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EVOS

™

M5000 Imaging System User Guide

Analyze saved images

Analyze and
annotate saved

images

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

The analysis and annotations tools function in the same way in the Capture and
Review tabs:

•

Click the

Histogram

button to open the Intensity Histogram plot.

The Intensity Histogram plot shows the Pixel count vs. Intensity data
of the image displayed in the Viewing area (see page 27).

•

Click the

Measurement and Annotations Tools

button (the arrow on

the Show Measurements and Annotations split button) to open the
measurement and annotations tools. For instructions on how to add
and show measurements and annotations to your images, see page 28.

•

Click the

Analysis

button to display the

Auto Count

Manual Count

and

Confluence

options in the Review tab.

For instructions on how to perform an Auto Count, see page 30.

For instructions on how to perform a Manual Count, see page 34.

For instructions on how to measure the confluence of your cell culture
sample, see page 36.

Содержание EVOS M5000

Страница 1: ...or Research Use Only Not for use in diagnostic procedures EVOS M5000 Imaging System For Fluorescence and Transmitted Light Applications Catalog Number AMF5000 Publication Number MAN0017563 Revision C...

Страница 2: ...yback EVOS Onstage Incubator and EVOS Image Analysis sections update GUI section and the screens throughout the document A 0 07 August 2018 New user guide for the EVOS M5000 Imaging System Important L...

Страница 3: ...apture images 17 Overview 17 Capture images in a single channel 18 Capture images in multiple channels 24 4 Analyze and annotate captured images 25 Display settings and analysis tools 25 Configure dis...

Страница 4: ...ight cubes 74 Change the objectives 75 Calibrate the objectives 76 Appendix A Troubleshooting 78 Image quality issues 78 Software interface issues 79 Mechanical issues 79 Appendix B Graphical user int...

Страница 5: ...6 Symbols on instruments 107 Safety labels on instruments 109 General instrument safety 110 Chemical safety 111 Chemical waste safety 112 Electrical safety 113 Physical hazard safety 114 Biological ha...

Страница 6: ...curate 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 WARNI...

Страница 7: ...S M5000 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 BMP JPG PNG and TIFF formats or co...

Страница 8: ...etwork drives USB 3 0 flash drive 16 GB for image storage and preloaded with user documentation EVOS Vessel Holder Two 25 75 mm slides Cat No AMEPVH001 EVOS Vessel Holder Universal Cat No AMEPVH009 EV...

Страница 9: ...slider slot Objective selection wheel Condenser head Vessel holder Mechanical X Y stage Objective Stage X axis positioning knob Phase annuli selector Stage Y axis positioning knob Stage positioning kn...

Страница 10: ...onitor Network port Mechanical X Y stage USB A 2 0 ports 4 Focusing knobs USB 3 0 port 1 Power switch Stage X axis positioning knob 4 pin power input port 12 VDC 5 A Stage Y axis positioning knob Disp...

Страница 11: ...rm for image storage and analysis with the EVOS Image Analysis application Viewing area Displays the sample Capture tab Contains the controls for image capture Review tab Allows you to review and anno...

Страница 12: ...a run and cause a decrease in instrument performance The EVOS M5000 Imaging System should be installed away from direct light sources such as windows Ambient light can enter the imaging path and affec...

Страница 13: ...instrument 1 Open the shipping box and remove the accessory box 2 Carefully lift the instrument out of the box by grasping it firmly with both hands under the support arm 3 Place the instrument on a...

Страница 14: ...tage back to obtain access to the light cube shipping restraint which is centered under the back of the stage Note The light cube shipping restraint is secured with the light cube tool to the blank li...

Страница 15: ...uture use in the accessories box provided with your system Always re secure the X Y stage with the stage lock pin and re install the light cube restraint before moving the instrument IMPORTANT Before...

Страница 16: ...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 l...

Страница 17: ...can connect the EVOS M5000 Imaging System to a network via an Ethernet cable or Wi Fi adaptor and save captured images directly to shared folders on the network You can also connect to your Connect ac...

Страница 18: ...o open the Connect to the Cloud dialog 2 Enter your Connect user name and password then click Sign In When you connect to your Connect account the Sign In button changes to display your user name 3 To...

