Home

IncuCyte™ ZOOM Users Manual

1. Numeric Precision co C1 Ci 3 Grid Lines Color Both CY CX C None Gidin front of data Undo Zoom Maximize Customization Dialog Export Dialog Figure 19 Graph Window displaying Customization Features Look at the graph in Figure 19 Because Region Custom Group Replicates was selected two traces appear that represent the mean of all replicates in the selected wells When multiple wells traces appear on a single graph each one is listed at the top of the window The wells sectors will be assigned default titles indicating the vessel label and the well A1 A2 A3 etc unless a plate map was applied in the Properties of the vessel When a plate map is linked to a vessel titles will be defined by well information within the plate map 7 6 1 1 Graph Metric Window The Graph Metric Window aides Users in defining the metrics and output of the generated data Time Plot Select Metric When plotting kinetic traces select the radio button for Time Plot Depending upon the Analysis Job performed Basic Analyzer Angiogenesis Scratch Wound or NeuroTrack different metrics will be available for graphing and exporting NOTE Focus Position Phase Exposure Time Uncorrected Red Image Mean and Uncorrected Green Image Mean are diagnostic tools used to confirm Page 49 that the software is functioning within the specified parameters These Metrics will not be relevant to t
2. The Utilities drop down menu allows Users to export images movies or an image set refer to section 7 7 Exporting Images and Movies The View drop down menu allows Users to display or remove vessel properties from the vessel image located at the top center of the screen Time Tree The Time Tree lists only the times and dates for all completed scans for that vessel By selecting a time the corresponding image will be displayed View images from a specific scan time by clicking directly through the Time Tree or by using the forward or back buttons located below the Time Tree Vessel Image The selected flask dish or microplate is represented to the right of the Time Tree Use the representative vessel display to view the images in different sectors wells by selecting them with the mouse Image Tab Displays the image from the scan time selected from the Time Tree and the sector well selected from the vessel display Under the Image display tab Users are able to select de select or adjust the image channels Adjust Image Controls Use the brightness and contrast sliders to adjust image quality for the Phase channel The Fluorescence sliding Weight bars allow the User to adjust the display characteristics of fluorescence components within an image Adjusting the display settings will affect which components of an image will be displayed as fluorescent and also the brightness of those fluorescent images relative to
3. Apply button had been selected Place Tray and Vessel in IncuCyte Place the appropriate tray refer to section 7 8 1 Selecting and Placing Trays and vessel in the pre determined position within the IncuCyte Allow time for the vessel to equilibrate to the temperature of the incubator prior to initiating scans approximately 15 20 minutes It is important to allow the vessel to equilibrate to the temperature of the incubator prior to initiating scans Condensation in the vessel may cause focal issues with the microscope rA e Terminating Scan Times and Deleting Vessels 1 To terminate scanning right click on the Scan Bar to be deleted and either select delete for that specific scan time or delete all to remove all scan times Select the Apply button at the bottom of the screen A scan can also be manually terminated by pushing the Stop button on the front of the IncuCyte Pushing the Stop button will abort the scan that is ongoing at that time However if scanning is terminated in this way the IncuCyte will start scanning again at the next scheduled scan time The ONLY way to terminate all future scans is by deleting all the Scan Bars and then selecting Apply 2 To remove a vessel from the schedule simply highlight the desired vessel and click on the Remove Vessel button Select the Apply button on the bottom of the screen Page 33 N 7 3 2 Scan on Demand In addition
4. Scan button Finding and Viewing Scanned Vessels After scans have been generated there are two ways to search and view your vessel of interest using View Scans option or the Search option Once you have located your vessel using either the View Scans or Search tools simply double click on the vessel or use the View Vessel button on the lower right corner of the window 7 4 1 Finding Scanned Vessels 7 4 1 1 View Scans Tab When the ZOOM IncuCyte software is launched it opens by default to the View Scans screen The first time the software is opened the screen will be empty However once scans have been performed the software will open to show the very last scan that was completed as well as a Time Tree that lists the times and dates for all completed scans NOTE The View Scan screen is recommended for recently scanned vessels 1 Click through the Time Tree to find completed scans generated on the device 2 The center of the View Scan screen will display the Drawer Setup for that particular scan time Highlight the vessel of interest to display vessel properties to the right side of the screen 3 Once your vessel of interest is located either double click on the vessel or highlight the vessel and click on the View Vessel button in the lower right hand corner of the screen Page 35 N ESSEN BIOSCIENCE 7 4 1 2 Search Tab Scanned vessels can also be found by using the Searc
5. Z Al All the sectors within the flask will be combined into a single trace Z None any of the sectors will be grouped No error bars are available for graphs of single sectors Region Single Sector If the Region Single Sector is selected a new drop down menu will appear Choose the desired sector for graphing Error bars are not available for graphs of a single sector Page 52 Graph Export Histograms of Processed Jobs It is possible to generate histograms from both Phase contrast and fluorescent data see Figure 20 Histograms can only be generated for a single time point so select Single Time instead of Time Range and then select the Histogram radio button If the Histogram radio button is selected without first choosing Single Time the selection will automatically be reset from Time Range to Single Time Metrics available for Histograms are based on the Analysis Job Performed Basic Analyzer Phase Object Area Fluorescent Object Area Fluorescent Object Mean Intensity Fluorescent Object Integrated Intensity Angiogenesis Network Area Network Length Tube Width Uniformity Nucleus Area Nucleus Mean Intensity Nucleus Integrated Intensity Migration Invasion Wound Object Area NeuroTrack Cell Body Cluster Area Nucleus Area Nucleus Mean Intensity Nucleus Integrated Intensity These same Metrics are available for Time Plots except that in the case of Time Plots the plot represents average values over time Histogr
6. button 2 The vessel type Scan Setup and Properties have now been duplicated NOTE Users have the option to modify the Properties or Scan Setup Page 31 N ESSEN BIOSCIENCE 7 3 1 5 Timeline After completing vessel properties Users must set acquisition times using the Timeline displayed at the top of the screen The Timeline represents a 24 hour repeating time period Once all the scans for the first 24 hour period have been completed the IncuCyte will automatically start over for the next 24 hour period This pattern will repeat until scanning is terminated by the User ile The current time will be displayed as a dotted blue line within the Timeline Figure 14 To create a scan time left click on the Timeline A gray Scan Bar will now be displayed Hold the mouse over the Scan Bar to display the exact scan start time and scan length 2 The scan start time can be adjusted by right clicking on the bar to adjust the start times by small increments or by left clicking and dragging the bar to move the start time by large increments 3 To set multiple scans at regular intervals right click on the gray Scan Bar and select Set Interval Select the interval times desired for your assay e g every 30 minutes or every 3 hours Scan Bars will now appear for each scan time Refer to Figure 14 4 Right click on the gray Scan Bar to delete a single scan or all scans To delete a scan you may also left click and drag the S
7. IncuCyte ZOOM Users Manual 2013A Essen BioScience Inc www EssenBioScience com Page 1 N Table of Contents IncuCyte ZOOM Users WANA visi iccatersavcssnccccaeseatwscabcanicenseseescsnoseecanave aeuctboesdacndeaaudoassencendeadadarwishedadatenwes 1 A 0 S eee ee en EA E A eee 1 Chapter 1 A QM a seer Sena ntsc ve gu ec scp soecrasnn gators osenssenspenoeeecubeiesnee snake senatseeuacteukeseueseeceneaanan 5 1 1 ESTA eh OTRO VV E y sarees cercectecc cs tues wes EE ect eseieee 5 1 2 EM CIUSIVE REIN CICS ai A E E E E E E E 5 1 3 Warnings and DISCIAIMELS cccccessccccesseccccenecccceusececsuecccceescceseusececsuseceesenecessunecetsuneceessneeesseneses 5 Chapter 2 Getting to know your INCUCYte ZOOM ssssssesesssersssreresrrrsssreresrrerssrersssrerssrereserersseereserereseeresene 6 2 1 I CS TVS Sa sce teens E E E E eee deeccesneet anes 6 2 2 incucyte ZOOM COT IC URGING seriinin ER 6 2 3 Seya HOMILY e E E E 6 2 4 incuCcyte ZOO IVS DEC IMC AION seisine E 7 2 5 Unpacking and Checking the INCUC le Messenien arine E REE E 8 Chapter 3 Installation of the IncuCyte ZOOM HardWare essssssesessesesresesrrrsssreresrresssreresrrressrereserersseerese 10 3 1 IncuCyte ZOOM Hardware Features csscccssscccsececseeccscceeenceceuececeneceseeeeeenceseeeceseuecessueeeeees 10 3 2 Removing TNE SNIP DINE PIM escerai AS 12 3 3 Installing the Filter MOCUIE cccccesccccsssecccceseccceeseceesesececseseceeseeecceee
8. To establish new User accounts click on the Administer IncuCyte task bar in the upper left corner of the main window The available Administrator functions are organized within a series of tabs Select the Accounts tab to access password and accounts information The Change Password menu allows Users to set and change their own passwords This function is available to Users at all permission levels The Create User and Delete User functions however are only available to Users with the Administrator permission level New Users must have a unique User name Passwords are case sensitive and new Users should be encouraged to change their password the first time they log on When creating a new User it is important to select an appropriate Permission Level Users with an Administrator Permission Level have all of the functions of the IncuCyte control program available to them A User with a User Permission Level is able to change his her password view device information configure the device to collect data and perform all post collection analysis view plot annotate etc A User with Limited User Permission level can perform all tasks that the User can do except configuring the device to collect data The Guest Permission level is one step below the User level in that guests cannot configure the device to collect data The software functions available at the various permission levels are summarized in Table 1 Page 2
9. e Choose Sequence Type Set of Individual Images e Select the desired Image Type e Enter a Name Prefix e Click the Export button Export Image Set Multiple Images with Customization Following the same setup guidelines discussed in the previous section Export Image Set Multiple Images it is possible to customize the set of exported images by customizing the image cropping and scaling and or adding legend and timestamp information to each image To accomplish this the Sequence Type must be set to Image set with customization Once this Sequence Type is selected click on Image Designer Crop Scale Legend etc above the Export button at the bottom right of the Movie Image Set Export window A new window appears that is virtually identical to the Export Designer As discussed in that section the designer is used to interactively crop and scale the image and to add a legend or timestamp to each image if desired There is an important preview tool found in the Export Designer and we strongly recommend that the designer and previewer be used prior to launching a full image set export with customization Page 71 N ESSEN BIOSCIENCE 7 7 1 4 Drag and Drop Images It is also possible to Drag and Drop Images directly from the Vessel View Screen directly into Microsoft Word Outlook Publisher PowerPoint Excel or WordPad To drag and drop images into other software be sure to
10. s ccceseeeeeees 21 5 3 COPS CCUG sorire E 21 5 3 1 Setting the Date and Time sseessssssssssrresssrrrssrrrrssrrressreresrrressreresrrressreresereessreresrrressrereseeresene 21 5 3 2 Establishing User ACCOUNTS nen nn ee ee 22 5 4 Remote Administration of the Controller ccsssssccccccccseessseecccccesseeeseeececeessaeasseeeeeeeeseuaeasess 23 Chapter 6 Calibrating the IncuCyte ZOOM SysteM cccccsssccccssseccccesecceeeesececeeseceeeeeeeceseusecessunecessenes 24 6 1 SV SUI Cail ON ANON ate csetatcas chen testes ones sac ctsns ace sasctean sean sanasaooaiienenuesaeacesentsenussanereaeanseseeecsaeeeaactsen dus 24 6 2 Puoro con eCa AGL OU eiutea cers cen seantensvcececen E E EER 26 6 2 1 FRU BOY RE es ontentae ta umeauatananteins eee saeaaacsnae nena ernaeet uke penpnivntectenaes 27 Chapter 7 Cell Culture Monitoring and Data AnallySis ccccccccssccccceseeceeeesececeeeceeeeeeeeeeeeecesseneceseunes 27 7 1 M OCI CON siaa E setae seid enna N E E E oeecewenceetieaar ett E 27 7 2 Te E E A OA A EE EE E A E E A EEE EEE 28 7 3 Scheduling Scans ana Loading Vessels iincc coscsadsvinssaiavonoduinsonesncdcosessievesaaicavianattieadonencesensnoteannnacseees 29 7 3 1 24 HOU RED SACS erna E E N 29 7 3 2 BO At OE Derna NG e E rasa aaa eowniaigenasneneamabeseteans 34 7 4 Finding and Viewing Scanned VEeSSEIS ccccccsssccccessecccssecccsesecccceecceeeuseceseuecessueeceeeeneceeteeeeetas 35 7 4 1 Finding Scanned Vesele nrnna AE 35 7 4
11. 3 Click on the Go button and select Yes to confirm that you want to start the warm up The warm up cycle will begin after a few seconds and will continue for approximately 45 minutes The green activity light on the front of the microscope next to the Eject Cancel button will be lit during the warm up A Warming up message will be displayed on the LCD in the top right corner along with information of time remaining until the warm up is complete 3 8 1 Auto Warm up In the event that the manual warm up process is omitted for instance if the IncuCyte ZOOM is temporarily removed from the incubator and the person replacing it forgets to run the manual warm up procedure afterwards then an auto warm up process will be initiated The auto warm up is NOT intended as a substitute for the manual warm up cycle but rather it is a backup process that occurs in those instances where the manual warm up is accidentally omitted When auto warm up starts the microscope is powered on and the front panel LED lights up until the camera achieves the requisite temperature If the microscope is placed into the incubator cold then the auto warm up process is automatically triggered Auto warm up keeps the camera powered on for up to 45 minutes until it reaches a temperature slightly above that of the standard incubator temperature Auto warm up cannot occur if the controller is not powered on The auto warm up cycle will be interrupted
12. ESSEN BIOSCIENCE When performing invasion and migration assays the Scratch Wound scan type can be utilized at any of the three objectives 4X 10X or 20X Additionally a Wide Mode scan type is available to ensure that both boundaries of the wound are imaged The Wide Mode scan type is mandatory when acquiring images using the 20X objective is optional when using the 10X objective and is not an available feature when scanning vessels at 4X To utilize the Wide Mode scan type select the check box located beneath the scan type in the Scan Mode task pane For better sampling and shorter scan times when performing migration and invasion assays use the 4X j objective at 1 image per well For better resolution and sufficient sampling use the 10X objective at 1 image per well with the Wide Mode option selected Cell Migration Invasion Module The Cell Migration Invasion Software Module is used to identify the Relative Wound Density of cells invading or migrating into a scratch created using the Essen Woundmaker Depending on your type of assay the IncuCyte ZOOM used in conjunction with the Cell Migration Invasion Module can measure the rate and extent of cell movement into that wound over time In the first step of the process the cell migration invasion algorithm analyzes each image and assigns a well specific wound mask that corresponds to the Initial Scratch Wound From here a Scratch Wound Mask fo
13. Timeline Within the Scan on Demand scheduler the 24 Hour Repeating scan schedule including all of the time bars will be faded to the background but will remain clearly visible 1 The current time will be indicated as in the 24 Hour Repeating scheduler but will be followed by the yellow Scan on Demand Time Bar NOTE If the Scan on Demand Time Bar is red this means that the Scan on Demand scan time overlaps with a scan time in the 24 Hour Repeating scheduler Clicking on the red Scan button will result in an error 2 Similar to the 24 Hour Repeating schedule the width of the time bar will correlate to the amount of time required to scan the vessel inserted into the Scan on Demand Tray NOTE The estimated time indicated by the width of the yellow bar to scan in the Scan on Demand mode is 20 longer than the estimated Page A4 N 7 4 ESSEN BIOSCIENCE time in the 24 Hour Repeating scheduler This additional time is required so that the User has time to replace the Scan on Demand vessel with the 24 Hour Repeating vessel Scan on Demand can be used even when all positions within the IncuCyte are scheduled under the 24 Hour Scheduler As long as there is either a period between regularly scheduled scans or a cooling the user may temporarily remove scheduled vessels to perform a Scan on Demand 7 3 2 4 Initiate Scan Scanning using the Scan on Demand Scheduler is initiated by clicking the red
14. each other It will NOT affect the absolute calibrated fluorescence values Pagea sa e e eee Calibrated fluorescence values are measured in terms of pixel brightness using Calibrated Units CU s The fluorescence Min Max Intensity settings can be adjusted either using the Auto Scale function or via Manual Adjustment Z M Auto Scale tries to assign optimum Min Max settings for a particular image Auto scaling is easy just press the Auto Scale button and in many cases the Auto Scale settings will be adequate There are times when the auto scale derived Min Max settings for one image will not represent the best settings for other images If the Always box next to the Auto Scale button is checked then every time a new image is selected it will be automatically Auto Scaled rather than using the values from the previous image If a movie is generated each image within the movie can be individually auto scaled refer to section 7 7 2 To manually adjust the fluorescent intensity values either type new values directly into the Min Max intensity boxes and press the Enter key or use the arrows to scroll through values Selecting a Minimum and Maximum Intensity clips the brightness at which fluorescent components will be displayed Minimum Intensity Any portion of the image with a pixel intensity value less than or equal to the Minimum Intensity will be assigned a relative brightness equal to the da
15. for instance when generating a movie use the Essen ImageLock plate When utilizing an ImageLock plate Users can be run either the Scratch Wound or Standard with lock scan type options Because the scanning process is so precise in ImageLock mode the scan length will be longer than that for a plate run in Phase Contrast mode The scan patterns available for the ImageLock 24 and 96 well plates differ from those available for standard 24 and 96 well plates A maximum of three images is possible per well and the scan pattern is arranged in a straight line through the center of each well dictated by the grid on the bottom of the wells 8 1 1 Standard with Lock By selecting an ImageLock plate and running it in Standard with lock mode the highest fidelity movie will be generated When an ImageLock plate is selected as the vessel type the Scan Type drop down box located under the Scan Mode in the Scan Setup tab will include the Standard with lock option 8 1 2 Scratch Wound The grid on the bottom of the wells of an Essen ImageLock plate allows the IncuCyte ZOOM to find an exact location on the plate for imaging which is necessary when performing migration and invasion assays using the Essen Woundmaker When an ImageLock plate is selected as the vessel type the Scan Type drop down box located under the Scan Mode in the Scan Setup tab will include the Scratch Wound option Page 85 N 8 2 8 3
