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Essen BioScience IncuCyte ZOOM User Manual

Continuous live-cell imaging & analysis system
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IncuCyte™ ZOOM Users Manual

2013A

Essen BioScience, Inc.
www.EssenBioScience.com
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  • Page 1: Incucyte™ Zoom Users Manual

    IncuCyte™ ZOOM Users Manual 2013A Essen BioScience, Inc. www.EssenBioScience.com Page 1...

  • Page 2: Table Of Contents

    Table of Contents IncuCyte™ ZOOM Users Manual ........................1 2013A ................................1 Chapter 1: Warranty ..........................5 Limitation of Warranty ........................5 Exclusive Remedies ......................... 5 Warnings and Disclaimers ....................... 5 Chapter 2: Getting to know your IncuCyte™ ZOOM ..................6 Intended Use ...........................
  • Page 3 IncuCyte™ Control Software Installation using Windows XP ............20 IncuCyte™ Control Software Installation using Windows Vista and Windows 7 ......21 Connecting ............................ 21 5.3.1 Setting the Date and Time ....................21 5.3.2 Establishing User Accounts ....................22 Remote Administration of the Controller ..................23 Chapter 6: Calibrating the IncuCyte™...
  • Page 4 Archiving ............................74 7.8.1 Archiving Vessels ........................75 7.8.2 Opening an Archive ....................... 76 Hardware ............................77 7.9.1 Selecting and Placing Trays ....................77 7.9.2 Changing Objectives ......................79 7.10 Plate Map Editor ........................... 81 7.10.1 Selecting Wells ........................82 7.10.2 Adding Well Items .........................

  • Page 5: Chapter 1: Warranty

    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.

  • Page 6: Chapter 2: Getting To Know Your Incucyte™ Zoom

    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) Safety has Priority Please note the following directions for safe and problem-free operation of your IncuCyte™...

  • Page 7: Incucyte™ Zoom Specifications

    Should you have problems that require service, please contact: US Contact Information UK Contact Information Japan Contact Information Essen BioScience, Inc. Essen BioScience, Ltd. Essen BioScience, K.K. 300 West Morgan Road BioPark, Broadwater Road...

  • Page 8: Unpacking And Checking The Incucyte

    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 personnel. The process is described in the sections below and should be followed if you ever move the system to a new location.
  • Page 9 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.

  • Page 10: Chapter 3: Installation Of The Incucyte™ Zoom Hardware

    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 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 11 Figure 3. Controller front view with access door open Figure 4. Microscope front view Page 11...

  • Page 12: Removing The Shipping Pin

    Removing the Shipping Pin The IncuCyte™ ZOOM ships with objectives and filter modules packed 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.

  • Page 13: Installing The Filter Module

    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.

  • Page 14: Installing An Objective

    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.

  • Page 15: Placing The Microscope Into The Incubator

    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™...

  • Page 16: Switching The Incucyte™ Zoom On And Off

    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 for the system to save any settings and data before it powers down.

  • Page 17: Auto Warm-Up

    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...

  • Page 18: Chapter 4: Establishing Network Connections

    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.

  • Page 19: Verifying The Incucyte™ Controller Network Connection: Windows Vista Or Windows 7

    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™...

  • Page 20: Static Ip Based Networks

    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 on the installer file.

  • Page 21: Incucyte™ Control Software Installation Using Windows Vista And Windows 7

    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).

  • Page 22: Establishing User Accounts

    Figure 10. 5.3.2 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.

  • Page 23: Remote Administration Of The Controller

    Installing Essen-recommended Windows updates 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.

  • Page 24: Chapter 6: Calibrating The Incucyte™ Zoom System

    Run the “Remote Desktop Connection” software and type in the name of the IncuCyte™ ZOOM controller in the “Computer” edit box. If connecting 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).
  • Page 25 Figure 12. Calibration NOTE: Calibration can ONLY be performed by 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™...

  • Page 26: Fluorescence Calibration

    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.

  • Page 27: Flr Calibration 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”...

  • Page 28: User Interface

    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.

  • Page 29: Scheduling Scans And Loading Vessels

    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.
  • Page 30 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).
  • Page 31 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 Vessel Properties Tab Name of the vessel. Label Cell type used in the vessel.
  • Page 32 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.
  • Page 33 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.

  • Page 34: Scan On Demand

    7.3.2 Scan on Demand In addition 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.

  • Page 35: Finding And Viewing Scanned Vessels

    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. 7.3.2.4 Initiate Scan: Scanning using the Scan on Demand Scheduler is initiated by clicking the red “Scan”...
  • Page 36 7.4.1.2 Search Tab: Scanned vessels can also be found by using the Search 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.