Страница 19: ...ing the sample setting optimal focus and capturing and saving images are performed in the Capture tab which is the first screen after start up Workflow Select objective and light source Adjust brightn...

Страница 20: ...for low magnification objectives Olympus 4 PH 4 10 Used for medium magnification objectives EVOS 4 10 PH 20 40 Used for high magnification objectives EVOS 20 40 PH Brightfield phase contrast off The a...

Страница 21: ...e 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 captu...

Страница 22: ...t page 7 Click Enable focus knob then turn the focusing knob until the region of interest is in focus 3 Repeat the focus procedure for each channel you want to capture 4 If you want to preserve the Z...

Страница 23: ...e Image Display Settings button again to collapse the controls Find the region of interest 1 Use the X axis and Y axis positioning knobs page 7 to move the sample stage and bring the region of interes...

Страница 24: ...remains selected as indicated by the blue line underneath the channel button 2 Click Capture to acquire the image The viewing area shows the newly captured image A thumbnail of the captured image is d...

Страница 25: ...imposed 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 5 To...

Страница 26: ...images captured in each selected channel A thumbnail of the captured image is displayed for each captured channel in this example DAPI GFP and Texas Red IMPORTANT Captured images are stored in the mem...

Страница 27: ...elect Save individual channels option when saving captured images see page 39 Configure display settings Configure image display settings 1 Click the Image Display Settings button to expand the contro...

Страница 28: ...e 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 Display scale bar 1 Click the Scale Bar button to sup...

Страница 29: ...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 7 Click Scale Bar Settings button again to save y...

Страница 30: ...3 If needed change the Color and Thickness of the annotation to make it more visible over the image 4 If desired select to display the Dimensions Area or Perimeter information for the annotation from...

Страница 31: ...single 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 assig...

Страница 32: ...ly when you are counting from the Capture tab Available options depend on the LED light cubes installed and you can only select channels that contain captured images In this example only DAPI and TX R...

Страница 33: ...r 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...

Страница 34: ...unts the objects based on your criteria Viewing area identifies the objects that were counted with colored circles and the Object Count field displays the number of objects included in the count 8 To...

Страница 35: ...per 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 11...

Страница 36: ...n the Viewing area for manual count You can select multiple channels provided that they contain captured images In this example DAPI and TX Red channels contain captured images and both are selected f...

Страница 37: ...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 all tags for a label check the Delete box for the label...

Страница 38: ...e 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...

Страница 39: ...gets and 5 background areas Increasing the number of targets and background areas improves the accuracy and reproducibility of the confluence measurements 6 Refine the sensitivity of the Confluence me...

Страница 40: ...s made in the Auto Count tool For Manual Count the screenshot shows the labels counts and percentages as shown in the Manual Count tool For Confluence the screenshot shows an image of the culture sele...

Страница 41: ...JPG PNG and TIFF 3 Select Save to cloud to save a copy of your captured image to your Connect account To save the copy of your image to another account in the Connect platform click Switch Account th...

Страница 42: ...en in addition to the captured image Leave the Save screenshot option unselected to save only the captured image Note Save screenshot function allows you retain an image of the annotations and image d...

Страница 43: ...lect Folder 4 Enter a Base Filename and Starting number for saved images Note The default base filename is Image Base filename is appended by a numerical suffix and channel name Starting number determ...

Страница 44: ...be saved recalled and edited Run a time lapse routine Run a new time lapse routine 1 On the Capture tab click Automate then select Time Lapse to open the Time Lapse tool 2 Select the Capture Channels...

Страница 45: ...fore using the EVOS Onstage Incubator in your time lapse experiments ensure that The EVOS Onstage Incubator has been set up for use page 95 The gas inputs have been configured page 102 The oxygen sens...

Страница 46: ...locate the boundaries and the In Focus position of the Z Stack b To accept the Current Z as the In Focus position click Set To change the In Focus position move the Coarse and Fine focus sliders on t...

Страница 47: ...stem calculates the number and Z positions of the images based on your entry Manually Set Number of Images Select to enter the Number of Images Z distance in m between focal planes in the text box The...

Страница 48: ...et 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 Images are collected at the end of an interval For...

Страница 49: ...y 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 expe...

Страница 50: ...tton navigate to the desired destination folder for the Time Lapse images then click Select Folder 19 Select the File Type PNG or TIFF 20 If desired select Save Time Lapse Movie to create a video from...