16. 8 bit TIFF 7 7 1 1 Export Current Image Single Image To export only the SINGLE image that is currently displayed in the Vessel View window following the steps specified below refer to Figure 23 1 Select Export Current Image A new window will open 2 Select the appropriate image type in the Export Single Image window using the Image Type drop down menu 3 After the image type has been selected browse to the file destination 4 Select either the keep full size image dimensions or Customize with image designer radio buttons Page N ESSEN BIOSCIENCE By selecting the Keep full size image dimensions option the entire image acquired at its native size is exported using the current brightness and contrast settings Customization of the export image is available by using the Customize with image designer tool This button launches a new Export Designer window that allows the User to customize the cropping and zoom level as well as the option to add a legend and timestamp if desired With the legend option a scale bar and field of view dimensions will appear in the exported image as they do in the Vessel View In the Export Designer window the full size image is cropped as it appears in the Vessel View window whether viewed full size or zoomed in The cropping boundaries can also be changed in the current window by clicking on and dragging the red boundary line
17. Allen wrench supplied Replace the plastic cover in the module mount screw hole See the diagram below Figure 7 Page 13 3 4 3 5 j N z m 2 L Objective Filter Module i Bracket Figure 7 Microscope with filter module installed Installing an Objective The IncuCyte ZOOM ships with up to three objectives with magnifications of 4X 10X and 20X The system will need to be calibrated for all of the purchased objectives To get started the 10X objective should be installed if it is available otherwise the 20X or 4X should be used Objectives are installed on a bracket that is shipped already installed in the system See the diagram above With the filter module access cover removed pull out the drawer halfway to provide access Remove the objective bracket from the system by loosening the screw using the supplied 2 5mm T handle hex wrench and then pulling the bracket straight out Finger tighten the objective into the bracket Install the bracket and objective back into the system on the two guide pins and retighten the screw using the 2 5 mm T handle hex wrench If you are installing a 20X objective verify that the corrective ring on the objective is set to 0 8 Close the drawer and replace the filter module access cover Positioning the Controller It is important to put the controller into position before placing the microscope into the incubator The controller should be placed on a level surface within 3
18. IncuCyte software be sure that the Graph on Graph selection is chosen from the Drag and Drop pull down menu If a graph overlay is attempted with the wrong Drag and Drop selection a window will appear indicating that the overlay cannot occur There are 3 commonly made errors that can prevent creating a graph overlay from occurring If you are not able to drag your graph be sure that Z The correct Drag and Drop option is selected The right mouse button is selected and not the left Z Youare not trying to drag the same graph onto itself ry Dragging and Dropping Graphs from the IncuCyte Software into Other Software Packages Page 55 It is also possible to drag and drop a graph created within the IncuCyte software directly into a Word document EXCEL file PowerPoint file or e mail In these cases the graph can be dragged as either the raw data or the image To drag the image into Word select Drag and Drop Image to Word or Outlook To drag the graph image into EXCEL PowerPoint or email select Drag and Drop Image to Other Document To drag the raw data from the graph select Drag and Drop Raw Data to Document If the graph is dragged as Raw Data into EXCEL the data will appear neatly within the spreadsheet cells If the raw data are dragged into Word they will appear within columns The format in which the data will be dragged and dropped into the target software will be determined by the format in which
19. Mode to make linear measurements within the image To turn Measurement Mode on or off click on the ruler icon Spectral Unmixing Spectral unmixing may be required to remove fluorophores that produce signal in both color channels These properties should be saved prior to adding images to an image collection or performing an analysis NOTE It is important to perform the spectral unmixing in small increments to determine the appropriate value Figure 15 below illustrates spectral unmixing Green Red Blend Green Channel 8 of Red Removed from Green p je s olny syun VIUBISZION 4 Green Channel Green Channel Green Channel 0 of Red Removed from Green 8 of Red Removed from Green 100 of Red Removed from Green S XId PIPIO IIAO MOYS 0 p s mo spun adUadSaJON 4 UI219 Figure 15 Spectral unmixing of a mixed population of A549 cells expressing NucLight Green or Red Top Row Appropriate removal of the red fluorophore from the green channel using the spectral unmixing tool Bottom Row We lowered the green fluorescent channel to show the difference between optimized and overcorrected spectral unmixing Using the NucLight Red A549 cell circled in yellow as a guide the red fluorophore is visible in the green channel with an average Green Calibrated Unit GCU of 7 5 and an average Red Calibrated Unit RCU of 11 2 Optimization of the red fluorophore in the IncuCyte ZOOM showed that an 8 spectral unmixing of the red removed from gre
20. Region and Group refer to Figure 18 1 44 PM on 4 27 KONEX Fie Utilities View 28 Sat A549 Green Nuclei Count 1 mm x A549 Green Nuclei Count 1 Wel 416 PM Avg A549 Green Nuclei Area m all 6 15 PM l gt A549 Green Nuclei Confluence Percent 8 15 PM Avg A549 Green Nuclei Integrated Intensity GCU x m image 10 15 PM Total A549 Green Nuclei Integrated Intensity GCU x ym Image H 28 Sat Avg A549 Green Nuclei Eccentricity 5 s nf ous HT1080 Red Nuclei Metrics BS fey _ Sean Metrics _ el 5 01 Tue Sasic Mean v Histogram B A549 Green Nuclei Metrics amp HT1080 Red Nuclei Metrics B Figure 18 The Metric Graph Export Window Press the Graph button A graph will now appear in a new window Figure 19 The graph will be assigned a default title in this case All Custom Region Mean vs Time Page 48 LN ESSEN BIOSCIENCE File HT 1080 Red Nuclei Count 1 mm 1000 800 600 z Viewing Style gt x x 400 Border Style gt a k z Font Size gt 200 z V Show Legend Op A k Att Numeric Precision gt Plotting Method d 26 Sat Data Shadows gt 30 Mon Apr 2012 Grid Options d Include Data Labels Drag Drop and Alignment Settings Mark Data Points _ gt S z Edit View Tools HT1080redA549green Custom Region Mean ys T vs UL HT1080 RedNuc 1 2K well A HT1080 RedNuc 1 1K well a
21. advantage of the Manual Alignment slider over the Auto Alignment function is that Manual Alignment facilitates alignment of individual traces one at a time whereas Auto Alignment resets all traces simultaneously The Manual Alignment selection also facilitates deletion of selected traces First select the trace to be deleted by using the Trace Selection pull down box Now simply click on the Delete button The Delete function can be used to delete a trace from any graph containing multiple traces not just graphs created by dragging There are 2 circumstances under which traces from a graph CANNOT be deleted These are Z lf the graph contains only a single trace Z lf all the wells or sectors from a vessel are graphed simultaneously then the trace for the very first well or sector cannot be deleted If all wells from a microplate are graphed simultaneously this will be the trace for Well A1 The above two deletion rules also hold true for the target graphs onto which plot overlays are created Selecting an Individual Point on the Trace Use the mouse to select an individual point on a trace Hold the mouse over the point and the time and date of the scan represented by that point will be displayed Double left click on a selected point and it will open the Vessel View window for that scan The Vessel View window cannot be opened by double clicking on the points in an Average Graph Page 59 7 6 1 8 The Graph Pull D
22. available on all models of IncuCyte Zoom and others are available depending on the configuration of your system Outlined below are the steps to make a processing definition using the Basic Analyzer to train the IncuCyte ZOOM software for phase confluence 1 Once an Image Collection is saved start a New Processing Definition by clicking on the New Processing Definition link within the Vessel View found just underneath the Add to Image Collection link A new window will open Add New Processing Definition that contains a drop down menu for Users to select the Image Collection that they would like to use in this case to train phase confluence their processing definition on Users are also able to use the Search button to locate an Image Collection NOTE The default image collection will be the most recently made Image Collection Page 42 N ESSEN BIOSCIENCE 3 Click the Continue Button An un previewed window will open 4 The Basic Analyzer phase confluence requires Users to select a Training Image Collection red circle Figure 16 to determine the parameters that will differentiate between the Background and the Cells within the image The Training Image Collection should be the Image Collection that you made containing 3 6 representative images of your cells of interest refer to 7 5 2 1 5 The images previewed are part of the Image Collection black circle Figure 16 that you
23. be dragged to create overlays in the Calendar Mode setting This overlay facilitates a comparison of cell growth in absolute time However what if the same experiment was performed on different days In this case if both graphs are dragged together and then displayed in Calendar Mode they will not overlap at all because they have no dates times in common To display these graphs properly overlapped view the graph overlay with the Calendar Mode button NOT checked Now both graphs will be displayed as confluence relative to hours irrespective of the specific days and times and they will be overlapped Auto Alignment At the bottom of the Graph Window find the Auto Alignment tab This tab can always be opened but the Auto Alignment function is only active when the Calendar Mode button is NOT checked When the IncuCyte software is first opened this value will be set to the default number 22 However if the value is changed from this default the new value will be displayed until it is changed again by the User even if the program is closed and then opened again at a later date If the Auto Align button is checked then whenever graph overlays are generated by dragging they will be aligned at this set value When multiple traces are included on a single graph in Calendar Mode their relative positions are constrained by the absolute times and dates of each point within the graph However when Calendar Mode is no longer checke
24. below If no results are visible press the Refresh button at the bottom of the screen to update the log Check to be sure the test passed for all three tray positions before proceeding to Chapter 7 If the calibration was not successful cell culture monitoring will not produce quality results Fluorescence Calibration A fluorescence calibration kit will be provided when a dual fluorescence filter module is purchased with the IncuCyte ZOOM and the instrument will be calibrated at the time of installation It is currently recommended that the instrument be recalibrated twice a year The fluorescence calibration kit includes reagent sufficient for multiple calibrations as well as three calibration slides These calibration slides are disposable and should be used only once New calibration kits can be purchased from Essen BioScience Follow these steps to perform the fluorescence calibration 1 Place a calibration tray in the front position of the IncuCyte ZOOM Fluorescence calibration requires use of the front tray position and the remaining positions can be occupied with trays at the time of calibration 2 Load a calibration slide with supplied dye Use 40 ul dye per slide Place the pipet tip at one end of the slide window and expel the liquid dye The liquid will wick into the slide window After all the dye has been expelled use the pipet to transfer a small amount of dye to the other end of the window to be sure it is completely
25. by scan However auto warm up will resume if the camera has not achieved its target temperature after the interrupting scan has been concluded 3 8 2 IncuCyte Controller Air Filter The filter for the IncuCyte ZOOM controller is located behind the front panel see Figure 9 Although this filter is easily accessible it is NOT a consumable part and Users should not attempt to exchange it themselves If there are any questions about this filter please contact Essen BioScience for more information Page 17 Air Filter Figure 9 IncuCyte Front Panel Chapter 4 Establishing Network Connections The IncuCyte controller houses an embedded control computer that must be connected to your network in the same way that ordinary PC s running Microsoft Windows are connected We recommend that you consult with your IT department during the network connection configuration and verification processes A standard Ethernet cable is used to connect the controller to a network hub or switch The unit is equipped with two network ports The leftmost port is used as the primary network interface to connect the IncuCyte controller to the network The jack on the right is a port for the microscope camera Ethernet cable See Figure 8 for details The controller is configured at the factory for automatic assignment of its IP address on a network using DHCP Dynamic Host Configuration Protocol Follow the procedures described
26. click Add _ to add it to the plate Because the Growth Conditions category is used for conditions where no concentration or dilution series is specified clicking Add __ will add the condition directly to the plate with no intervening options Adding Multiple Well Items To sequentially add multiple items to a grouping of wells i e cells growth conditions compounds select the plate map icon second to the right on the top menu that reads Keep Current Selection After Adding to Wells Normally after adding an item to a well or group of wells that grouping will automatically be deselected With the Keep Selection icon activated the selected grouping will remain selected after addition of a well item Deleting Editing Well Items from Lists Once the three lists are populated the User may edit or delete items from the lists Editing an item will open the same window as when adding an item to a list only the fields in the window will already contain the information for the selected item Edit the desired fields Short Name Description Color and hit OK to save the changes To delete an item from a list simply click on the item hit Delete and OK NOTE This action does not delete any instances of the item already added to the plate map Editing Well Contents Once a well or group of wells are populated the User can edit its contents and appearance To edit well contents first
27. coded to reflect change in the current 6 Click the OK button 7 Data will begin to be processed A status window will open displaying the time until job completion 8 Once complete the Analysis Job will be displayed at the bottom of the Vessel View window Double click on the Analysis Job or highlight and click on the Launch button to open the Analysis Job For Established Assay Mode Once the parameters are defined in the Assay Development Mode Users can apply an Analysis Job at the time of assay initiation scheduling NOTE We recommend scheduling Analysis Jobs at time of vessel scheduling for real time analysis of the Live Content Imaging Page 45 ESSEN BIOSCIENCE N 1 Add a New Vessel into the Scheduler refer to section 7 3 2 Under the Analysis Job Setup heading select the Analysis Job type Basic Analyzer Angiogenesis or NeuroTrack by using the Job Type drop down menu 3 Under the same heading select the Processing Definition by using the drop down menu or the Search button Refer to Table 5 Analysis Job Rules for Analysis Jobs that are available at time of scheduling 4 Data will be processed following each scan to provide analysis in real time Table 5 Analysis Job Rules Processing Definitions will only be available if the IncuCyte ZOOM is using the same objective that the Processing Definition was developed with and if the vessel being scheduled contains ch
28. desired area of the plate in the Plate Map Editor and click on Save Selection as Region under the Regions tab on the left side of the window Enter a name for the region and hit OK to save the region To create a graph of or export data from a saved Region go to the Graph Export window from an opened Job Analysis in the View Vessel window and find the Region in the Regions drop down list Regions created from the Plate Map Editor will be highlighted in blue Select Replicates from the Group drop down list to view each group of replicates e g a specific drug concentration as its own plot on the graph or to arrange exported data into replicates Printing a Plate Map At any time during the creation of a plate map the User may print a hard copy of the map to put in a lab notebook or for reference use while in the lab The Print and Print Preview buttons are both found in the menu bar at the top left of the Plate Map Editor In addition to the plate map the hard copy will also include any notes written in the Plate Map Notes section below the Well Item lists in the Plate Map Editor Exporting an image of the Plate Map In addition to simple print functions Users can also export Plate Map images using the Export Plate Map button at the bottom left hand corner of the editor This brings up a selection menu where the User can select from 4 different file formats PNG TIFF JPEG BMP and 5 differ
29. filled Avoid getting any dry spots in the window as this will interfere with proper calibration Place the calibration slide in slide cutout of the calibration tray The orientation of the slide within the cutout position is not important 3 Select the Tests tab under the Administer IncuCyte tasks bar Then select the Fluorescence Calibration test Press the Run button A new window will open to confirm that the IncuCyte is properly configured to run the test Press the Yes button to start the calibration See Figure 13 The calibration procedure will begin Fluorescence Calibration requires 8 minutes Calibration status will be indicated at the top of the IncuCyte software window and in the status box at the bottom left Page 26 N ESSEN BIOSCIENCE Optics Reconfiguration Device Accounts Tests update Delete Logs Optics Test Magnification Filter Module Date Time Result i 20x Dual Color Model 4459 1 21 2013 2 50 00 PM 10x Dual Color Model 4459 2 11 2013 1 10 00 PM 4x Dual Color Model 4459 1 21 2013 3 01 00 PM O Spatial Calibration Magnification Position Confirm Test Date Time Result 20x Front 1 15 2013 5 52 00 M 20x Middle 9 10 2012 4 31 00 PM CEN 20x Rear oO 9 10 2012 4 41 00 PM 10x Front oO 2 11 2013 1 33 00 PM 9 10x Middle oO 1 14 2013 5 01 00 PM Pas O O Tray Position 10x ier Front A Calibration Tray Loa
30. for the system to save any settings and data before it powers down If the system fails to power down press and hold the power switch for 10 seconds IMPORTANT The IncuCyte ZOOM should be powered on immediately after installation into the incubator It is recommended that the unit be powered on at all times when it is in the incubator to minimize the formation of condensation on the system Proceed to the next Section Warming up the Microscope to learn how to warm up the microscope Warming up the Microscope A warm up cycle should be run to get all of the components in the system up to temperature quickly and avoid condensation After the system has been on for 2 minutes the system will start an auto warm up This will be indicated by a Monitoring Temperature message on the controller LCD The auto warm up is NOT intended as a substitute for the manual warm up cycle but rather it is a backup process that occurs in those instances where the manual warm up is accidentally omitted Page 16 CANCEL the auto warm up by pressing the Stop Eject button on the front of the microscope and start a manual warm up Use the LCD panel located on the front of the controller to start the manual warm up and follow the steps indicated below 1 Click on the Menu button on the LCD screen and select the Tools option from the list on the left 2 Click on the Warm up button from the newly displayed list of options
31. in Section 4 1 DHCP Based Networks to connect to this type of network If the network is not configured to assign IP addresses dynamically then a static IP address must be assigned to the controller See Section 4 2 page 20 Static IP Based Networks to reconfigure the IncuCyte controller for a fixed IP network 4 1 DHCP Based Networks Turn off power to the unit and connect the controller s primary Ethernet port to the network with a standard Ethernet cable Power on the controller and wait a few minutes before verifying the controller is networked correctly If connected correctly the controller will appear as a computer on the Microsoft Windows Network as a member of the Workgroup group of computers Assume that the controller is assigned the name ZOOMXXXXxX in the factory where XXXXX is the 5 digit serial number of the controller This serial number is found on a label on side of the controller Page 18 To verify that the ZOOMXXXXX controller has been successfully added to the network a Windows based PC hereby referred to as the PC on the same network is required These verification methods are discussed separately for Windows Vista 7 and Windows XP PC s 4 1 1 Verifying the IncuCyte Controller Network Connection Windows Vista or Windows 7 1 Under the Windows Start menu select Network Vista or Computer Windows 7 a Windows 7 only On the left side of the window there is a l