  • Page 37: Vessel View

    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. 7.4.2 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.
  • Page 38 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. Auto Scale tries to assign optimum Min/Max settings for a particular image.
  • Page 39 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. The Tool task pane also includes a measurement tool. Use Measurement 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...

  • Page 40: Data Processing

    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”...

  • Page 41: Analysis Job Utilities

    7.5.1.1 Assay Development Phase: In 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).
  • Page 42 Wound, 3) Angiogenesis, and 4) NeuroTrack . The Basic Analyzer is 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.
  • Page 43 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.
  • Page 44 7. When the Preview Status is complete, select the “Confluence Mask” and evaluate your phase segmentation by previewing all images within your image collection (black circle Figure 17). Figure 17. Processing Definition Preview 8. 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.
  • Page 45 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.
  • Page 46 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”...

  • Page 47: Graphing And Exporting Data

    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). The “Processing Definition Search” window opens, allowing Users to search for their desired Processing Definition. Double-click the Processing Definition or click the “Select”...
  • Page 48 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”...
  • Page 49 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.
  • Page 50 that the software is functioning within the specified parameters. These Metrics will not be relevant to the 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.
  • Page 51 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.
  • Page 52 When Custom Region is selected, the vessel color will change from light orange to green, thus alerting the User that this is a special graphing 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.
  • Page 53 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.
  • Page 54 Figure 20. Histogram 7.6.1.2 Graph Customization Initial default labels along with other graph attributes can be personalized. Left- click 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.
  • Page 55 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.
  • Page 56 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.
  • Page 57 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”...
  • Page 58 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.
  • Page 59 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.
  • Page 60 7.6.1.8 The Graph Pull-Down 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.

  • Page 61: Microplate Graphing

    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.

  • Page 62: Exporting Experimental Data

    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.
  • Page 63 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”...
  • Page 64 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.
  • Page 65 Include Error Measurements 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.

  • Page 66: Saving And Printing Graphs

    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.
  • Page 67 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: Blended Composite using CURRENT DISPLAY SETTINGS or Fluorescence (Green or Red) using CURRENT DISPLAY SETTINGS Additional image types are available for images that have been processed.
  • Page 68 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”...
  • Page 69 Figure 23. The Export Current Image Window with Export Designer Open 7.7.1.2 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”...
  • Page 70  Deselect one or more individual wells/sectors by holding the “Alt” key and then clicking with the mouse.  Deselect a region of the vessel by holding the “Alt” key and then clicking and dragging with the mouse.  Right-click on a well/sector to activate additional options for selection/deselection.
  • Page 71 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.  Choose Image Type  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.

  • Page 72: Exporting Movies Overview

    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 select the correct destination software from the “Preferences”...
  • Page 73 Migration/Invasion: Confluence Mask Initial Scratch Wound Mask Scratch Wound Mask NeuroTrack™: 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. 1.

  • Page 74: Archiving

    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.

  • Page 75: Archiving Vessels

    Important concepts to remember while creating an archive are listed below: 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.

  • Page 76: 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”...

  • Page 77: Hardware

    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”...
  • Page 78 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.

  • Page 79: Changing Objectives

    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.
  • Page 80 Use a 2.5mm Hex T-handled driver to loosen and remove the 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.

  • Page 81: Plate Map Editor

    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.

  • Page 82: Selecting Wells

    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.
  • Page 83 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. 7.10.2.2 Cells To add a cell type, select the “Cells”...
  • Page 84 7.10.2.7 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”...

  • Page 85: Chapter 8: Special Modules And Scan Types

    Special Modules and Scan Types are described in further detail below. 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.

  • Page 86: Cell Migration/Invasion Module

    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”...

  • Page 87: Neurotrack Tm Module

    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”...

  • Page 88: Chapter 9: Troubleshooting

    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: Vessels that have scratches on the bottom or top (lids for microplates/dishes).

  • Page 89: Images And What They Mean

    Images and What they Mean PROBLEM SOLUTION The edges of the wells seem to be out of Visual edge effects can be eliminated by focus at the 4X objective. increasing the total media volume in each well. However, this will not dramatically change the proliferation data.
  • Page 90 PROBLEM SOLUTION 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 this is a re-occurring problem or affects data analysis.
  • Page 91 PROBLEM SOLUTION Fluorescent cells are out of focus using Users planning to image in fluorescence the 20X objective. with a 20X objective should investigate the best setting for their system and their vessel. Adjusting the corrective ring to 1.4µM in this case, was the optimal setting.

  • Page 92: Contacting Technical Support

    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).

  • Page 93: Chapter 10: Performance Optimization

    Figure 31: The IncuCyte Log Chapter 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.
  • Page 94 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.

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