Страница 51: ...he Time Lapse routine The system provides information about the progress of the Time Lapse routine as it captures the images based on the routine specifications 24 When the Time Lapse routine is compl...

Страница 52: ...hich you can then recall and run with a new sample 1 On the Capture tab click Automate then select Time Lapse to open the Time Lapse tool 2 Click Load File to open the Choose Routine dialog 3 Select t...

Страница 53: ...our 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...

Страница 54: ...ing System User Guide 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 Pl...

Страница 55: ...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 6 To zoom in on the image use the Zoom slider 7 To anno...

Страница 56: ...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 Capture Z stack images 1 On the Capture tab click Automate then selec...

Страница 57: ...oundary of the Z Stack then click Set for Bottom Note You can click the Z number button for the Top In Focus and Bottom positions to jump to those positions along the Z axis 7 Define the Z Stack param...

Страница 58: ...age to generate a projection image When selected the system uses the most in focus pixels from images captured at different focal planes to generate a composite in focus image Each image in the Z Stac...

Страница 59: ...EVOS M5000 Imaging System User Guide 57 13 When the Z Stack routine is completed the Review tab opens to the files captured in the routine...

Страница 60: ...e 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 EVOS Image Analysis page 10...

Страница 61: ...tings and to analyze and annotate captured images Hover the pointer over the Viewing area to reveal the buttons for Display Settings and Analysis Tools The display tools function in the same way in th...

Страница 62: ...e image displayed in the Viewing area see page 27 Click the Measurement and Annotations Tools button the arrow on the Show Measurements and Annotations split button to open the measurement and annotat...

Страница 63: ...Adjust White Balance for the color camera page 64 and set saturated pixel options page 65 General Set general instrument options such as savings options for TIFF files focus wheel action Saved Settin...

Страница 64: ...rret position For instructions on how to remove an objective see Change the objectives page 75 1 Go to the Settings Objective Selection and Calibration tab 2 Find the newly installed objective in the...

Страница 65: ...jective you want to calibrate then click Calibrate Objective 3 Follow the onscreen instructions to calibrate the objective magnification for the selected objective For more information see Calibrate t...

Страница 66: ...tab then click White Balance Calibration to open the White Balance Calibration tool 2 Select a 20X or higher objective then set the phase ring to Brightfield using the Phase annuli selector page 7 3...

Страница 67: ...vides a visual aid for optimal illumination when adjusting the brightness settings 1 Go to the Settings Visuals tab then click the Highlight Saturated Pixels checkbox 2 To assign a specific color to t...

Страница 68: ...he Settings General tab 2 If you want to assign specific names to each saved setting select Prompt for a name when adding Saved Settings 3 If you want to see a confirmation dialog before applying save...

Страница 69: ...hared folders on the network You can also connect to your Connect account Thermo Fisher s cloud based platform to store your image files and analyze them with the EVOS Image Analysis application Conne...

Страница 70: ...k Drive Map Network Drive dialog opens 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...

Страница 71: ...drive and folder or connected to the website click Finish Note When Reconnect at logon is checked and a successful connection is established the system will attempt a reconnection to any previously c...

Страница 72: ...view the Service tab click the Settings button then select Service Copy error logs 1 On the Service tab click Copy Error Logs The instrument automatically saves a copy of the error logs in the previo...

Страница 73: ...ys the update progress When the update is complete the system restarts and the M5000 software automatically boots up again Update from USB 1 Download the latest software from thermofisher com evos to...

Страница 74: ...ty 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...

Страница 75: ...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 t...

Страница 76: ...ight cube you want to change 2 Move the stage back to allow access to the light cube which is centered under the back of the stage 3 Use the light cube tool to loosen the two slotted screws white arro...

Страница 77: ...o 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...

Страница 78: ...nless they are reinstalled after removal from the instrument Calibrate objective magnification Calibration procedure requires the use of the EVOS M5000 calibration slide supplied with the EVOS M5000 I...

Страница 79: ...e 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 th...

Страница 80: ...t lies flat on the stage ensure that the sample s thickness is even Be sure vessel holder is mounted flat with respect to stage Difficulty focusing on coverslipped sample on standard slide Place the s...