32. the Microplate Graph function to graph all wells or selected wells in a microplate format That is all wells are plotted individually as they would appear in a microplate 6 well through 384 well The colors of the Microplate Graph directly correlate to the colors selected in the Plate Map Editor For more information on Microplate Graphs please refer to Section 7 6 2 If individual graphs are desired Users can divide the plate into regions and graph on separate axes Regions of a microplate vary from large to small encompassing the entire plate single columns single rows or individual wells With the exception of individual wells graphical results can be viewed as grouped or not grouped Page 50 Regions and Grouping Select the Region pull down menu to display all possible microplate regions All Wells Select All Wells to simultaneously graph the Mean or Median for all wells within a microplate The Region All Wells meaning the entire plate provides the largest number of grouping possibilities See below Z Group All All the wells of the microplate will be combined into a single trace Z Group Columns All the wells of the microplate will be graphed as columns For example a 24 well plate will generate a graph of 6 columns Group Rows All the wells of the microplate will be graphed as rows For example a 24 well plate will generate a graph of 4 rows Group None In this case none of the wells will be grouped A 24 wel
33. the final movie will be played Careful consideration should be given to the final size of each movie frame which will have a huge impact along with the number of frames on the ultimate size of the output movie file 9 Once you are satisfied with the customization click OK to exit the Export Designer 10 When all the parameters have been chosen press the Export button The Export button will indicate that number of movies files that will be exported under the selected conditions 11 A new window will appear indicating the status of movie generation Multiple movies will be exported one immediately after the next until all movies have been exported and compressed Zall Slideshow Mode As a quick alternative to creating a movie click the Slideshow button below the time tree in Vessel View to scroll through time points of the selected image without exiting Vessel View While in Slideshow mode you can switch to any image in the vessel by clicking on its location at the top of the window Also switch to the Graph Export tab to view changes in any selected metric over time To change the speed at which IncuCyte cycles through images click on the Preferences Menu at the top of the main IncuCyte screen 7 8 Archiving Vessel image data plate maps analysis jobs and all associated data are stored on the IncuCyte controller While the 10TB of controller hard drive space is sufficient for retaining a si
34. to the 24 Hour Repeating scan schedule Users also have the ability to scan in the Scan on Demand mode Simplistically this means that the IncuCyte can be used to scan individual vessels at times when the IncuCyte is not scanning This includes both periods in between regularly scheduled scans in the 24 Hour Repeating scheduler as well as in designated Cooling Times Scan on Demand allows Users to scan only one vessel in one tray position at a time rE A rE D rA PA Properties Once the Scan on Demand vertical tab is selected the following properties must be entered into the scheduler prior to scanning Scan Pattern Channel Selection Z Tray Type Z Tray Position Z Cutout Position Z Vessel Type Z Scan Type Zz Z Label and Unique ID As with the 24 Hour Repeating schedule vessel Properties label cell type are used to both describe the vessel and searching for the vessel In addition to a Label Users can also supply a Unique ID to each vessel This Unique ID can either be generated manually by typing or pasting information into the Unique ID field or a Unique ID correlating to the current year day time will be assigned to the Scan on Demand vessel This information can be used to link subsequent scans together However it is important to note that if the User fails to type in or select using the Load button the correct Unique ID scans will not be linked together and cannot be accomplished post scanning
35. 2 5 4 Table 1 User Account Access Function Guest Limited User User Admin _ Scheduling Access No o No O Yes Yes Admin Access No _ Limited limited AT Edit Vessel Documentation No Yes Yes Yes Yes Delete Archives from ZOOM Their own Their own Any Attached Storage Delete Archives from browsed Yes Yes Yes to from My Computer with warning with warning with warning with warning ny S S Yes DeleteVessels No No Theirowmn Any launch AnalysisJobs No Yes Yes Yes _Delete Analysis Jobs No Theirown Theirown Any Add Processing Definitions No Yes Ye Yes Edit Processing Definitions No Theirown Theirown Any Delete Processing Definitions No Theirown Theirown Any Add to Image Collections No Theirown Theirown Any Edit Image Collections No Theirown Theirown Any Delete Image Collections No Theirown Theirown Any View Diagnostic Scan Metrics No No No Ys Remote Administration of the Controller The IncuCyte ZOOM controller consists of a single board computer running a Windows based embedded operating system This computer is dedicated to controlling the IncuCyte ZOOM hardware and providing remote access to the data collected by the instrument To insure reliable and proper operation of your IncuCyte the factory settings of the controller operating system should not be altere
36. 2 Select components you want to install 3 Choose a destination folder and Install 4 Install the Windows Media Player Codecs Required to save movies Page 20 SA 5 3 u of J Z NOTE It is possible you will get a message titled Windows Media player 10 stating that This version of Windows Media Technologies is incompatible with this version of windows This message can be disregarded Just click Cancel The incompatibility message communicates that the machine already has a newer version of Windows Media Codecs for movie generation It will be followed by a message entitled Windows Media Player Codecs Setup stating that The Codec package for the 7 Player has been installed successfully 5 If prompted install the Windows Media Encoder Required to save movies 6 Close the installer when completed IncuCyte Control Software Installation using Windows Vista and Windows 7 1 Review the license agreement To continue installation you must agree 2 Select components you want to install 3 Choose a destination folder and Install 4 If prompted install the Windows Media Encoder Required to save movies 5 Close the installer when completed Connecting 5 3 1 Setting the Date and Time The IncuCyte ZOOM controller settings will be synchronized with the local computer at the time of installation It is unlikely that any further synchronization will be required after th
37. 2 Ja a A Reset Crop Cick and drag the red guides to choose the area to be exported Figure 23 The Export Current Image Window with Export Designer Open Export Image Set Multiple Images It is possible to export all or a subset of all the images obtained for a particular vessel in bulk format by selecting the Export Movie or Image Set from the Utilities pull down menu This window displays the selected Vessel and its corresponding Time Tree refer to Figure 24 The window is divided into two main sections Export Time Area Range and Save Location and Options Note that one or more possible images from each well or sector can be selected for export The image number in the check box refers to which image will be exported It does NOT refer to the total number of images that will be exported Thus if only the checkbox for image 3 is selected then only image 3 will be exported for each selected well sector and or time point That is only ONE image will be exported per sector or well and that image will always be image number 3 To export all three images for each well sector the checkboxes for all three image numbers must be selected 1 Select Wells Sectors for Image Export e One or more wells sectors can be selected for image export e Select one or more wells sectors by clicking on them individually with the mouse the wells sector chosen will be highlighted in yellow e Select a region of t
38. 2 SSS E E E P O E A AE A E E A E 37 7 5 Data PrOCOSSINE ncevactges eoaniiesienaeneesctasiieuscante stat uniasaecombacactadiuernvenscoiavoncanvonsucrctaiiaevecdaguerdueniaceecrnineens 40 75 1 Assay Mode vs Established MOde c ccsscccccsseccccensececeeseceecescceeeuseceeseneceeeensceeseusecesseneses 40 7 5 2 PVA SUS gt TN OS s 41 7 6 Graphing and EXDOrting Dala een eae en rn ee eee 47 7 6 1 Bae RE ae e d seescctin ev E PEAS EAEE EAEE TEREE PEE EE A 47 7 6 2 Microplate Sele a lg ee ec eee ee ne eee ee ne eee eee eee eee 61 7 6 3 ExpOrUNE e 4 OL hg 1 A l10cc DA gt ee ae ree a ce ne ne en en ee eee 62 7 6 4 Saving ANC Printing Graphs ceecccccsseccccesscccceesececsusececceueceeeeesecessueceseuneceesenseeessusecetseneses 66 7 7 Exporting Images and MOVIES ssciseieasiao nanan eee e siatecetnaneaeneosneeneeees 66 7 7 1 Exporting Images Overview esssssessessessessessessessessesseeseesersersersersresresressrssroreoseesesseeseeseeseese 66 7 1 2 Exporting Movies Overview sssssessessessessessessessessersresersrrsrrsersrrerrsresressreseoreereeseeseeseeseeseest 72 Page 3 N ESSEN BIOSCIENCE 7 8 AKCII E ee era pecs eigen ete neste anomie avai eey enn core o cosnan ces scuccess 74 7 8 1 PVCU OS SC Seatac E E E cnt eactsi ote aaeseco se aeeucsueatae 75 7 8 2 OS US anA E ee E 76 7 9 POW AG Soot sons recacee nace E s2cteeaseat ss gatsasssuaeacease teanteeseeadsteass 77 7 9 1 Selecting and Placing WANS eccaiccc
39. Dragging and Dropping Graphs There are two different circumstances under which graphs can be dragged and dropped using the IncuCyte software Dragging and Dropping Graphs within the IncuCyte Software Create Plot Overlays Under some circumstances it will be convenient to combine traces created in separate graphs onto one single graph in order to facilitate a direct comparison For example the same cells were plated in two separate flasks at the same starting confluence at the same time How does their growth compare Unfortunately the mean growth curve of each flask must be generated separately creating two separate graphs However it would be optimal if both traces could be combined onto a single graph As indicated in the drag and drop box at the bottom of the graph window simply right click and hold on the graph to be dragged and then use the mouse to drag it onto the target graph Release of the mouse button will cause the graph to be dropped The new graph still contains the title from the original Growth Curve for vessel 1 however it now contains two traces labeled as vesel 1 and vessel 2 If only a single trace for example a single well from a 12 well plate needs to be dragged and dropped then it the desired well must be graphed alone and dragged and dropped individually Alternatively if the entire graph is dragged and dropped individual traces can be deleted To drag and drop graphs onto each other within the
40. Established Mode section 7 5 2 3 Page 30 N ESSEN BIOSCIENCE 7 3 1 3 Vessel Properties Tab Vessel properties are used to describe the assay being scheduled refer to Table 3 These properties will be used when searching for and viewing scanned vessels Table 3 The Vessel Properties Tab Name of the vessel Cell type used in the vessel Cell Type and Passage number of the cells can be selected by scrolling Passage up and down or a number can be typed directly into the passage box Use this button to open the Plate Map Editor and create a plate map for microplates Once a plate map has been Plate Map created and applied to a vessel a small plate map icon will appear next to the plate map button Refer to section 7 9 Plate Map Editor Any extra information desired Vessel properties are used when searching for a vessel are displayed when viewing a scan and can be y edited under the properties tab when a vessel is viewed 7 3 1 4 Scheduling Identical Assays When scheduling identical vessels that have the same Scan Setup and vessel Properties simply complete information that defines one vessel and use the Copy Vessel and Paste Vessel buttons L Complete the Scan Setup and Properties of one vessel Highlight the vessel in the Drawer Setup pane and click the Copy Vessel button Click on an empty tray position that you would like to copy that vessel to and then click on the Paste Vessel
41. X objective in standard mode NeuroTrack Module The NeuroTrack Module is used to identify neurite dynamics for the study of neurotoxicity neurodegeneration neuronal development and differentiation The NeuroTrack Module can quantify multiple label free assay metrics such as neurite length cell body clusters and branch points In the first step of the process the NeuroTrack software analyzes a HD phase image of neurons by first differentiating the cell bodies from the background based on texture and or brightness and masks them as cell body clusters From here linear features are detected based on width and brightness and are masked as neurites The segmentation mask can be refined and filtered by adjusting a few parameters to tailor the mask to the specific cell type used in the assay Page 87 Chapter 9 Troubleshooting The quality of images and subsequent confluence measurements obtained using the IncuCyte ZOOM depend on having a clear unobstructed light path through the particular well or flask Therefore before use carefully inspect the vessels selected for the experiment The following is a list of important issues to avoid Z Vessels that have scratches on the bottom or top lids for microplates dishes Z Drops of media or other liquid on the outside tops lids of vessels If this occurs carefully clean with ethanol and a Kimwipe Z Media hanging on the inside of tops lids of vessels Condensation on t
42. acteieeseateteacnedsitetecessccooteierseetteacsesuitatecrcieconeietecensiatecesetnncedes 77 7 9 2 Changing ODJectVES arsson nea rar eae ET 79 FAQ PAN D EGU GO I arrra A E 81 PMOL See VV ING eE E E S 82 FAO Anae N eE ee a nn eee ee 82 Chapter 8 Special Modules and Scan Types sssccccesseccccessccccesececeesececeeecceeeeseceseugecesseaeceeseueeeeesenecs 85 8 1 Scan Types available with Essen Imagelock cccccssccccsseccccesececcesecceceesececeunececeuaeceeseusecessuness 85 8 1 1 Standard With LOCK esens a E AE 85 8 1 2 Seratch WOUN sessir a E EE 85 8 2 Cell Migration INVASION Module sisiutctcssnssscorsiutuateroccanwnedtiesdunaessdsessduheebanascduinnes Waoduanencicadsduaennnmnteets 86 8 3 Angiogenesis Analysis MOUule ccccsescssossssccssssecccnssscncnssscsecussecceusssencusssessoussscueuesseneousuesseness 86 8 3 1 Tiled Field of View Mosaic Imaging Mode cccccccsssecccessececceseccceensecceeusececsueceeeeusecessuneeesseness 86 8 4 NeuroTrack Module a cerarne aces seosaguatiees acevunasesteraeavan spy neseropisansadpacaratuseatoeuneqeosequedteiartersseneetset 87 Chapter 9 TOUDICSNOOUNE eens nee etme mr eS Oe ST Pr A 88 9 1 images ona Whatthey NS Nico cents cirescrs neers E 89 9 2 Contacting Technical SUPPOT cccccceseccccsecccceesececsesececcuececceeeceeseusececsunecessenececsuuneeeseuesessuness 92 Chapter 10 Performance OptiMiZation cccccecsssesccsosssscncosssccccussscueus
43. allowing Users to search for their desired Image Collection Double click on the desired Image Collection or click the Select button located on the lower right of the window to open 3 Users can review images that are contained within the Image Collection by using the Image Stack forward and reverse buttons Users can remove an image by clicking on the Remove Image Stack button found on the lower left corner of the window 4 Use the OK button in the lower right hand corner to save changes Alternatively use the Cancel button to undo any changes made to the Image Collection 7 6 Graphing and Exporting Data 7 6 1 Create a Graph After scanned images have been analyzed they can be used to generate graphs of the selected metric over time The IncuCyte graphing feature can be accessed in the Vessel View window by either selecting the Graph tab and then click on the Graph Export button or by choosing the Graph Export option from the Utilities pull down menu Page 47 N ESSEN BIOSCIENCE NOTE No assay metrics will be available until an analysis job is complete and opened Open the Analysis Job at the bottom of the Vessel View window notice that the color banner at the top of the Vessel View window changes from blue to green Once Graph Export is selected the Metric Graph Export window will open allowing the User to choose the graphing Metric the Start and End times and the
44. am and then save to that program as desired Other Options Additional options are available when exporting data Py Py Include Experiment Details in the Header Fill Holes in the Data with the Following Characters Paged Z Include Error Measurements Z Break Data Into Individual Images Include Experiment Details in the Header Choose this box to include export of the following additional details along with the data the selected Metric usually confluence cell type passage number and any notes If multiple files are exported the additional details will appear in every file Fill Holes in the Data with the Following Characters It is possible to set a scan pattern that does not include all the possible wells or sectors in the vessel If the data are exported using the Layout option Show each Scan as its Own Table the table will have empty spaces where sectors or wells were omitted from the scan pattern Rather than leaving these spaces empty it is possible to select characters e g BLANK to fill the spaces Include Error Measurements Use this option to include either standard error SE or standard deviation SD for each measurement Note that SE and SD are not available for individual images but are calculated for the selected grouping all rows columns none When None is selected error measurements are available only if there are multiple images per sector Break Data Into Individual Ima
45. ams display the distribution of the Metric at a single time point over the designated Spatial grouping As for Time Plots Histogram spatial distribution can be anything from a single image to every scanned image To customize your Histogram press the Histogram Bin Settings button A new window will open allowing the User to adjust the Min Max Values of the x axis as well as the Bin Count The software will assign recommended settings based upon the data but it is possible to substitute Custom values as desired After Bin Settings have been selected press the Graph button Pressing the Graph button directly will generate a Histogram even if Bin settings are not viewed adjusted first The Histogram will simply use the settings assigned by the software Increasing the number of Bins results in finer resolution of the distribution across the selected Metric Page 53 X ESSEN BIOSCIENCE File Utiites View a 27 Fri 9000006090909906 9000090000000 2 39 PM d 000000000000 5 900000000006 ke amp HT1080 Red Nuclei Metrics image Proper Scan Metrics PT ode Gel Co co ese gen teh a i VP ee AT Pp x EE mmm tom lon 4 ji gt Sector _ Custom Region 4 27 201 Green Nt ten togam IE _ _ w od o O eee ee Figure 20 Histogram 7 6 1 2 Graph Customization Initial default labels along with other graph attributes can be personalized Left clic
46. annels that match the Processing Definition Phase Green i Phase Red Red only Phase Green Red Green Red 7 5 2 4 Edit Processing Definition Once a Processing Definition is created the User may edit values within the Parameters Clean up and Filter settings Page 46 1 To edit a Processing Definition click on the Edit Processing Definition link within the Vessel View found just underneath the Launch New Analysis Job link 2 The Processing Definition Search window opens allowing Users to search for their desired Processing Definition Double click the Processing Definition or click the Select button located on the lower right of the window to open 3 Users can edit and Preview as outlined in 7 5 2 2 Once satisfied with the changes Users can either Save the Processing Definition or rename it as a New Processing Definition Save As NOTE If a Processing definition has been edited Users may need to update their Analysis Jobs performed using the edited Processing definition This will ensure that data is consistently masked with the same parameters 7 5 2 5 Edit Image Collection Once an Image Collection is generated the User can edit its contents 1 To edit an Image Collection click on the Edit Image Collection link within the Vessel View found just underneath the Edit Processing Definition link 2 The Image Collection Search window opens
