Parameter Overlays in FCS Express allow you to display multiple parameters from a single file onto a histogram as Overlays (Figure 5.30). Parameter Overlays can be used to explore gated populations over multiple channels, and are often coupled with Transformations such as tSNE for exploratory data analysis (see image at bottom).
Figure 5.30 Parameter Overlays
To add Parameter Overlays to a histogram, right-click on the histogram and select Add Parameter Overlays (Figure 5.31).
Figure 5.31 Add Parameter Overlays to Histogram
In the Available Parameters section of the Add Parameter Overlays window (Figure 5.32), check the box next to each parameter to add. You may also choose to add a legend to the plot with the parameter names, or switch the Overlay display from Overlaid to Stacked by checking the appropriate box.
If the Switch to stacked check box is selected, the Stacked Y Axis Title Type options become available. You may choose to display on the Y Axis the Counts or Legend Text (parameter name) for each parameter on the histogram. Note the Overlay display (stacked or overlaid) and Stacked Y Axis Title Type options can also be changed from the Specific Options formatting category.
Figure 5.32 Add Parameter Overlays Window - Select Parameters, Include Legend, Set Overlay Display, and Stacked Y-Axis Title Options.
In addition to providing a simple and fast tool to explore antibody staining patterns of gated populations, the Parameter Overlays can also be used with Transformations such as tSNE (Figure 5.33). In the figure below, a single tSNE population has been gated and its staining pattern visualized over multiple parameters using the Add Parameter Overlays feature.
Figure 5.33 Using tSNE with Parameter Overlays for Exploratory Data Analysis