Before you create a histogram overlay, you must have already completed these steps:
- Uploaded experiment files (or cloned those of another experiment)
- Set your scales and compensations appropriately
- Annotated your staining panels (channels)
- Drawn your gates (created populations)
- Annotated any conditions, individuals, or other parameters that you want to use in your plots
From the Illustration Editor navigation bar, under Plots, select Plot type>Histogram, and toggle the Overlay button to the on position. A histogram overlay figure is automatically generated using the default Layout settings.
Make sure to select the appropriate variables for your histogram overlay in the Layouts window. The default histogram overlay Layout for a new illustration has Conditions in the Overlaid dimension, Populations in the columns position, and Channels set to the Table 1 dimension. In the event that you need to change the parameters of your overlaid dimensions, click in the Conditions box to switch the Overlaid variable to Populations, Channels, or another Sample tag. You can also click and drag the overlaid position variable to the rows, columns, or tables position by mousing over the 12 dots (grip) in each variables bubble, clicking and then dragging the bubble to the appropriate position.
The plot statistics area is located below the illustration window. In the plot statistics area, you can select to color your overlay using a particular statistic or normalize against an "Unstimulated" or control sample. The histogram overlay is automatically re-populated by selecting the box for the statistic or normalization method that you want to use to create your overlay.
This table can be exported as tab-delimited data by clicking on the csv icon located in the statistics table navigation bar.
Clicking the Export tab opens the Export illustration to file window, which allows you to export your illustration in your choice of format. You can change the name of the illustration, export it as a PDF, PNG, or SVG file, and include a number of exporting options such as including a QR code link and a table of statistics. The autosave feature will save your histogram overlay by default unless you choose to turn off the autosave feature.
Changing the Style of Histograms
Additional changes can be made to the histogram overlay plot either by clicking Plots in the Illustration Editor navigation bar or by mousing over a plot in the figure. In either case, selecting More plot settings will open the plot setting window, which allows the user to fine-tune the figure by adjusting the color palette, outline color, scale range, background color, and plot size. Options to apply a highlight to the histogram outline, as well as to smooth the outline can be toggled on or off.
Overlaid histograms have the same styling options as non-overlaid and also gain a few unique parameters. For each histogram in an overlay, the precise vertical spacing can be defined and the presence or absence of a baseline can be toggled. Additionally, a Statistic option is added as a way to fill each histogram in an overlay. The histograms are filled based on the statistic value calculation defined in the Fill color box.