47. aphing Mode When Custom Region is selected the User can select deselect a subset of individual wells with the mouse as desired Normally when a graph is generated it is automatically assigned a Label based upon the selected Region and Statistic Because in this case the Region is variable it is possible for the User to assign a custom Label If the Label box is not populated the default Label will simply read Custom Region accompanied by the Statistic To save a custom region for later use click the Save button to the right of the Label box The custom region will then be saved under the Region pull down menu for the current vessel type To delete a saved custom region select it from the Region pull down menu and click the X next to the Region box The selected region will then appear as a Custom Region giving the option to edit it or permanently delete it by choosing a different saved region NOTE If a plate map is associated with a vessel microplates only any Region created in the Plate Map Editor will be available for graphing To create a plate map or assign regions see Section 7 9 Graphing Flasks and Dishes Flasks and Dishes contain only two possible regions Z All Sectors vr amp Single Sector Region All Sectors If All Sectors are selected the graphs can be viewed with or without error bars and the error bars will represent the inter sector Mean or Median as selected in the Statistic drop down menu
48. application specific software modules requires the user to purchase a license This will be indicated in the manual where applicable Current Filter modules available are 4460 IncuCyte ZOOM HD Filter Cube High Definition Phase Contrast only 4459 IncuCyte ZOOM HD Dual Color Filter Cube High Definition Phase Contrast Green and Red Fluorescence 2 3 Safety has Priority Please note the following directions for safe and problem free operation of your IncuCyte ZOOM Read through these operating instructions carefully Z t is essential to follow the instructions in Section 3 page 10 when putting your new IncuCyte ZOOM into operation The unit must only be connected to a receptacle outlet with a grounding connection Use only the AC power cord supplied or an equivalent cord with an IEC 320 C13 standard connector approved for operation at 5 amps and the appropriate voltage for your local power Also ensure the local line voltage falls within the range printed on the IncuCyte ZOOM Controller Z NEVER open the IncuCyte ZOOM controller It does not contain any parts that need to be maintained repaired or changed by the User Z f the IncuCyte ZOOM is not used in the manner as described in this manual provided protections may be impaired Page 6 2 4 Your IncuCyte ZOOM is of rugged construction but it is still a precision instrument If you treat it with the appropriate care it will reward you with many years o
49. at point However if for some reason synchronization is required at a later date the IncuCyte software will issue a prompt to this effect The prompt will appear after login but before the software is launched The exact verbiage of the prompt will depend on which settings are in a state of disagreement Figure 10 displays the window that will appear if an individual at the User or Guest permission level logs onto the software and Time Zone synchronization is required A slightly different message will appear if the individual is at the Administrator permission level The Time on controller zoom40006 needs to be synchronized 4 Please use Administer IncuCyte gt Device gt Synchronize as soon as possible Figure 10 Time Zone Synchronization Window at the User or Guest Permission Level Page 21 NOTES We strongly recommend that ONLY individuals at the Administrator permission level implement changes to the controller time settings Tine or such as 5 3 2 Time Zone synchronization is ONLY required if the IncuCyte software issues a prompt the example displayed in Figure 10 Establishing User Accounts Essen BioScience will establish the initial Administrator account Additional Users can be created using the Create User function All IncuCyte Users must have an account on each instrument they wish to use However we recommend that the number of Users with Administrator level permission be kept to a minimum
50. attempt to find the controller on the network by continuing to the next step Verify that the Address toolbar is displayed at the top of the window Under the View menu time at the top of the window verify that the Toolbars gt Address Bar menu item has a check mark next to it Type the following in the Address box at the top of the window ZOOMXXXXX Page 19 7 Click on the Go button to the right of the address bar If the controller is found on the network you will be prompted for a User name and password Press the Cancel button to exit If the controller is not found then a Cannot Find message will be displayed after a brief search period 4 2 Static IP Based Networks In order to reconfigure the IncuCyte ZOOM controller for a static IP address a direct connection must first be made to the controller In order to accomplish this you will need to connect a monitor keyboard and mouse to the controller To gain access to the monitor keyboard and mouse ports you need to unscrew the two screws on the back of the controller and remove the back cover See Figure 8 With the controller powered down connect the monitor keyboard and mouse to the controller After connecting power up the controller Once Windows has booted up you can log in with a User name ZoomAdmin and the Essen provided password Recall that the ultimate goal is to configure the controller s primary Ethernet po
51. cal Note hm i iy iy i Please refer to these documents for a detailed outline on how to process images using the Analysis Job Utilities Below is a brief overview of the data processing modes and the Analysis Job Utilities used in the IncuCyte ZOOM software 7 5 1 Assay Mode vs Established Mode As described in the figure below we separate the data processing flow into two distinct phases Page 40 N Completed only once per assay cell type or magnification Every Vessel Established Assay Phase ESSEN BIOSCIENCE 7 5 1 1 Assay Development Phase n this phase the User defines the image analysis parameters that will be used to analyze all images within an established assay These parameters will be applied to all current and future experiments vessels that use the same experimental conditions Assay Cell Type Objective Future experiments will skip the Assay Development Phase Collect Representative Data Make Image Collection 1 The first step in Assay Development Phase is to collect representative images that form a data set called an Image Collection For example if a User wants to monitor cell confluence over time the User would collect phase contrast images of cells every 3 6 hours as they proliferate over the course of 3 5 days Train if Phase Assay Development Phase Test and Refine 2 This image collection is then used to train phase and test phase and or fluorescence a Processing Defi
52. can Bar off the timeline oaaae ceeeaceecs reeeaeeeces seeeaeeeee 1 eeeeeea Figure 14 24 Hour Scheduler Page 32 LN ESSEN BIOSCIENCE 7 3 1 6 Multiple Vessel Scheduling When scanning multiple vessels the Vessel Scheduling button at the bottom of the Schedule Scans screen allows the User to insert cooling idle times between vessel scans and customize the order in which IncuCyte scans vessels Using the Auto Cool function is recommended and allows for the most efficient vessel scheduling NOTE The Multiple Vessel Scheduler is ONLY needed if Users plan to introduce cooling times or need to change the order of scanned vessels The microscope can heat up during excessive scanning In order to prevent overheating it is recommended that total continuous scan time not exceed 45 minutes and that the duty cycle be kept under 50 As an example 40 minute long scans scheduled every three hours results in a duty cycle of 22 2 40 min 180 min add 7 7 3 1 8 Apply Schedule When the Scan Setup vessel Properties and Scan Time s have been set select the Apply button at the bottom of the screen A window will appear confirming that scanning parameters have been applied NOTE Pressing the Reload button at the bottom of the Schedule Scans screen will reset all settings physical layout properties scan times etc back to the way they were prior to the last time the
53. ched to ZOOM Archiving using these two options is discussed in further detail below 7 8 1 1 Archiving to My Computer Data archived to My Computer allows users to save archives to their local computer or to specify a location on a networked server by simply clicking the Browse button identifying the location and following the on screen instructions Local archives are immediately accessible which is advantageous if continued access to the archive is anticipated However archiving to a local computer or to a networked server is significantly slower is susceptible to network interruptions requires your local computer to be on for the duration of the archive process and is generally less efficient than archiving to a Storage Device Attached to the IncuCyte ZOOM Controller 7 8 1 2 Archiving to Storage Attached to ZOOM Archiving to Storage Attached to ZOOM uses a dedicated storage device that is directly attached to the IncuCyte ZOOM controller through either an eSATA port preferred or a USB port Archiving data to Storage Attached to ZOOM using the eSATA port is the fastest method for archiving data An optimized IncuCyte ZOOM Archive Drive can be purchased from Essen BioScience to perform this function ZOOM attached archives are immune to network failures computer reboots or log offs as well as reboots of the IncuCyte controller However ZOOM attached archives can only be accessed when the sto
54. cked in a separate box There is also a shipping pin installed to lock the microscope in place It must be removed before the system installation can proceed Remove the filter module access cover by pushing it down and pulling the cover out The pin will be visible as in Figure 5 below To remove the pin depress the button in the center of the T handle and pull the pin straight up until it is free Store the pin with the other ZOOM accessories so that it can be reused if the system needs to be shipped to another location or returned for service Shipping Pin Figure 5 Microscope with access cover removed Page 12 3 3 ESSEN SCI BIOS NCI Filter Module Connector Figure 6 Microscope with shipping pin removed Installing the Filter Module There are currently two types of filter modules available for your IncuCyte ZOOM one is for systems that collect phase images only no fluorescence and the other allows collection of dual wavelength fluorescence One of these modules will have been shipped in the accessories box with your ZOOM With the filter module access cover removed pull out the drawer about halfway in order to get better access Remove the plastic cover from the filter module mount screw hole and place the module into the microscope Press the module into place such that the connector on the module engages the connector in the microscope The module can then be secured in place using the 2 5mm T handle hex
55. ckground together less than 3 CU it may be helpful to increase the acquisition time Scan Type For most vessel types the software will automatically choose a standard scan type Special scan types are available when Essen Bioscience Image Lock plates are selected or when special module have been purchased e g Angiogenesis or Scratch Wound Scan Pattern The Scan Pattern describes the number and locations of images that are generated for each well microplates or sector flask dish The Scan Pattern is displayed within the software as a pattern of green squares where each square represents one image Create new scan patterns by using the edit scan patterns button on the lower left corner of the screen Scan Patterns can be edited or created for all 3 magnifications however only the scan pattern that matches the current IncuCyte ZOOM configuration can be chosen when scheduling a scan May be required to remove fluorophores that produce signal in both color channels Un mixing can also be completed in the Vessel View Refer to section 7 4 2 4 for more information on spectral unmixing Once parameters are defined for your specific assay type cell type and magnification Users can apply those to future assays at the time of assay scheduling You must complete an image collection and processing definition at least once during assay development before launching an analysis job setup during the scheduling step Refer to Analysis Job Rules in
56. concentration is desired click OK and the compound will appear on the plate map 5 To create a dilution series check the box next to Create a dilution to activate dilution options In the drop down menu immediately to the right select divide by or subtract and enter value by which to dilute Alternatively select manually enter to enter a range of concentrations separated by spaces or semicolons 1000 300 100 30 To arrange the dilution series on the plate select the direction in which the dilution series runs by selecting a direction under Dilute wells and the direction in which the replicates run If for example a dilution series runs from left to right and the User wants the replicates to be arranged Page 82 7 10 2 2 7 10 2 3 7 10 2 4 7 10 2 5 7 10 2 6 horizontally a box will appear allowing the User to specify how many wells exist at each concentration singlets duplicates triplicates etc Once the dilution series is specified click OK and the compound will appear on the plate map in the specified series Cells To add a cell type select the Cells tab then click the New button Once a region of the plate is selected click Add ___ to open up a new window Here specify passage number and seeding density and click OK to add the cell type to the plate map Growth Conditions Once a growth condition is specified in the list simply
57. cted to an IncuCyte ZOOM the User can easily maneuver throughout the distinct parts of the software The Task List located on the left side of the screen enables access to administrative tasks ns device control logs User accounts and vessel deletion vessel scheduling fae search SR search options and vessel archiving The software also displays the status of the connected IncuCyte ZOOM device including objective configuration device temperature drive space indicator and scan status Page 28 N 7 3 ESSEN BIOSCIENCE The IncuCyte ZOOM Software was designed to allow Users to efficiently navigate throughout the program by clicking on text as well as graphical icons and images Users should take the time to investigate the functionality of the IncuCyte software interface as well as moussing over the information icons found throughout the window screens J Information icons are found throughout the software Mouse over the to find useful hints and tips Scheduling Scans and Loading Vessels To initiate any assay begin by selecting the Schedule Scans s eduesens Option located on the left side task list Once selected the scheduling interface will enable the User to either schedule scans on a 24 Hour Repeating schedule or a single Scan on Demand 7 3 1 24 Hour Repeating The 24 Hour Repeating Schedule allows Users to define a set interval for acquisition times Once the 24 Hour Repeating vertica
58. cur the default value is 22 Whatever alignment value is set under this tab will apply to all future graph overlays To change the value select a new value and then press Apply See Auto Alignment under Other Graph Features section 7 6 1 7 for more information 7 6 1 7 Other Graph Features Error Bars When multiple wells or sectors are combined to generate a single trace Error Bars can be added that represent the standard deviation between the wells or sectors Standard Error and Standard Deviation Error Bars are available The Standard Error Bars are the default selection To change or remove Error Bars select the View drop down menu from the top of the Graph window When Error Bars are chosen then all graphs generated subsequent to that time will automatically be assigned Error Bars Deselecting the Error Bars will eliminate all Error Bars from subsequent graphs within a single well or sector 9 Error Bars are ONLY available for multiple wells or sectors They are NOT available for multiple images page 57 a ESSEN BIOSCIENCE N Calendar Mode At the bottom right corner of the graph is the Calendar Mode check box If the Calendar Mode box is selected the x axis of the graph will be displayed as year month day and time If Calendar Mode is NOT selected the x axis will be displayed in units of hours When should Calendar Mode be used If multiple graphs were created over the same time period they can
59. d However we recognize that there may be instances when it is desired or even required to log onto the embedded controller desktop Some examples include Renaming the controller from the factory set name Configuring the primary Ethernet port with a static IP address Installing anti virus and or firewall software Installing Essen recommended Windows updates hy Fy Fy Fy The IncuCyte controller ships with a unique Administrator password for accessing the controller s Windows desktop Essen BioScience maintains a record of this password so we strongly discourage you from renaming the Administrator password We also recommend that this password be protected and that the controller operating system be accessed only out of necessity Altering the controller operating system as little as possible is the best way to guarantee stability of the software that controls the IncuCyte ZOOM The easiest method for accessing the controller desktop is through the Windows Remote Desktop Connection tool Windows XP Users can access this utility through the Windows Start menu under the Programs gt Accessories gt Communications menu Users of Windows Vista and Windows 7 will find the utility in the All gt Programs gt Accessories menu Page 23 ry f gy 0 fa Ww Z Run the Remote Desktop Connection software and type in the name of the IncuCyte ZOOM controller in the Computer edit box If conn
60. d and the traces are displayed merely as confluence relative to hours of growth individual traces can be aligned with each other according to a specific selected percent confluence point that is common to all of them When multiple traces need to be aligned onto a single graph it is important to select an alignment If an alignment value is set that is outside the range of one or more traces then the program cannot generate an accurate alignment To set a new Auto Alignment value scroll through the values in the alignment box or simply type in a new value Then press Reset or hit Enter on the keyboard Page 58 Another useful application of the Auto Alignment function is the comparison of multiple traces at different stages of growth Manual Alignment Deletion This function enables manual alignment or deletion of each individual trace on a graph When the Manual Alignment tab is selected the graph will initially be aligned at the value set in the Auto Alignment box While the Auto Alignment function is used to align all traces on a graph to the same point Manual Alignment facilitates the alignment of individual traces upon a single graph at different points Select each trace to be manipulated using the Trace Selection pull down box to the right of the slider The selected trace can be pulled to the left and right using the slider Use the slider to realign all three traces on the graph NOTE The
61. ded 4x E Confirm Test 4x ra The Fluorescence Calibration uses a Calibration Tray populated with an pe Essen BioScience Fluorescence Calibration Slide Please verify that Run The Calibration Tray is placed in the front position Fluorescence Calibratij An Essen BioScience Fluorescence Calibration Slide is placed Magn in the slot at the top gt 20x Are you ready to start the test 10x E View Test Results Figure 13 Fluorescence Calibration 6 2 1 FLR Calibration Results When the calibration is complete the status of the Fluorescence Calibration completed successfully will be displayed in the far right column of the table labeled Result pass fail status is reported for each magnification You might need to click on the green Refresh button on the bottom to see the updated status A summary of calibration details and accompanying images can be accessed via the View Test Results button located at the bottom of the screen The calibration results will be displayed in a new window The detailed results can also be viewed under the Logs tab Log Type Test The log will also state whether or not the calibration passed If the calibration failed the log will include recommendations to avoid potential issues the next time through Chapter 7 Cell Culture Monitoring and Data Analysis 7 1 Introduction The IncuCyte ZOOM introduces an innovative platform to acquire analyze and quantify images