Страница 81: ...icked Click capture first it is only possible to save an image that is captured Unable to connect to network Verify physical cable connections confirm the Ethernet jack is active for Ethernet connecti...

Страница 82: ...e about the current objective click 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 im...

Страница 83: ...u can limit the autofocus algorithm to the selected region along the Z axis Saved Settings Allows you to save and edit Z axis focus positions and brightness settings for future use Press the button to...

Страница 84: ...ea on and off Grid Settings allows you to set the grid size 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...

Страница 85: ...lows 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 34 Confluence Allows you to select up to 5 referenc...

Страница 86: ...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 al...

Страница 87: ...Opens the Incubator Controls which allow you to control the EVOS Onstage Incubator and monitor its status during your experiments Save Opens the Save dialog which allows you to select a save location...

Страница 88: ...play size of the folders or image files Browse Allows you to navigate to the folder containing your saved images and to select the image to display in the viewing area Currently selected folder or ima...

Страница 89: ...lows you to assign and unassign objectives on the objective turret and to calibrate objective magnification Visuals Allows you to calibrate color channel lighting White Balance Calibration and to assi...

Страница 90: ...Wi Fi network and to map network drives Service Displays the EVOS M5000 hardware and software information allows you to update the EVOS M5000 firmware and software from Connect Thermo Fisher s cloud b...

Страница 91: ...ence and transmitted light brightfield phase contrast Objective turret 5 position front mounted control Objectives not included Wide selection of high quality LWD and coverslip corrected objectives Co...

Страница 92: ...and filters In addition to the channel dedicated to the transmitted light from the condenser for brightfield contrast applications the EVOS M5000 Imaging System can accommodate up to four fluorescent...

Страница 93: ...me software and user interface that controls the EVOS M5000 Imaging System Standard items included Stagetop Environmental Chamber Control unit Master Stage Plate also available separately as EVOS Onst...

Страница 94: ...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 ra...

Страница 95: ...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 temperature Control unit Hose heater connector Heated ho...

Страница 96: ...r 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 M5000 Imaging System see A...

Страница 97: ...ate 1 Remove the X Y stage base plate from the X Y stage by unscrewing the four 3 0 mm screws indicated by red arrows on the base plate If necessary unscrew and remove the vessel holder adaptor before...

Страница 98: ...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...

Страница 99: ...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 completel...

Страница 100: ...e 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 regula...

Страница 101: ...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...

Страница 102: ...has been calibrated see Calibrate oxygen sensor page 103 Turn ON the EVOS Onstage Incubator 1 Turn ON the EVOS M5000 Imaging System as described on page 15 2 Turn ON the power switch to the EVOS Onst...

Страница 103: ...ified atmosphere in the incubator chamber Incubator status will change to Running and the control panel will display the Target and Actual values for Temperature Humidity CO2 and Oxygen 8 Click Close...

Страница 104: ...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 9...

Страница 105: ...gen Sensor Calibration to open the Incubator Setup window 3 Verify that your gas connections have been correctly configured 4 Verify that the Oxygen has been set correctly If not enter the correct val...

Страница 106: ...m However you need a current Connect account to sign in To create your Connect account online go to thermofisher com connect Note This sections provides an overview of the EVOS Image Analysis applicat...

Страница 107: ...d 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 25 Ana...

Страница 108: ...f 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 prac...

Страница 109: ...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...

Страница 110: ...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 pre...

Страница 111: ...mination des d chets DANGER High voltage DANGER Haute tension WARNING To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer...

Страница 112: ...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 tw...

Страница 113: ...the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles General safety guidelines To minimize the hazards of chemicals Read and...

Страница 114: ...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 additi...

Страница 115: ...place 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 e...

Страница 116: ...infectieuses Suivre tous les r glements municipaux provinciaux provincial et ou nationales en vigueur Porter des lunettes de protection appropri des v tements et des gants In the U S U S Department o...

Страница 117: ...CSA CSA America ANSI ASME ASSE ASTM NSF and UL European safety and EMC standards The CE Mark symbolizes that the product conforms to all applicable European Community provisions for which this marking...

Страница 118: ...m support for the latest in services and support including Worldwide contact telephone numbers Product support including Product FAQs Software patches and updates Training for many applications and in...

Страница 119: ...thermofisher com support thermofisher com askaquestion thermofisher com 14 January 2020...

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