et conformes aux exigences locales, nationales et communautaires en matière de confinement des récipients.) ·... -
Page 158: Appendix G Documentation And Support
Documentation and support Customer and technical support Visit thermofisher.com/support for the latest service and support information. • Worldwide contact telephone numbers • Product support information – Product FAQs – Software, patches, and updates – Training for many applications and instruments •... - Page 160 EVOS_M5000_Imaging_System_MAN0017563-v1-GUID-F34BD420-299B-4855-A319- C1EA0A40CE68-2023/06/20 22:18:48 en 23:34:33.218Z thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 16 November 2023...
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