62. dminister IncuCyte button on the left hand side of the software Click on the Logs tab on upper part of the screen Then click the Export button Click Yes to export the entire logs file ce YS p Export All x Q You are not currently viewing the entire log Do you want to export the entire log Yes No Cancel 6 Name the text file after you company or entity 7 Email the text file along with a brief description of the issue to local support Local support can be found at http www essenbioscience com support html Page 92 N ESSEN BIOSCIENCE File View Preferences Help zoom40004 Admin Ready To Scan gt gt Start 11 09 2012 12 32 30 Front Cal Magnification 20x Filter Module Dual Color Model 4459 Mfg Date 05 07 2012 01 25 PM Coord sys info x off 15 62mm y off 19 18 mm Rot 0 01 deg Global plane tipped 0 02 deg 0 32 um mm Local plane tipped 0 10 deg 1 82 um mm Tray offset 1 46496 mm from home at center Windows 1 2 0 09 x fit error Windows 2 4 0 31 y fit error gt gt End 11 09 2012 12 36 43 gt gt Start 11 09 2012 12 36 43 Objective Magnification 20x Filter Module Dual Color Model 4459 Mfg Date 05 07 2012 01 25 PM Confirm Results Trials 20 Overshoot 0 550 0 550 Device Status ELA 24 27 10 7 S 2 23 5 S 2 0 0 11 9 320 0 0 Figure 31 The IncuCyte Log Chapt
63. e objective bracket Loosen and rotate the objective counterclockwise to remove it from the mount Place the new objective on the mount and rotate clockwise Be sure to rotate the objective until it is tight in the mount NOTE The 20X objective has a corrective ring Figure 28 red circle used to compensate for aberrations Users planning to image in fluorescence with a 20X objective should investigate the best setting for their system and their vessel We recommend setting the corrective ring at 0 8uM and image the vessel If focusing issues result at that setting continue to adjust the corrective ring until optimal images are acquired Replace the objective bracket into the ZOOM by using the two guide posts Tighten the objective bracket into place using the Hex T handled driver Replace the panel door and return to software Cyte Zoom Filter Module Figure 28 20X Objective It is important to warm the new objective at 37 C for 30 minutes prior to placing it in the IncuCyte ZOOM to eliminate condensation issues on the objective during image acquisition Select the Configure button Select the drop down for New Magnification Be certain to select the Nikon objective you have just placed into the IncuCyte ZOOM An Optics Reconfiguration warning message is displayed stating that changing objective and accepting changes in the Optics Reconfiguration menu will delete the current schedule Users have t
64. ecting for the very first time the name used will be the factory set name Make sure that you have connected and verified that the controller is on the network see Sections 4 1 1 and 4 1 2 Press the Connect button and wait for a Windows login prompt It may take as long as 15 seconds before the prompt appears Log into the controller with a User name ZoomAdmin and the Essen provided password The controller operating system should contain the necessary components required to rename the computer configure the primary Ethernet port install security software etc Please consult with your IT department for answers to questions related to your local network configuration and security software IMPORTANT Always use the Disconnect option and not the Log Off option when terminating the Remote Desktop Connection via the controller Start menu Chapter 6 Calibrating the IncuCyte ZOOM System 6 1 System Calibration Before using the IncuCyte ZOOM for the first time a calibration procedure must be performed This procedure ensures that the motion of the IncuCyte ZOOM microscope is accurate and repeatable such that images are obtained within their proper target locations This is especially important when acquiring images in 96 and 384 well plates The calibration procedure requires up to 1 5 hours and it utilizes a special calibration tray that is shipped out with the unit Figure 11 The calibration tray is the sa
65. en was required GCU RCU x 0 08 amount of red signal removed from the green channel If too much of the red signal is removed from the green channel overcorrected pixels will develop giving the appearance of holes within the image Overcorrection of spectral unmixing may affect assay metrics Page 39 7 9 7 4 2 5 Properties Tab Includes the information associated with the vessel that was entered when the vessel was schedule Some vessel Properties can be edited These include the vessel Label Cell Types and Passage number New Notes can be added but previously added Notes cannot be edited To edit properties enter the new information and press the Update button at the bottom of the window 7 4 2 6 Graph Export Tab Displays metrics regarding image acquisition No assay metrics will be available until an analysis job is complete Data Processing Data processing analyzes and summarizes qualitative images into quantitative data The IncuCyte ZOOM software separates data processing into two distinct modes the Assay Development Phase and the Established Assay Phase Data Processing Technical Notes are available on our website http www essenbioscience com supportdocs for the special modules listed below Angiogenesis Data Processing Technical Note Confluence Data Processing Technical Note Object Count Data Processing Technical Note NeuroTrack Data Processing Technical Note Scratch Wound Data Processing Techni
66. ent graph This option can also be used to average plots from different vessels by first combining plots from different graphs using the Drag Drop feature and then averaging the resulting graph NOTE Error Bars are NOT available on graphs created using the Average function Each of these functions opens a new graph window preserving the original Page 60 7 6 1 9 Zooming the Graph Zoom Once a graph has been created it is possible to zoom into any particular area for closer inspection To zoom left click the mouse and then highlight the portion of the graph to be expanded Release the mouse to see the Zoomed portion of the graph Undo Zoom To reverse the zooming Z Right click on the expanded graph A menu will appear Select Undo Zoom from the menu and the graph will return to its full size format Z Type the character Z on the keyboard 7 6 2 Microplate Graphing The Microplate Graphing selection provides another option for graphing all experiments conducted in a microplate format Using the Microplate Graphing function users can easily graph all wells individually in high or low density microplate refer to Figure 21 Unless selected otherwise the Microplate Graphing color scheme directly mirrors the information in the Plate Map editor Furthermore Microplate graphing can be customized using the Menu options These options include Editing Titles Subtitles Axis Range Control Show Hide x and y axis labels Logari
67. ent predetermined sizes Page 84 Chapter 8 Special Modules and Scan Types In addition to the Standard Basic Analyzer special Image Processing Software Modules are available with enhanced processing features These Modules allow Users to customize processing for migration and invasion Scratch Wound angiogenic tube formation Angiogenesis and neurite dynamics NeuroTrack The Invasion Migration and Angiogenesis at 10X objective require additional scan types other than the standard scanning types used for HD phase contrast green channel fluorescence and red channel fluorescence imaging Using Essen ImageLock plates Users have the ability to analyze invading or migrating cells using the Scratch Wound scan type When users want to visualize a larger more integrated view of the angiogenic network without compromising image resolution the Tiled Field of View FOV mosaic image scan type option is available when using a 10X objective Special Modules and Scan Types are described in further detail below 8 1 Scan Types available with Essen ImageLock Essen BioScience manufactures a special 24 and 96 well plate type called the Essen ImageLock plate These plates facilitate very precise repeated imaging within each well Under standard imaging conditions slight variations can occur in the location of imaging from one scan to the next resulting in small jumps between frames When very precise repeated imaging is required
68. er 10 Performance Optimization The IncuCyte is a phase contrast microscope and as such requires an unobstructed path to the specimen from both above and below Keep in mind the following warnings to avoid image degrading problems Keep vessels clean Avoid condensation Avoid bubbles Avoid media hanging on top of flasks Don t scratch vessels Avoid writing labels on the tops of vessels Avoid touching the tops and bottoms of vessels Handle them from the side as much as possible Page 93 N Vibration is another factor that can negatively affect image quality The incubator that contains your IncuCyte ZOOM should be on a sturdy stand If possible try to schedule scans at times when the incubator will not be accessed as opening and closing the incubator door can cause image blurring even on a sturdy stand Although it is desirable to not have large gaps in data collection it is not necessary that the sampling be perfectly uniform Scheduling daytime scans for just before work begins at lunchtime break times and again just after working hours may produce the best results This also ensures that samples in the IncuCyte will be available during normal working hours in the lab Another useful rule to follow is don t over scan your samples We recommend keeping scan times under 45 minutes and duty cycles under 50 As an example 40 minute long scans scheduled every 3 hours results in a duty cycle of 22 2 Under these co
69. ere are two possible destinations for exported data Z M Export to Clipboard Choose export to Clipboard to export the data directly to a new location such as EXCEL PowerPoint or MS Word Simply select export to Clipboard press the Export button and then navigate to the destination software Then choose Paste The data should appear in the previously selected format Export Data to One or More Files The data can be exported to a single file by selecting the radio button for All Scans in One File For example suppose a 96 well plate was scanned at 10 different time points If the data are exported to a single file when the file is opened it will contain the data for all 10 scan times Alternatively the data from each time point each scan can be saved to a different file In this case select Each Scan in a Separate File Continuing with the same example as above a 96 well plate that was scanned at 10 time points will generate 10 individual files and each file will contain the data for a single time point To save scans to separate files browse to a destination file and then enter a file prefix The Preview box will be populated automatically as the File Prefix is entered Press the Export button Whether the data are exported in a single or multi file format the file s will be saved in text format with a txt extension Open the data as a text file or open the file in an alternate software progr
70. f trouble free operation Should you have problems that require service please contact US Contact Information UK Contact Information Essen BioScience Inc Essen BioScience Ltd 300 West Morgan Road BioPark Broadwater Road Ann Arbor Michigan 48108 USA Welwyn Garden City Hertfordshire Tel 1 734 769 1600 AL7 3AX United Kingdom Fax 1 734 769 7295 Tel 44 0 1707 358688 ussupport essenbio com Fax 44 0 1707 358687 eusupport essenbio com IncuCyte ZOOM Specifications Power 100 240 VAC 50 60 Hz 3 5 Amps max 120 Volts 1 75 Amps max 240 Volts Network Connection Ethernet 10 100 1000 Mbps Controller Dimension Height x Width x Depth 5 5 x 17 0 x 21 5 14 0 cm x 43 2 cm x 54 6 cm Microscope Dimension Height x Width x Depth 12 4 x 17 7 x 15 5 31 5 cm x 45 0 cm x 47 0 cm Controller Weight 38 Ibs 17 2 Kg Microscope Weight 44 Ibs 20 Kg Page 7 Japan Contact Information Essen BioScience K K Cerulean Tower 15F 26 1 Sakuragaoka cho Shibuya ku Tokyo 150 8512 Japan Tel 81 3 5456 5481 Fax 81 3 5456 5511 josupport essenbio com 2 5 Controller Operating Environmental Conditions O to 33 Celsius 5 to 90 RH Non Condensing Microscope Operating Environmental Conditions O to 42 Celsius 5 to 95 RH Non Condensing Unpacking and Checking the IncuCyte The installation of a new IncuCyte ZOOM system will be done by trained Essen BioScience field service pers
71. feet of the incubator s Page 14 3 6 cable port The top of the incubator often works well Do not however connect any of the controller cabling at this point Placing the Microscope into the Incubator When choosing a shelf for the microscope keep in mind that flasks and plates are placed into the IncuCyte ZOOM cell drawer from above For incubators that are at table height a shelf installed near the bottom of the incubator is usually most convenient Approximately 12 5 inches of height will be used by the instrument In most incubators this usually allows room for at least two shelves to be installed above the microscope Remove all shelves above the targeted shelf before setting the microscope into the incubator Feed the two cables from the microscope out through the cable access port on the back or side of the incubator The cables can usually be routed up a corner of the incubator so as not to interfere with the installation of shelves above the microscope A split stopper supplied with the unit can be used to plug the access hole around the cables Shelves can be reinstalled above the unit as close to the incubators as the mounting brackets allow WARNING The top cover of the microscope should NEVER be used as a shelf and should NEVER be used to support a shelf IMPORTANT After the microscope is placed in the incubator Proceed IMMEDIATELY to the next two sections To avoid large amounts of condensation on the unit it is des
72. file on the hard drive and clicking on it it will not open If however a graph is created and then saved into another software program for example MS Word it can be opened directly through that program In this case however it will be a static image that cannot be manipulated 7 7 Exporting Images and Movies 7 71 Exporting Images Overview The image export function is accessed through the View Vessel window by using the Utilities pull down menu Images can be exported either individually or in bulk The Image can be exported as one of twelve image types depending upon the channel used to acquire the images Z Blended Composite using current display settings JPEG Z Phase Contrast using the current display settings JPEG will export an image that maintains the brightness and contrast settings currently displayed in the Vessel View Phase Contrast Original JPEG Phase Contrast Original 8 bit TIFF Green using the current display settings JPEG Green uncalibrated 16 bit grayscale source PNG Green uncalibrated 16 bit lossless TIFF Green calibrated 32 bit raw floating point values Red using the current display settings JPEG Red uncalibrated 16 bit grayscale source PNG Red uncalibrated 16 bit lossless TIFF Red calibrated 32 bit raw floating point values my i i i i i i iP i iPS The two image types identified with an asterisk might not be as common as the other types but they should be interp
73. formed Page 44 N ESSEN BIOSCIENCE For initial assays or currently scanning vessels Analysis Jobs can be launched once a processing definition has been established If an Image Collection was made containing representative data and a Processing Definition was defined prior to the completion of the scheduled assay an Open Ended Job Analysis can be launched However if Users must include representative images for an Image Collection at time of assay completion an Analysis will be performed post data acquisition 1 Click on the Launch New Analysis Job link within the Vessel View found just underneath the New Processing Definition link or the Launch button in the bottom left corner of the Vessel View 2 Select the Job Type from the drop down menu in this case the Basic Analyzer 3 Select the Processing Definition refer to 7 5 2 2 Use the Search button to quickly locate a saved Processing Definition NOTE The Processing Definition parameters will be displayed on the right hand side of the Analysis Job window 4 Give the Analysis Job a Unique Name for example User Name Date 5 Define the Processing Scope by selecting a defined time range a single time or open ended Also highlight the wells sectors to be analyzed viewing mode When an Analysis Job is opened the vessel view banner changes from blue to green The color banner at the top of the Vessel View window is color
74. from living cells that remain unperturbed by the detection method for repeated measures over long periods of time features we refer to as Live Content Imaging In addition IncuCyte ZOOM has a powerful database designed to make it simple for Users to search and retrieve experimental data using a unified search tool The design of the IncuCyte ZOOM software provides flexibility in designing cellular assays as well as providing easy to understand sophisticated analysis tools These analytical tools allow Page 27 N Tide ESSEN BIOSCIENCE Users to translate qualitative observations to quantitative data in order to facilitate data driven decision making Simple image export options channel blending and unmixing and movie making tools provide a means to easily integrate IncuCyte ZOOM generated data into presentations posters and publications The basic workflow of setting up an experiment from scheduling to experimental results for both assay development and performing established assays is represented below Schedule Scans 24 Hour Repeating or Scan on Demand Search and View Scanned Vessels Schedule Scans Data Processing e Image collection User defined phase and fluorescence processing Graph or Export Data Launch Analysis Job on Vessel Graph or Export Data All aspects of live content imaging using the IncuCyte ZOOM software are described in detail within this chapter User Interface Once conne
75. ge Page 41 N ESSEN BIOSCIENCE V Once a representative image is chosen click the Add to Image Collection link under Analysis Job Utilities in the top right corner The Add Current Image to Image Collection window opens Users can create an image collection under four Analysis Job Types using the drop down menu Basic Analyzer training for HD fluorescent object testing Angiogenesis NeuroTrack or ScratchWound Use the radio buttons to add the image to an Existing collection or create a New collection Select the Required Image Channels that will be used to train phase analysis or test fluorescent object count NeuroTrack Angiogenesis or ScratchWound the desired processing definitions Once all fields are completed click the OK button Each image collection should be limited to 3 6 representative images NOTE Adding too many images will prolong the development of the Processing Definition Image Collections may contain images from multiple tissue culture vessels but these images must be taken at the same magnification Fine New Processing Definition A processing definition is a stored set of parameters that completely specifies how to process acquired images Currently there are four types of processing definitions 1 Basic Analyzer which includes confluence phase and fluorescent object analysis 2 Scratch Wound 3 Angiogenesis and 4 NeuroTrack The Basic Analyzer is
76. ges When there are multiple images per sector you have the option to break data down into measurements from each image 7 6 3 2 Dragging and Dropping Metrics It is possible to export the Metrics associated with a particular vessel directly to another document or to a selected file directly from the Launched Analysis job within the Vessel View NOTE It is NOT possible to drag and drop a time range Only data from a single scan time can be dragged and dropped To export data displayed in the Vessel View window follow the steps specified below Select the Graph Export tab located within the Vessel View Select the View menu at the top of the screen and uncheck Condensed Metrics 3 Select the desired assay metric from the drop down menu to be exported oll Graph Export Located next to the icon Page 65 4 Select the desired time from the Time Tree located at the top left of the screen 5 Right click and hold anywhere within the table displaying the assay metrics 6 Use the mouse to drag it into the target software and release the mouse button 7 6 4 Saving and Printing Graphs Graphs can be saved or printed using the File pull down menu at the top of the plot If a graph has been created and saved within the IncuCyte software it can ONLY be opened again through the IncuCyte software using this menu selection If the graph file is selected by another method for example just locating the
77. gnificant amount of data users will eventually be required to clear space in order to continue acquiring and processing data All vessel information can be backed up through a process called Page 74 N ESSEN BIOSCIENCE archiving Once vessels have been archived they should be deleted from the IncuCyte controller in order to free hard drive space for future scans Archiving is a data management tool used to keep historical records of your vessels Important vessels that have been fully analyzed should be archived for emergency back up purposes Importantly image data contained within archives cannot be re analyzed While regular archiving is essential for data management it is recommended that vessels remain on the controller as long as they are being actively evaluated The archiving process copies the selected data from the controller to a new location Data is not deleted from the IncuCyte controller as part of the archiving process Once archiving has completed successfully without errors the original Vessel data should be deleted from the controller to clear hard drive space for future scans Once deleted Vessels CANNOT be retrieved Image data contained within archives CANNOT be re analyzed 7 8 1 Archiving Vessels To initiate archiving select the Archives option located on the left side task list Once selected the Create New Archive table is active displaying all vessels available for archivi
