The wide world of histograms

Before you create a histogram, you must have already completed these steps:

From the Illustration Editor navigation bar, under Plotsselect Plot type > Histogram. By default, the Overlay option is switched on and a histogram overlay figure is automatically generated using the default Layout settings.

(Default histogram illustration.)

Make sure to select the appropriate dimensions for your histogram overlay in the Layout menu. Check out this article on how to set up the illustration Layout. The default layout for a new illustration has FCS files in the Overlaid dimension, Channels in the X axis in the Columns position, and Populations set to the Table 1 dimension. Modify the layout dimensions as needed to create a meaningful histogram. You can use sample tags as layout dimensions to organize the files, for instance by condition. Check this article to learn how to annotate your files using sample tags.

By default, the histogram overlay will be colored by the raw median signal intensity per channel. You can modify the desired statistic from the Plots menu and the statistic and equation from the Statistic menu under the plot. You can select to color the histogram overlay using a particular statistic or normalize against an "Unstimulated" or control sample using, for instance, fold change of the medians.  When using a comparison equation, make sure to set the control in the appropriate position. By default, the control cells value will be highlighted in yellow in the statistic table.

(The statistic coloring the histogram overlay can be edited from the Plots menu.)

(Top panel: default statistic and equation displaying raw median signal intensity per marker. Bottom panel: median signal intensity per marker is represented as a fold change with respect to the “Unstim” cells value. The cells of the table highlighted in yellow indicate the normalizing value used in the fold change equation.) 

The table of statistics can be exported as tab-delimited data by clicking on the CSV icon at the right of the Statistics menu. Read more about statistics and equations in the Illustration editor. 

Clicking the Export tab opens the Export illustration to the 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. Learn more about the exporting options in the Illustration editor in this article.

The autosave feature will save your histogram overlay by default unless you choose to turn off the autosave feature. Check this article to understand the saved illustrations.

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 you to fine-tune the figure by adjusting the color palette, fill and 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 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.


(More plot settings showing all settings specific to histograms.)

When the Fill color is set to Overlaid dimension, the color legend can be customized by clicking on the color box and selecting from the color palette or entering a customized RBG code.


(Customize the color legend by clicking on it and selecting a new color.)

By default, dot coloring has a Fill opacity of 100%. You can configure the opacity from 0 to 100 from the Plots menu or by hovering over the plot. 

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                (Opacity access from the navigation bar.)       (Opacity access by hovering over the plot.)

Histograms and virtual concatenation tool

The virtual concatenation tool is available when working with histograms when they are not overlaid. To access this tool, first navigate to the Plots menu and toggle off the Overlaid option. You can now Concatenate FCS files from the Plots menu by selecting the desired Concatenate option or from the Layout menu by selecting the Trident symbol to concatenate Rows or Columns.


(Left panel: Plots menu showing the Concatenate options: none, Rows, Columns or All multi-file positions; right panel: Layout menu showing the option to concatenate the Rows dimension.)

You can virtually concatenate the experimental variables selected in the Rows or Columns position in the Layout menu except the Channels and Populations. If you configured the Layout dimensions using the experiment Sample tags, you may want to virtually concatenate the FCS files by clicking in the Plots menu and selecting the option All multi-file positions under the Concatenate tool. This would virtually concatenate the FCS files without the need for reconfiguring the Layout positions.

(Histograms displaying four samples “Unstim” and four samples “Stim” on the left. Histograms showing All multi-files positions virtually concatenated on the right.)

Check this article to learn more about the virtual concatenation tool.


*For Research Use Only. Not for use in diagnostic procedures.

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