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iCyte® or
iCys® Cytometric Analysis Software |
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The iCyte®
Automated Imaging Cytometer and the
iCyte® Research Imaging Cytometer are based on an inverted platform utilizing lasers, photomultiplier tubes, and a scatter detector to measure constituents and other properties of cells resulting from multicolor fluorescence detection. The accompanying iCyte or iCys Cytometric Analysis Software allows users to configure application-specific protocols, define a carrier, and analyze the resulting data.
With easy-to-use tabs, you can quickly set up an iCyte or iCys Run, including:
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- Selecting lasers and adjusting the laser power
- Selecting channels and creating virtual channels
- Defining the segmentation contours
- Defining the desired areas or wells to scan on a specimen carrier
- Setting up complex runs, such as repetitive scans
- Setting up multiple carrier runs (with the optional Robot feature)
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| Both iCyte and iCys feature a number of unique capabilities, including : |
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| Segmentation |
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| iCyte and iCys process events to obtain cell features. Data from one selected channel (the contour channel) is used to draw a contour around all of the adjacent pixels above a preset threshold value set by the user to define an event. Contours are displayed in the Scan View window as geometric shapes that surround the data defining each event. For example: |
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| Figure 1: Contour Lines |
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| Values can be set to define the following types of contours on which to count and evaluate events: |
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- Threshold (Figure 1, Red)—For each event, the threshold contour is the contour developed from the contour channel data to calculate features from all other channels.
- Integration (Figure 1, Green)—The integration contour defines the boundary within which the total fluorescence of the event will be calculated (integrated).
- Background (Figure 1, Blue)—The two background contours are used to calculate the local background for an event.
- Peripheral (Figure 1, Yellow)—The peripheral contours define two boundary lines that are used to determine the properties peripheral to an event.
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| Figure 2: Types of Phantom Contours |
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- Phantom (Figure 2, Pale Green)—Segmentation on cellular events cannot always generate the necessary data. In this case, phantom contours provide a non-cellular analysis approach based on random sampling. Phantom contours create user-specified circular contours that can be useful when mapping tissue constituents or for cellular analysis.
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| Figure 3: Subcontours |
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- Subcontours (Figure 3, Light Blue )—iCyte and iCys can generate subcontours within each data contour, using a channel that was not used to establish the threshold contour. This allows analysis of specimens that contain localized constituents, such as the nuclei within a cell.
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| Figure 4: Multi-component Segmentation |
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- Multi-component segmentation (Figure 4)— With the iCyte and iCys product lines, the optional software allows separate segmentation on different channels and relates the independently segmented events to each other.
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| Virtual Channels |
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| Virtual channels may be defined using mathematical or logical operators on one or more real channels to obtain a resultant processed image. For example, two channels can be added together, one channel can be subtracted from another, or the greater pixel value from two constituent channels can be retained. In addition, virtual channel operators can be used again on existing virtual channels, thereby allowing data display and collection for very specific events. Virtual channels are useful in far-ranging applications, for example: |
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- Correcting for autofluorescence in tissue sections.
- Performing spectral overlap compensation.
- Functioning as segmentation channels in experiments where the cell types do not share common features, such as live/dead cell assays.
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| Event Data |
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| Event Data may be displayed in the iCyte and iCys in a variety of different ways, shown in Figure 5: Generating Event Data: such as scattergrams, histograms, and expression maps (2-parameter frequency distribution plots) Gating regions can be drawn on these graphs to isolate populations. Any event features (or ratios of event features) can be displayed in these graphs. .Event data is also available in statistical tables. |
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| Figure 5: Generating Event Data |
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Scattergram |
Histogram |
2-Parameter Histogram |
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| Statistics of event features or their sub-populations may be displayed in a statistics table. Detailed Event feature information about a particular event may be displayed by selecting the event (or set of events) from a graph. |
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| Well Features |
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| Well Features are summary statistics derived from the event features on a per-well or per-tissue-array element basis. |
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Well Feature values can be viewed using iBrowser™ Data Integration Software.
Well Features can be calculated on the entire population of events or on any gated sub-population. When working with microplates, the Well Feature values can be displayed as color-coded expression levels during and after an iCyte or iCys run.
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| Data Reanalysis |
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| Storage options allow saving all of the image data as a raw data image file for each of the selected hardware channels during scanning. Analysis parameters not directly related to data acquisition (such as the PMT or scatter sensor settings) can then be modified and the existing images reanalyzed using the new parameters. As a result, once data from a specimen has been acquired at optimum settings, the analysis itself (selecting threshold levels, gallery images, etc.) may be optimized, by simply reanalyzing the raw data files, without rescanning the actual specimen. |
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| Repetitive Scanning |
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| iCyte and iCys can be set to perform Repetitive Scans or Rescans of a given scan area, either for observing specimens at time points separated by significant time intervals or for manipulating the specimen (such as restaining or incubating) between scans. |
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| File Merging |
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| iCyte and iCys provide the ability to merge image data files so that the hardware channels from two separate runs may be treated as though they are from a single run. Segmentation may be done on a channel from either run (or, in the case of sub-contours or the iNovator, both runs). Virtual Channels may be defined utilizing constituents from either run. |
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| Additional Software |
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| The iCyte and iCys Cytometric Analysis Software comes with the iCyte or iCys instrument. Additional copies can be purchased to allow scientists to analyze data on a stand-alone PC. In addition to the basic iCyte and iCys software, the following applications are also available: |
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- iBrowser™ Data Integration Software. iBrowser allows users to view and analyze the iCyte or iCys data in new and different ways. Use iBrowser to see all of the scan areas in a run, or generate a report that highlights important findings. iBrowser is standard with the iCyte instrument. Additional copies may be purchased for remote workstation use.
- iNovator Application Development Toolkit. iNovator extends the iCyte or iCys application to perform tissue microarray analysis, use complex image filtering, and generate runs that use multiple analysis pathways.
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