78. h option found in the left task list pane Once the Search screen is displayed Users can search the entire Scanned Vessels table using the Find field or search within individual columns The searching tools within the IncuCyte ZOOM software auto filters the table as soon as Users begin typing at the beginning and within words thus quickly narrowing down to a limited list of scanned vessels Users can also place quotation marks around search terms in order to search the complete string NOTE Searches are NOT case sensitive Use the green Refresh button located at the lower left corner of the screen prior to searching to ensure that the table is updates with the most recent data 1 Using the Find field searches the entire Scanned Vessels table Use the Clear button located next to the Find field to remove filter results Search a specific column by clicking on the first row of the column Column searching offers the auto sort when typing as well as multiple other functions which are listed in the table below Refer to Table 4 for details NOTE Because the column search tools use the and function to filter Users are able to filter scanned vessels using multiple columns For example you can use the User Column to narrow down to an individual and then sort using other column fields such as Label Color Data or Cell Type Table 4 Column Search Allows you to sort and customize the column For exa
79. have selected at the top of the screen Preview Image Collection This will likely be the same Image Collection used as the Training Image Collection NOTE The Processing Definition can be tested on several Image Collections within the editor if desired m Ty Sec nj s Toa pol foh Preview image Colection Detads Job Type Basic Analyzer Required Image Channels Vessel Label HT1080_ParVsRed_FBS image Stacks 3 Phase 1392 x 1040 1 22 un pocel SEE ara is oe Tene 5 29 2012 259 PM Fiter Module Dual Color Model 4459 Red Not Required aea Gai a Cleanup l Hole Fil m3 0 0 Adjust Size pixels 0 Filters Area um F min 0 000 max 0 000 anon fr ny aaa a f F min 0 000 F max 0 00 i 5 Figure 16 Establishing a New Processing Definition 6 Click the Preview button NOTE The best way to begin setting up the Processing Definition is to use the preset values already contained within the Processing Definition Editor i e do not change Segmentation Adjustment Cleanup or Filters at this time Page 43 N ESSEN BIOSCIENCE The color banner at the top of the Processing Definition window is color coded to reflect change in the processing mode The un previewed window will have a yellow banner while the previewed window will have a beige banner 7 When the Preview Status is complete select t
80. he Confluence Mask and evaluate your phase segmentation by previewing all images within your image collection black circle Figure 17 Prevew Zooms00 a Zoom User Sean Type Stated Tee 5 29 2012 259 PM Segrertmon T mage Ste H1 image 1 Mew Vessel a fal Show Legend X al Groen remowed from Ped Lot Red semoved from Green ee S eae Avg Integrated Total Integrated Image Channel Confluence Percent Count 1 Image Avg Area ym Avg Eccentricity vg cu tensity Intensity CU x Intensity CU x pm 2 Image pm Image gt Phase Contrast 14 45693 312 998 4173 0 38086048 N A N A N A Figure 17 Processing Definition Preview To refine the mask use the Segmentation Adjustment slider to bias the segmentation more towards Background to eliminate background or towards Cells to include more cell area Use the Cleanup options to further manipulate your mask Hole Fill removes any holes in the cell mask that are greater than the area specified Adjust Size grows or shrinks the mask by the number of specified pixels When satisfied that the Processing Definition appropriately segments all images within your Image Collection save the Processing Definition File gt Save and give it a unique name 7 5 2 3 Launch New Analysis Jobs Once a processing definition has been defined for an assay cell type or magnification Analysis Jobs can be per
81. he User under normal circumstances Select Statistic Image Mean Median When multiple images are available within each well then selecting the Mean vs Median will select the image Mean or image Median respectively for graphing If a single well is graphed then each point on the graph will represent the Mean or Median for all images in that well at that time Trace Mean Median When multiple wells are represented by each point on the graph for instance if all wells within a column are combined to generate a graph of that column then the Median or Mean refers to the combination of the individual wells Select Start and End Time The Time Range over which the data are to be graphed can be selected using the Start Time and End Time radio buttons to the right of the Time Tree In order to graph the Time Range option must be selected in the box above the Start Time radio button The alternate selection Single Time is only applicable to data export and Histograms The default settings are the times and dates of the first and last scans in the series The chosen Start and End times will be highlighted in blue and pink respectively After the Start or End Time radio button is selected the time can be changed by Directly selecting a time with the mouse zd Using the arrow buttons to sequentially scroll through the times until the chosen set point has been reached Graphing Microplates The Microplate Graph Users can use
82. he option to Accept Changes and Clear Schedule which changes the objective magnification in the software and completes the activity Alternatively Users can Cancel without changes but doing so will leave the objective change process incomplete NOTE The User can confirm the change in magnification by checking the Device Status log on the lower left section of the screen The reconfigured objective should be listed Page 80 UN ul _ N 2 A L ps gt gt co j 3 LOr kt 7 10 Plate Map Editor To assist in experimental design and data analysis the IncuCyte ZOOM software includes a Plate Map Editor that allows the User to custom design a plate map for any microplate based experiment The User may design and save a plate map at any point before or during an experiment or after the experiment has been completed Access to the Plate Map Editor is available from four locations Main IncuCyte screen Select Plate Map in the File menu at the top of the window From this location the User may save a plate map to a file but not to an individual vessel Z Schedule Scans screen under the Properties tab Vessel View window under the Properties tab amp The User may open the Plate Map Editor independently of the main IncuCyte software by browsing for the IncuCyte GUI program group via the Windows Start menu in the lower left of a Windows desktop MN With the Plate Map Editor
83. he vessel by clicking and dragging with the mouse Page 69 N ESSEN BIOSCIENCE e Deselect one or more individual wells sectors by holding the Alt key and then clicking with the mouse e Deselect a region of the vessel by holding the Alt key and then clicking and dragging with the mouse e Right click on a well sector to activate additional options for selection deselection It is possible to individually select up to 9 images per well sector for export if the scan pattern includes 9 images or less However it is also possible to set a scan pattern that includes more than 9 images per well sector In this case there will be two options for image export Export all Images or Export Image ___ of Total os a KAA A A OG K L L A A O a KOK OKOK A KOK K eeaaaagd DOOD Oz SOOLOGOD DD ADADDA 4 5 A e a a D amp o g D A a m e Save Location and Options Target folder C Users Example Desktop Browse Example Sequence type Set of individual images i Example_A1_1_2012y04m27d_13h44m jpg So ee Figure 24 Movie Image Export Window 2 Single Time Image Export e Select Single Time instead of Time Range Then use the arrow buttons to scroll to the appropriate time or use the mouse to select a time e Select the sectors or wells for image export Remember only the selected images for the highlighted time point will be exported e f multiple images were obtained per wel
84. irable to connect and power the unit within a few minutes of installation For connecting the cabling see the rear panel of the IncuCyte ZOOM controller Figure 8 There are two cables that connect the IncuCyte ZOOM controller to the microscope These cables which are fixed to the microscope should be connected to the controller before the unit is powered on The larger circular style plug on the microscope main cable should be connected to the mating socket on the back of the controller The second cable is a gigabit Ethernet interface to the digital camera in the microscope and it should be plugged into the mating connector labeled camera on the rear of the controller The power cord for the unit can now be installed Use only the AC power cord supplied or an equivalent cord with an IEC 320 C13 standard connector approved for operation at 5 amps and the appropriate voltage for your local power Page 15 3 7 3 8 Microscope Main Cable Camera Ethernet Connection ABBR BeBe Uomo GUO e ME p OO ico al HO OO conoid aeoOO0 AOmUL Network Connection Power Input SAS Drive Expansion Figure 8 Controller Rear Panel Switching the IncuCyte ZOOM On and Off The power switch on the IncuCyte ZOOM can be found under the front panel cover The system can be started or stopped by quickly depressing and then releasing the power switch When the system is running it may take 30 to 60 seconds
85. is is a re occurring problem or affects data analysis Images appear blurry Vibration can negatively affect image Za A z Ael quality The incubator that contains your IncuCyte ZOOM should be on a sturdy stand If possible try to schedule scans at times when the incubator will not be accessed as opening and closing the incubator door can cause image blurring IncuCyte photographed condensation Be sure to avoid condensation on the present on the outside bottom of the insides and outsides of all vessels prior to plate instead of the cells placing them into the IncuCyte Allow J 3 AN time for the vessel to equilibrate to the temperature of the incubator prior to initiating scans approximately 15 20 minutes Page 90 N ESSEN BIOSCIENCE PROBLEM Fluorescent cells are out of focus using the 20X objective SOLUTION Users planning to image in fluorescence with a 20X objective should investigate the best setting for their system and their vessel Adjusting the corrective ring to 1 4uM in this case was the optimal setting Page 91 Contacting Technical Support If you are experiencing technical difficulty with the IncuCyte or there is a Device Error in the status bar follow the instructions below to export and email a log file to Essen BioScience technical support refer to Figure 31 Log into the IncuCyte software GUI with an account that has administrator permissions Click on A
86. ist of destinations Favorites Libraries etc Look for the Network destination and select this by left clicking Look for the computer named ZOOMXXXXX recall that XXXXX is the 5 digit controller serial number If you find it in the window then the controller was successfully added to the network and you are done If not you should attempt to find the controller on the network by continuing to the next step At the top of the window there is a text area that reads Network Left click in an empty area to the right of this text and type the following ZOOMXXXXX followed by the Enter key If the controller is found on the network you will be prompted for a User name and password Press the Cancel button to exit If the controller is not found then a Cannot Find message will be displayed after a brief search period 4 1 2 Verifying the IncuCyte Controller Network Connection Windows XP 1 Open My Computer from the PC s Windows Start menu Under the heading Other Places on the left side of the window open My Network Places Under the heading Network Tasks on the left side of the window click once on View workgroup computers Look for the computer named ZOOMXXXXX recall that XXXXX is the 5 digit controller serial number If you find it in the window then the controller was successfully added to the network and you are done If not you should
87. k twice on the graph background area and the Customization Window will appear as shown in Figure 19 This window can be used to set a variety of graph attributes including graph style axis font etc Select the various tabs to explore all available graph related features Title Subtitle and individual well sector names however CANNOT be changed using the Customization window To assign new labels to wells or sectors select the Edit option at the top left of the graph window This will open the Titles and Labels window This window provides an opportunity to change the Title and Subtitle as well as the means to assign new well and sector labels All default or previously set labels will appear in the Plot Labels box while the currently highlighted label will appear below To change a label simply highlight the desired Plot Label with the mouse and then type the new label into the box underneath Do not click OK until you are ready to close the Titles and Labels window Clicking on the Advance button will bring up the Customization window The Customization Window can also be accessed by double left clicking on the graph Page 54 7 6 1 3 Drag and Drop Alignment Settings Task Pane The Drag and Drop Alignment Settings can be visualized by opening the Tasks Pane at the bottom of the Graph Window Click on the arrow on the blue bar at the bottom of the Graph Window to open or close the Drag and Drop Alignment Tasks Pane 7 6 1 4
88. l be discussed here Page 56 LN ESSEN BIOSCIENCE Outlier Removal This function facilitates the increasingly stringent removal of outlying vessel data Selecting Outlier Removal will reduce the standard deviation of the Metrics The outlying data are selected automatically by the software according to an algorithm points cannot be excluded manually Select from None no outlier removal Standard or Aggressive Whenever a graph is created it will conform to the currently selected degree of stringency None is the IncuCyte default setting and it is recommended To improve the appearance of a noisy curve try using the Smooth Feature Increasing the stringency of the Outlier Removal will not necessarily improve the appearance of a curve Graph Drag and Drop Auto Alignment When graphs are dragged and dropped to create a plot overlay they can be set to automatically align at a specific point on the Y axis Select the Drag and Drop Auto Align at Y Value when Drag and Drop occurs check box in the Preferences Window The Drag and Drop Alignment selection selected or un selected chosen via the Preferences window will apply to all graph overlays but the numerical value at which alignment occurs MUST be set using the Graph Window To select the numerical value for alignment use the Auto Alignment tab at the bottom of the Graph Window and choose the value at which alignment will oc
89. l be discussed individually Layout Use this section to select the overall Layout of the exported data The data for each scan time can be displayed within a single long row or displayed as its own table Show Each Scan as a Single Row In this case each scan time will be listed to the far left and the data for each well will be displayed as a single row to the right In Figure 22 the data are displayed as a single row In this figure Region All Wells Group None were selected prior to opening the Export Metrics window NOTE Wells from which no data were obtained are omitted Experiment Corning 384 Date Time Elapsed Corning 384 Al Corning 384 E1 1 17 2008 16 30 0 4 28 9 21 1 17 2008 19 30 3 5 53 10 4 1 17 2008 22 30 6 6 57 12 6 1 18 2008 1 30 9 8 18 LAS Figure 22 Single Row Export Page 63 Show each Scan as its own Table Alternatively the data can be displayed as a single table one table for each scan time This option will only be available if Group None was selected in the Graph Export setup prior to pressing the Export button If each scan is displayed as its own table the columns labels can be displayed or not by checking the corresponding check box If data are exported in the form of a table then wells or sectors from which no data were obtained because they were skipped in the scan pattern will appear as blank spaces Destination Choose a destination for the exported data Th
90. l or sector select which images to export If the images are exported in bulk then the same image will be exported for every selected well or sector e Next use the Browse button to select the target file location e Select the Sequence Type Recall that images can be exported as movies or images In this case only images are desired so highlight Set of Individual Images in the Sequence Types drop Page 70 7 7 1 3 down menu The movie options will still be available in this menu but if you try to export single images as a movie you will get an error message e Choose Image Type e Enter the desired name into the Name Prefix box The prefix will be automatically assigned additional time and area information that will appear in the Example box below e Select Export The Export button will display the total number of images files to be exported 3 Time Range Image Export Time Range Image Export is similar to Single Time Image Export and a review of the Single Image export instructions will be useful e Select Time Range instead of Single Time in the drop down menu adjacent to the Time Tree e Use the mouse or the arrow buttons to select the scan times for export All images including and between the selected times will be exported NOTE It is NOT possible to select non consecutive times for export e Select the desired wells sectors and images e Use the Browse button to choose the destination file
91. l plate will generate a graph of all 24 wells ry ry Single Column or Row In this case the only region of interest is a specific Column or Row within the microplate Selecting one of these regions will open a new drop down menu to facilitate further selection of the specific column or row of interest Single Columns and Rows offer only two grouping options All or None Either all the wells within the selected Column or Row can be combined to generate a single trace or none of the wells within the selected Column or Row will be combined resulting in multiple traces one for each well Single Well No grouping options are available for graphing a single well Scroll through the possible wells to find the well that is desired for graphing This selection will generate a single trace for the selected well Error bars are not available when a single well is graphed Single Well 96 and 384 Well Plates High density microplates microplates containing 96 or 384 well wells have a modified selection format for graphing a single well Because scrolling through all 384 wells to select just the single well desired for graphing would be inconvenient in the case of high density plates it possible to independently select both the Row letter and the Column number Custom Region Page 51 When Custom Region is selected the vessel color will change from light orange to green thus alerting the User that this is a special gr
92. l tab is selected you will be required to enter information that defines your specific assay 7 3 1 1 Vessel selection In the Drawer Setup pane determine which tray position front middle or rear you would like to place your vessel and then use the Add Vessel button to select a vessel type 1 Once the vessel selection window is open you will be able to search a table for your vessel of choice The IncuCyte software automatically recognizes the tray type required for each vessel and will display the tray type below the vessel selection table 2 At this time highlight an empty vessel position within the tray and click the OK button Once the vessel selection is completed the Scan Setup and vessel Properties tabs become activated 7 3 1 2 Scan Setup Tab At this time select the channels required for image acquisition of your vessel as well as the scan mode scan pattern spectral un mixing and analysis job setup Refer to Table 2 for details Page 29 Channels Scan Mode Spectral Un mixing Analysis Job Setup Table 2 Scan Set Up Scan Set Up Tab Select phase green or red or a combination of the three NOTES Acquisition time is the amount of time that the LED will illuminate the specimen The default acquisition times for the green and red channels routinely work well If phototoxicity is a problem lower the acquisition times If very faint objects are being imaged ona low background objects and ba
93. llows invading and migrating cells as they move into the initial wound creating a measurement of Relative Wound Density Angiogenesis Analysis Module The Angiogenesis Analysis Software Module is used to identify the fluorescent signal from background in order to quantify multiple angiogenic assay metrics such as tube length and branch formation for each time point In the first step of the process the angiogenesis algorithm analyzes each fluorescent image and assigns a segmentation mask that closely resembles the in vitro network From here the mask can be refined and filtered to exclude non tube forming events specifically measuring angiogenesis over time 8 3 1 Tiled Field of View Mosaic Imaging Mode The Tiled Field of View FOV mosaic image scan type stitches together six images from a 2 x 3 grid to produce a larger more integrated view of the angiogenic network Figure 30 Because the scanning process acquires 6 images per well the scan length will be longer than that for a plate run using the 4X objective Page 86 3 4 One well of a 96 Well Plate Figure 30 Diagram of Tiled Field of View The Tiled Field of View FOV is available when scanning Corning 96 well plates at 10X If a 96 well plate is selected as the vessel type the Scan Type drop down box located under the Scan Mode in the Scan Setup tab will include a Tiled Field of View option NOTE The 10X FOV is similar to imaging a 96 well plate with a 4
94. me size and is inserted in the same fashion as the regular trays Each of the three tray positions front middle and rear must be calibrated individually What follows is an overview of the calibration procedure FA OKOK KoKo xe O CALIBRATION TRAY Figure 11 The Calibration Tray Page 24 N ESSEN BIOSCIENCE Z incuCyte ZOOM File View Preferences Help z00m40004 Admin Ready To Scan Task List A View Scans A sew Schedule Scans 3 Archives f Administer Optics Reconfiguration Device Accounts Tests update Delete Logs Filter Module Dual Color Mode 4459 Dual Color Mode 4459 Dual Color Model 4459 Date Time 1 21 2013 2 50 00 PM 2 11 2013 1 10 00 PM 1 21 2013 3 01 00 PM Confirm Test Date Time 1 15 2013 5 52 00PM et 9 10 2012 4 31 00 PM ieee 9 10 2012 4 41 00 PM Pass 2 11 2013 1 33 00 PM Pass 1 14 2013 5 01 00 PM Pas 1 14 2013 5 07 00 PM ize 1 15 2013 5 32 00PM ees 9 10 2012 4 23 00 PM ieee 9 10 2012 4 14 00 PM Pas Select a tray front middle or rear Select Confirm Test Tray Position Front X Confirm Test i Run Filter Module Dual Color Model 4459 Dual Color Model 4459 4x Dual Color Model 4459 Date Time 1 15 2013 6 01 00 PM 1 15 2013 6 25 00 PM 1 15 2013 5 46 00 PM View calibration results by clicking on View Test Results button Figure 12 Calibration NOTE Calibration can ONLY be performed b
95. mple Right Click you can sort edit the script language or remove or add a column Left Click ital on the funnel icon in the upper right corner to Ascending or descending sort tool Filtering using the Start Date Time column requires you to right click on the funnel icon in the upper right corner A calendar is displayed showing the current month and day To drill out simply click on the month This then displays a yearly calendar by month You can further drill out by clicking on the year To clear a filtered column search in order to begin a new search click on the X in the lower left corner of the Search screen Page 36 LN ESSEN BIOSCIENCE 5 Once your vessel of interest is located either double click on the listed vessel or use the View Vessel button in the lower right hand corner of the screen The search tools used to search for scanned vessels are found throughout the software Data such as image collections processing definitions and job analysis as well as searching for vessel types in the scheduling window all use the same unified search concept Vessel View Once the vessel of interest is opened the Vessel View window opens The View Vessel window allows Users to view images collected export images or movies and update vessel associated properties 7 4 2 1 7 4 2 2 7 4 2 3 7 4 2 4 Menu Bar The Vessel View menu bar includes drop down menus for Utilities and View
96. nditions any local temperature rises will typically be less than 1 degree C and relatively short lived The IncuCyte is capable of collecting nearly 900 images in 30 minutes in all three channels so another good reason to avoid such over scanning is to limit the amount of data collected This will keep the system more responsive We have found that between 0 5 and 1 images per square centimeter is usually sufficient to characterize the growth of cells in most tissue culture vessels For example 48 images will usually characterize a T75 flask very well With good technique that results in uniform seeding even fewer images can be used Also with some slower growing cell types scanning every 4 or even 6 hours may be sufficient 8400 0011 C00 Page 94
97. ng Users can search the entire Archiving Vessels table using the Find field or within individual columns Refer to 7 4 1 2 for details on the searching tools within the IncuCyte ZOOM software In order to save hard drive space and to maximize archiving efficiency itis recommended to review scanned vessels and their associated analyses jobs and eliminate any data that is unnecessary Important concepts to remember while creating an archive are listed below Z Due to the volume of data involved archives are more likely to be written successfully when the archive size is kept small Essen BioScience recommends archiving data on a monthly basis or more frequently Z Smaller archives are less likely to be interrupted by system reboots logoffs Windows updates or network interruptions when archiving to My Computer see below Z Asmall archive is more transportable Z Archives contain ONLY vessel images and their associated analysis jobs Image Collections and Processing Definitions CANNOT be archived Page 75 7 8 2 Once the vessels of interest are sorted e g by date or by project Users may select vessels individually or use the Select All button located in the lower left corner of the window Select the Create Archive button in the lower right corner of the window and you will be asked to specify the archive destination Users have the option to select either My Computer or Storage Atta
98. nition It is within this Processing Definition that Users set the parameters that will be applied to all images within current and future data Save Processing Definition 3 Once the Processing Definition is established it should not need to be created again The tested finalized and saved Processing Definition is then used in the Established Assay Phase Launch Analysis Job on a Vessel 7 5 1 2 Established Assay Phase Once the parameters are defined in the Assay Development Phase Users can apply those parameters to all current and future experiments at the time of assay initiation scheduling Analyze Export Results 7 5 2 Analysis Job Utilities In the Assay Development Phase Users will be required to define the analysis parameters for their vessel This task can be completed by using the Analysis Job Utilities found in the upper right hand corner of the View Vessel screen The steps required to transform qualitative images into quantitate data are discussed below 7 5 2 1 Add to Image Collection Under Vessel View Users must review the scanned vessel and find representative images to add to the image collection An Image Collection is a user defined set of image stacks from one or more vessels grouped together to act as a training set An Image Stack is a set of images from one location well or sector and time that could include multiple channels phase image green fluorescence image and or red fluorescence ima
99. om right of the Movie Image Set Export window Click the Movie Designer button and a new window appears that is virtually identical to the Export Designer shown in Figure 23 of the Export Current Image Single Image section As discussed in that section the designer is used to interactively crop and scale movie frames and to add a legend or timestamp to each image if desired If a Timestamp is added then a timestamp that displays the corresponding time days hours and minutes will appear on each frame of the movie If Show Legend is selected a scale bar and field of view dimensions will appear in the exported image or movie as they do in Vessel View In Page 73 addition to these customization options there is also a frames per second parameter that can be adjusted from a minimum of 1 to a maximum of 30 frames per second When the mouse is placed over this control a thumbnail preview of the movie at the selected frame rate is shown as an aid As mentioned when discussing the Export Design window perhaps the most important tool found in the Export Designer is launched via the Output Preview button in the lower left corner of the Export Designer We strongly encourage you to preview your customization settings prior to export The preview allows you to view a still movie frame at the exported size as well as in a full screen mode that fills your monitor This is often important when considering the size at which
100. onnel The process is described in the sections below and should be followed if you ever move the system to a new location The IncuCyte ZOOM System is shipped in two boxes one containing the automated microscope and the other containing the controller NOTE The IncuCyte ZOOM consists of 2 components the controller and the microscope The controller remains outside the cell culture incubator while the microscope is place inside the incubator and holds the cells for monitoring These two components are shipped in separate boxes To unpack the microscope start by setting the box with the correct side up on the floor or on a sturdy bench top Start removing materials from the top as shown in the exploded diagram Figure 1 Page 8 UN ESSEN z a See ee Figure 1 IncuCyte ZOOM Microscope Packaging The microscope weighs approximately 44 pounds so it is recommended that one person hold the box while a second carefully removes the microscope from the bottom foam piece and places it on a stable work surface The unit should then be removed from its plastic bag not shown in Figure 1 and inspected for any obvious signs of external damage The controller should be removed from its box and bag in a similar fashion see Figure 2 The controller box will contain the controller and under most circumstances it will also include a power cord appropriate for your local utilities However while Essen provides power c
101. op outside of vessel Touching either the tops or bottoms of vessels during manipulations Hold vessels by the sides Z Writing labels on the tops of vessels The writing will interfere with the images Label vessels on the sides or on the very edges of the tops where imaging will not occur Z t is VERY IMPORTANT that all lids are tightly fastened onto the flasks If the lids are loose it can cause the flasks to become displaced within the tray Z Besureall vessels are sitting COMPLETELY flat in the trays Page 88 N ESSEN BIOSCIENCE 9 1 Images and What they Mean The edges of the wells seem to be out of focus at the 4X objective Focus issues caused by air bubbles on the bottom and top of the well Visual edge effects can be eliminated by increasing the total media volume in each well However this will not dramatically change the proliferation data Sufficient proliferation data can still be generated with only 100 uL in each well Air bubbles must be manually removed by the user to obtain desired images Air bubbles should be removed by modifying pipetting technique or using a de bubbler before placing vessels in the IncuCyte ZOOM Page 89 N ESSEN BIOSCIENCE Striations and scratches are visible on the Striations and scratches are an artifact of bottom of the well the vessel being used and usually will not affect the data analysis Users should consider using a different vessel if th
102. open the default screen is set to a blank 96 well plate layout To change the plate layout click on the New Plate Map blank sheet icon on the menu bar at the top left of the window The Plate Map Editor supports plate layouts from 6 well to 384 well formats Refer to for Figure 29 for an overview of the Plate Map Editor To load a plate map already saved to a file click on the Open folder icon on the menu bar and browse to the file Z Plate Map Editor i o x Joe a alala Laul hect areas New Save Open ua wel Keep current selection miis op U Sus Ven tO Right click a selection to edt well contents Compounds Celis Growth Conditions Regions Compounds used in this plate Well items Add Compound to Plate ud Plate Map Notes Export Plate Map OK Cancel Figure 29 The Plate Map Editor Page 81 N 7 10 1 Selecting Wells To add well items cells compounds etc to a well or grouping of wells the desired area must first be selected on the plate layout Left click on a well or click and drag over a group of wells to select a specific area of the plate in which to work Wells may be deselected in the same manner To easily select or deselect entire rows or columns click the row column label along the border of the plate Select and deselect all wells in the plate by clicking on the All button on the top left of the plate Immediately to the left of the All button is a blank plate icon
103. ords compatible with most utilities there will be instances in which Essen does not stock the compatible cord In these rare circumstances the purchaser will have to provide their own cord IMPORTANT Use only the AC power cord supplied or an equivalent cord with an IEC 320 C13 standard connector approved for operation at 5 amps and the appropriate voltage for your local power Also ensure the local line voltage falls within the range printed on the IncuCyte ZOOM controller HH Page 9 u 2A L VAY a ne Figure 2 IncuCyte Controller Packaging If the device is damaged in shipping please call Essen BioScience immediately You may then be asked to return the unit Chapter 3 Installation of the IncuCyte ZOOM Hardware 3 1 IncuCyte ZOOM Hardware Features The diagrams below Figure 3 and Figure 4 identify many of the hardware features buttons connections indicators and displays that will be referenced throughout this manual Page 10 ESSEN BIOSCIENCE LN Power Button LCD Panel DDOOOOO DOO JOOUUUOUUL oggpgggggo noooooooo Reset Button Figure 3 Controller front view with access door open Eject Cancel Button Access Cover Figure 4 Microscope front view Page 11 a ESSEN BIOSCIENCE Removing the Shipping Pin The IncuCyte ZOOM ships with objectives and filter modules pa
104. osen The scans for which data will be available for export are selected using the Time Tree in the graphing box prior to selecting the Export button The default settings are the first and last scans available for the selected vessel The User has the option to select specific Time Ranges by using the mouse or arrow buttons on the Time Tree The User can select the entire time range or a subset within the range If only the data from a single scan need to be exported choose Single Time instead of Time Range from the drop down menu above the Start Time radio button If Time Range is selected then the data set that is exported will be a continuous set within the given time span It is NOT possible to simultaneously export data from individual temporally discontinuous scan times If discrete discontinuous Page 62 data sets are desired then each data set must be exported individually Single Time exports Which regions of the Export Metrics window are active are determined by selections made within the Export Metrics window as well as those Region and Grouping settings made prior to opening the Export Metrics window For example under the Layout heading Show Each Scan as its Own Table can only be activated if the Group option was set to None during the Graph Export Setup The Export Metrics window is divided into 3 main sections Z Layout Z Destination Z Other Options Each of these sections wil
105. own Menu Selecting the graph window pull down menu presents three additional graph related options File Use the File Menu to open save or print a graph or to close the window Edit The Edit Menu provides the option to edit the graph title and labels associated with traces on the graph View Use the View Menu to select different error measurements standard error or Standard deviation change the y axis to a log scale or view filters from an analysis Tools The Tools Menu provides options to edit how the data are viewed on the graph Smooth Lines Sometimes if the data are particularly noisy the Smooth feature might be more useful than Outlier Removal When Smooth is selected a new window will appear to facilitate selection of the smoothing size i e the number of points over which smoothing will occur If 3 data points are selected then each point on the graph will be averaged using its two neighboring points NOTE It is important to remember that smoothing is a tool for generating cleaner curves when excessive noise prevents useful interpretation of the results The smoothing feature should be used with discretion in order to prevent overt distortion of the data Estimate Rate of Change When rate of change may assist data analysis such as with cell migration assay this option will provide an estimated rate for each point on the graph Average Lines Together Create a graph showing the average of all plots on the curr
106. rage is removed from the IncuCyte controller and connected directly to a local computer Essen BioScience recommends that Users attach and detach the external storage ONLY when absolutely necessary NOTE The function of creating archives is for historical data management and immediate access should not be the main priority Opening an Archive Archives located on My Computer can be opened the same way as connecting to an IncuCyte ZOOM with a User account Simply click on the IncuCyte ZOOM icon and when the Open Connection window appears choose the Open an Archive radio button and select Open Archive and browse to the appropriate file location Alternatively if Users are already connected to the IncuCyte ZOOM they can access archives located on My Computer by simply selecting the Archives option in the task pane followed by the Browse Archives Attached to My Computer tab Page 76 Archives that reside on ZOOM attached devices can be viewed through the View Archives Attached to ZOOM tab but cannot be accessed If access is desired the ZOOM attached storage must be removed and attached to your local computer Prior to removal be sure to check the View Archives In Progress tab to confirm that there are no active archive jobs IMPORTANT When using ZOOM attached storage do not routinely attach and detach the drive to and from the controller The drive should remain connected to the IncuC
107. reted by most image processing software packages Users who wish to export images for presentation applications such as MS Office should avoid the less common formats The fluorescence intensity of the objects and or background can change over time when generating a sequence of fluorescent images If the User does not want these changes to Page 66 N ESSEN BIOSCIENCE be reflected in the images then the Auto Scale check box should be selected prior to exporting a fluorescent image set To apply the Auto Scale settings to exported images the User MUST select either Z Blended Composite using CURRENT DISPLAY SETTINGS or Z Fluorescence Green or Red using CURRENT DISPLAY SETTINGS When generating a sequence of fluorescent images the fluorescent intensity of the objects and or background can change overtime Users should use the Auto Scale Always check box prior to exporting a fluorescent image set Additional image types are available for images that have been processed These image types can only be exported from open Analysis Jobs Confluence Confluence Mask 8 bit TIFF Object Count Counting Color 1 Mask 8 bit TIFF Counting Color 2 Mask 8 bit TIFF Angiogenesis Network Mask 8 bit TIFF Skeleton Mask 8 bit TIFF Migration Invasion Confluence Mask 8 bit TIFF Initial Scratch Wound Mask 8 bit TIFF Scratch Wound Mask 8 bit TIFF NeuroTrack Neurite Mask 8 bit TIFF Cell Body Cluster Mask
108. rkest possible value in the image in this case black Maximum Intensity Any portion of the image with a pixel intensity greater than or equal to the Maximum Intensity will be assigned a relative brightness equal to the brightest possible value for the display in this case bright green All portions of the image with pixel intensities between the Minimum and Maximum Intensity values will be assigned intermediate degrees of brightness Whether Min Max settings are set manually or via the Autoscale function they will remain unchanged until manually re adjusted or re autoscaled on a different image Therefore the Min Max settings applied to the original image will be applied to all subsequent images that are viewed within the same vessel at all scan times or even to images from different vessels If images are combined to generate movies each frame of the movie will contain an image with the same Min Max settings Tools The Tool task pane located to the right of the image allows you to zoom in or out of an image by using the slider bar Dragging the slider bar will zoom to the center of the image however the area of the zoomed image being displayed can be changed by grabbing the image and dragging it with the Page 38 mouse Alternatively it is possible to zoom directly to a specific area within the image by scrolling the mouse wheel over the area of interest Z The Tool task pane also includes a measurement tool Use Measurement
109. rt with a static IP address selected for your corporate network Once on the desktop the controller s primary Ethernet port can be configured with a static IP address in the same way that the PC s primary port was configured Use the Windows 7 instructions for the controller configuration After configuring the primary Ethernet port log off of the controller s desktop Unplug the monitor keyboard and mouse from the back of the controller and reinstall the cover on the back of the controller Chapter 5 Installation of the IncuCyte Control Software The IncuCyte ZOOM control software is used to both configure the instrument to collect data and for post collection viewing and analysis of these data This software is designed to run on PC s running Windows XP Windows Vista or Windows 7 and requires an Administrator account to install Contact Essen BioScience Support refer to section 2 3 for support contact information to get the most recent version of the software Once downloaded run the installer by double clicking onthe installer file NOTE This version of the IncuCyte Control Software is not backwards compatible with IncuCyte microscopes that are running earlier versions of the software Contact Essen BioScience technical support for instructions for updating controller software 5 1 IncuCyte Control Software Installation using Windows XP 1 Review the license agreement To continue installation you must agree
110. s NO OTHER WARRANTY IS EXPRESED OR IMPLIED ESSEN BIOSCIENCE INC SPECIFICALLY DISCLAIMS THE IMPLIED WARRANTIES OR MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE 1 2 Exclusive Remedies THE REMEDIES PROVIDED HEREIN ARE BUYER S SOLE AND EXCLUSIVE REMEDIES ESSEN BIOSCIENCE INC SHALL NOT BE LIABLE FOR ANY DIRECT INDIRECT SPECIAL INCIDENTAL OR CONSEQUENTIAL DAMAGES WHETHER BASED ON CONTRACT TORT OR ANY OTHER LEGAL THEORY 1 3 Warnings and Disclaimers Doing any of the following may void your warranty Z WARNING DO NOT RUN THE STERILIZATION CYCLE ON AN INCUBATOR WITH THE INCUCYTE ZOOM GANTRY INSIDE Z WARNING DO NOT TURN OFF THE INCUCYTE ZOOM AND LEAVE THE GANTRY INSIDE THE INCUBATOR CONDENSATION BUILDUP CAN DAMAGE THE ELECTRONICS Essen BioScience is not responsible for data loss due to hardware failure Page 5 R ess Chapter 2 Getting to know your IncuCyte ZOOM 2 1 Intended Use The IncuCyte ZOOM is intended to facilitate live cell monitoring via customized imaging protocols It is intended for experimental purposes only Please read carefully through the operating instructions in this manual to ensure that you can properly and safely utilize all features of your new instrument 2 2 IncuCyte ZOOM Configurations The IncuCyte ZOOM is user configurable through the installation of objectives 4X 10X 20X filter modules and software modules The use of fluorescence filter modules and
111. s within the preview window Similarly the cropped rectangle can be moved by clicking and dragging within the current boundaries The final size of the cropped image in pixels is displayed below the Scaling control Use this control to adjust the size and resolution of the final image As the scaling increases the final size increases and vice versa This allows you to customize the final image size which can be very convenient when exporting data for presentation formats By increasing the scaling you can digitally zoom in on a small cropped area NOTE The sizes of both the scale bar and timestamp in pixels are fixed and will thus appear differently depending on scaling Experiment with the Scaling option to see how this affects both the timestamp and legend on the final image display options are applied The Output Preview allows Users to view the final exported result after cropping scaling and other We strongly encourage you to preview your customization settings prior to export via the Output Preview button found in the left hand corner of the Export Designer window Once you are satisfied with the customization click OK to exit the Export Designer 5 After the image type file destination and optional customization has been completed select the Export button Page 68 UN ESSEN BIOSCIENCE Litidee a Anaiyss Job Usites A 12 00 AM 1234667 8 8 10111
112. scent movies Additional image types are available for images that have been processed These image types can only be exported from open Analysis Jobs Confluence Confluence Mask Object Count Counting Color 1 Mask Counting Color 2 Mask Angiogenesis Network Mask Skeleton Mask Page 72 Migration Invasion Confluence Mask NeuroTrack Initial Scratch Wound Mask Scratch Wound Mask Neurite Mask Cell Body Cluster Mask 7 7 2 1 Export Movie Movie export functions are accessed through the Vessel Window by selecting the Utilities pull down menu To export movies follow the steps indicated below Select Time Range in the drop down menu adjacent to the Time Tree in the Movie Image Set Export Window Choose movie start and end times The movie will include all times including and between the selected start and end times Choose the wells or sectors and the desired image numbers as described for image export It is possible to simultaneously export movies for all images from all wells sectors at the same time Choose a target folder Select the Sequence Type Select Image Type Select a Name Prefix As discussed in Export Image Set Multiple Images the Example box will be automatically populated If movie Sequence Type Windows Media Video MWV9 or Windows Video AVI is selected a button with the text Movie Designer Crop Scale Legend etc appears above the Export button at the bott
113. schedule will automatically be deleted To change the objective follow the steps indicated below 1 As mentioned above only ZOOM Users with administrative privileges can reconfigure the optics The administrator should log in and select the Administer tab on the left side of the screen under Task List The first tab on the Administer screen is Optics Reconfiguration which displays a box in the center of the screen showing the 3 steps required to change the objective magnification refer to Figure 27 Optics Reconfiguration Follow this procedure to change the objective magnification and or the filter module Step 1 Reconfigure the device via the optics access panel in the front of the instrument See users manual for instructions Step 2 Press the Configure button below to specify the new configuration installed in the instrument Configure Step 3 Create a new scan schedule using the new configuration Figure 27 Optics Reconfiguration Screen 2 In order to change the objective Users must open the small square shaped optics access panel on the lower right hand side of the IncuCyte ZOOM instrument by pushing down and pulling forward on the panel door When this door is removed Users will have access to the objectives and filter cube The User can open the ZOOM drawer for better visibility as needed Page 79 N ESSEN BIOSCIENCE Use a 2 5mm Hex T handled driver to loosen and remove th
114. select the correct destination software from the Preferences menu on the main IncuCyte screen To drag and drop an image right click and hold on the image to be dragged and then use the mouse to drag it into the target software Release of the mouse button will cause a new window to open Selecting Move here will drop the image 7 7 2 Exporting Movies Overview Movies can be generated using a sequence of images within a selected time frame A single movie can be created and exported or multiple movies can be created and exported in bulk Because a movie by definition is comprised of a minimum of two images at consecutive time points movies CANNOT be generated if the Single Time option is selected in the pull down menu adjacent to the Time Tree Movies can be created in one of two common formats Sequence Type Windows Media Video or Windows AVI Video Fewer image types are available for movie export than for image export and the descriptions are fairly self explanatory Blended Composite using current display settings Phase Contrast using the current display settings Phase Contrast Original Green using the current display settings Red using the current display settings When generating fluorescent movies the intensity of the objects and or background can change over time If the User does not want these changes to be reflected in the movie s then the Auto Scale check box should be selected prior to exporting fluore
115. select the desired wells by left clicking over them then right click within the selected region to bring up the editing window From here the User can create a custom description of the selected well s delete items and change the color scheme of the well s Alternatively use the Undo and Redo buttons in the menu bar at the top left of the window if a mistake is made while editing a well s contents In addition text size font style can be changed using the font button in the menu bar Page 83 7 10 2 7 7 10 2 8 7 10 2 9 Creating and Using Regions Regions on a plate map allow the User to define a specific set of wells e g a dilution series which aids in graphing and exporting data from an experiment Once defined Regions appear in the Graph Export window in the Regions drop down list and allow graphing exporting of a custom grouping of wells Any replicates defined within the region are automatically detected and can be grouped by selecting Replicates in the Graph Export Group drop down list For example the User can create a region such that an entire dilution series along with negative and positive controls appears on the same graph without the need to drag and drop individual plots onto one graph In addition the labels on individual wells or groups of wells replicates will automatically appear in the figure legend of the graph To create a Region on a plate map simply select the
116. sscncusssessoussscecuesscneussscneousresseness 93 The manual is designed to allow the User to easily navigate throughout the document Use Ctrl F keyboard shortcut to easily perform keyword searches Hyperlinks are also embedded within the document When Users see a reference to a figure table or heading simply hover over the reference then use the keyboard shortcut Ctrl click to follow the link Page 4 Chapter 1 Warranty Your Essen BioScience Inc Essen IncuCyte product is warranted against defects in material and workmanship for a period of one year following delivery to the Buyer Essen warrants to its original Buyer only The Buyer must notify Essen in writing within fifteen 15 days following discovery of the defect During the warranty period Essen will at its option either repair or replace products that prove to be defective For warranty service or repair this product must be returned to the Essen factory Buyer shall prepay shipping charges to Essen and Essen shall pay shipping charges to return the product to the Buyer However Buyer shall pay all shipping charges duties and taxes for products returned to Essen from another country 1 1 Limitation of Warranty The foregoing warranty shall not apply to defects resulting from improper or inadequate maintenance by Buyer Buyer supplied software or interfacing unauthorized modification misuse or operation outside of the environmental specification
117. ssel is added to the schedule All trays need to be placed into the IncuCyte ZOOM drawer in the proper orientation To ensure proper orientation each tray contains three Alignment Windows at its outer perimeter one top left one top right one bottom right In Figure 25 the Alignment Windows are indicated by arrows Page 77 ESSEN BIOSCIENCE N Figure 25 Microplate Tray showing Alignment Windows The 3 tray Alignment Windows fit over 3 posts in the corresponding locations within the IncuCyte drawer When placing trays into the drawer make sure that the drawer Alignment Posts pass through the tray Alignment Windows see Figure 26 The tray should sit very flat and the posts should prevent any lateral movement Figure 26 Tray Placement Page 78 7 9 2 Changing Objectives The IncuCyte ZOOM provides researchers the opportunity for live content imaging at three different objectives 4X 10X and 20X Users with administrator privileges are the only users who can change the objectives within the software Changing objectives is manual therefore it is suggested that the User organize the workflow such that the objective is changed after a length of time as opposed to changing it on a daily basis In order to change the objective Users will want to be certain the current schedule is free of vessels and scans NOTE Once the objective is changed all vessels from the previous
118. that will also deselect all wells 7 10 2 Adding Well Items Before any information can be applied to a well on the plate map it must first be loaded into one of three lists to the left of the plate map Compounds are any treatments to a well where concentrations or dilutions must be specified Cells specifies type passage and seeding density for cells present on the plate Growth Conditions are additions such as growth medium where concentrations of a reagent will not vary or need not be specified on the plate map 7 10 2 1 Compounds 1 To add a compound to the plate map select the Compound tabs then click the New button below the blank compound list 2 In a new window enter a name to appear on the plate map and optionally a longer description to appear in the list of compounds Additionally select a color in which the compound will appear on the plate map when creating a dilution of a compound higher concentrations will appear as darker shades of the indicated color 3 After creating a compound description select the desired area of the plate and click Add __ 4 A new window will appear allowing the User to select a desired concentration and optionally a dilution series First select the units in which concentration will appear by clicking the drop down menu at the top of the window the default setting is mg mL and enter a numerical concentration to the left If only a single
119. the graph was created for example sorted by column vs row SUMMARY To drag one plot onto another within the IncuCyte graphing software select Drag and Drop as Graph on Graph To drag the graph image into MS Word or MS Outlook select Drag and Drop as Image to Word or Outlook To drag and drop the image into other software programs select Drag and Drop as Image to Other Document To drag and drop other raw data select Drag and Drop Raw Data to Document 7 6 1 5 Creating Averaged Graphs When multiple traces are included on a single plot either as a result of dragging or by graphing all wells sectors these traces can be combined to create a single Average Graph To do this select the Tools pull down menu from the top of the Graph window and then select Average Lines Together Select OK in the dialog box that appears and a new Average graph will be created in a new window Note that the Average Graph function does not generate a graph with error bars The Average graph function can be also used to combine graphs from different vessels and or from different days 7 6 1 6 Preferences Outlier Removal and Drag and Drop Auto Alignment When generating the selected Metrics the stringency of outlier removal can be adjusted by selecting the Preferences option from the top of the main IncuCyte screen to open the Preferences Outlier Removal Window Two features of the Preferences window wil
120. thmic y axis scaling S M L marker sizes hm iy iy im is In addition users can export images of the Microplate Graph by selecting one of 4 different file types PNG TIFF JPEG or BMP and selecting one of 5 different predetermined file sizes Print and Print Preview and Drag and Drop functionality have also been added to the Microplate Graphing option Page 61 N ESSEN BIOSCIENCE File Edit View HT1080 CMP intra assay All Wells Mean vs Time Object Count per mm over 2 days EOC C OCL OE SIII II II SSS F ASSA LUA AAA N LKKAAA L KAASS KKAARA LKAAAT LUA AAA LK KAAAR LK KANAB L A AASA Fa L L ALL At lai TEERESES 3 l E ae F a a a El Figure 21 Example Microplate Graph 7 6 3 Exporting Experimental Data Data can be exported by selecting the Export button underneath the graph settings Export parameters can be adjusted according to the desired Layout Destination and Other options 7 6 3 1 Exporting It is possible to export the Metrics associated with a particular vessel directly to another document or to a selected file There are a variety of options regarding the format in which the data are exported To export vessel Metrics select the Export button rather than the Graph button at the lower right corner of the graphing window Choosing the Export button will open a new Export Metrics window in which details of the selected export format can be ch
121. useceseeecessuneceeseneceetaeeeetas 13 3 4 WS eet PANTY Ah CIN E EEE PEET AT PEE E PETE 14 3 5 Po ONNE ne CO RU OCG rea acsteasadaute ceeds notea tense isesonenaatenseaeue acnsieenee dae 14 3 6 Placing the Microscope into the INCUDAtOL c cccseccccesseccccesececceecceceeseceeeeececeueceeseneceesenaeeetas 15 3 7 Switching the IncuCyte ZOOM On and Off cccccceseccccsseccccesscceceesececeesececceeceesensecessuseceseuneses 16 3 8 Warming up the Microscope cesricranansrnas iiie o anaE T S 16 3 8 1 PO NV I rai a E E E E E E 17 3 8 2 IncuCyte Controller Air Filter cccccccsseccccessccccesececeesececeesecceeeneecessuecesseneceeeeaeceesegeeeetes 17 Chapter 4 Establishing Network CONNECTIONS siciscssiusecscsacedndssardesenencccsioadtiounnnsavesnbsscteuannaiauanonedeaenenadedans 18 4 1 DACP B sed NETWORK orina AA A EE E E E E A E E E TS 18 4 1 1 Verifying the IncuCyte Controller Network Connection Windows Vista or Windows 7 19 4 1 2 Verifying the IncuCyte Controller Network Connection Windows XP cccccseseceeeeeeees 19 4 2 Static IP Based NETWOFK S cepere 20 Chapter 5 Installation of the IncuCyte Control SoftWware ssssssesessresesrrresrrreesrrresrrerssrrresereresrrresene 20 Page 2 N 5 1 IncuCyte Control Software Installation using Windows XP ccccccsssececeesececeeseceeeeeeeeeseeeeeeees 20 5 2 IncuCyte Control Software Installation using Windows Vista and Windows 7
122. y a User at the Administrator Permission level Place the Calibration Tray into one of the tray positions the order in which tray positions are calibrated is not important but all 3 must be calibrated Navigate to the Tests tab under the Administer IncuCyte task bar The Front Middle and Rear tray positions can be selected using the pull down menu Each Tray Position must be individually calibrated and confirmed The first time the instrument is calibrated select the Confirm Test check box to run the Calibrate and Confirm Test refer to Figure 12 The status if the Calibration and Confirm test completed successfully will be displayed in the far right column of the table labeled Result pass fail status is reported for each magnification and tray position combination You might need to click on the green Refresh button on the bottom to see the updated status A summary of calibration details and accompanying images can be accessed via the View Test Results button located at the bottom of the screen The calibration results will be displayed in a new window The results will be displayed as coordinate values The Log will also indicate whether the test passed or failed The detailed results of the Calibration and Confirm test can also be viewed in the Logs tab Select Logs and then use the pull down Page 25 6 2 menu under Log Type to select Tests The calibration results will be displayed in the large window
123. yte ZOOM until full WARNING If the ZOOM attached storage requires removal Users MUST check the View Archives in Progress tab to determine if archives are being processed or are in queue DO NOT power off or unplug the drive while in the process of archiving 7 9 Hardware 7 9 1 Selecting and Placing Trays Tissue culture vessels are held within the IncuCyte drawer in trays The IncuCyte drawer can hold a maximum of three trays and each tray will hold different numbers of vessels depending on the tray type A summary of all available compatible vessels and trays can be found at http www essenbioscience com support html under the document titled IncuCyte Supported Vessel List The IncuCyte can be configured with any combination of trays and vessels from this list While the document contains all vessel types and trays available at the time this software version was released it is likely that new vessels and trays will be added to the list as they appear on the market and as customer demand dictates Tray and vessel database update files are obtained by request from support refer to Customer Support contacts listed in Section 2 3 and will be delivered as a zip archive along with database update instructions via e mail In order to further facilitate tray selection each tray will have its Tray Type number stamped on the bottom portion of the tray Additionally the shape of each tray will be displayed when a ve

IncuCyte™ ZOOM Users Manual

Contents

Download Pdf Manuals

Related Search

Related Contents

IncuCyte&trade; ZOOM Users Manual

Contents

Download Pdf Manuals

Related Search

Related Contents

IncuCyte™ ZOOM Users Manual