In a dot plot colored by channel, each event in a typical biaxial plot is colored by its expression on a third channel, called the coloring channel, or the Z-axis channel. This functionality is available in the Gating Editor and the Illustration Editor. Click the links below to jump to the relevant section.
- Dimensionality reduction (DR) example of dot plots colored by channel
- Dot plots colored by channel in the Gating Editor
- Dot plots colored by channel in the Illustration Editor
- Dot plotting method-binned pixels versus stacked dots
- Stacked dots with custom size and shape
- Color bar range
- Coloring palette
- Virtual concatenation
Dot plots colored by channel have an important role in dimensionality reduction (DR) workflows. When looking at a DR map, coloring by channel allows you to correlate spatially collocated groups of events with biological populations:
(The same sample with viSNE map colored by different channel; this can be applied to any other DR analysis.)
Dot plots colored by functional channel
Consider the example below of a simple signaling experiment. The coloring channel is pStat3. The two samples are unstimulated (Unstim) and IL10-stimulated PBMCs (Stim). The higher intensity of pStat3 in CD3+ T cells, shown in the red-colored population, indicates that T cell signaling is occurring in the sample stimulated with IL10.
(Dot plots colored by channel reveal signaling.)
Dot plots colored by channel can help with gating
Coloring dot plots by channel can help identify where to gate, by displaying more information to interpret while drawing the gate. This can especially help in situations where drawing a clean gate is challenging.
In the Gating Editor, click on the setting icon next to the plot to show the plot settings window, choose a plot type of Dot – Color by Z-axis Channel. Other important settings are indicated but discussed in later parts of the article.
(Gating Editor settings for dot plots.)
The coloring channel selection will show up above the plot below the population and file selection. Each dot on the plot is colored according to its data value in the coloring channel, for example, pStat3, resulting in the plot below.
(Gating Editor configuration for dot plots colored by channel.)
The channels utilized for any advanced analysis appeared highlighted in the Gating Editor and the specific icon is displayed next to the channel name.
(An example of UMAP. The channels used for UMAP analysis are highlighted in the Gating Editor. All advanced analyses label the channels similarly using the corresponding analysis icon.)
In the Illustration Editor, choose Plot type as Dot, then choose to Color by Z-axis channel. After this, use the selector for Z-axis to define which channel to use for coloring.
(Plot settings for dot plots colored by channel in the Illustration Editor.)
To generate a figure that colors plots with a selection of multiple channels, open the Layout menu and select as one of the dimensions Channels on the Z axis. If you already had Channels selected as a dimension for varying along the X- or Y-axis, set the dropdown selector to change the axis to Z. You can also add another dimension for the Z-axis coloring.
(Plot settings for dot plots colored by channel in the Illustration Editor.)
(Resulting illustration with dot plots colored by selected Z-channels.)
Note that you can also change the channel selected on the Z-axis by clicking on the Z-axis channel labeling directly in line with the plots.
Within an advanced analysis experiment, the channels utilized for such analysis appeared highlighted in the Illustration Editor. When you open the Select Z-axis channels window, a label with the name of the analysis is displayed next to the channel name. You can use the analysis type as a search term or click on the label to automatically filter the channel list and quickly select all channels used for that analysis.
(Channels used for UMAP show UMAP analysis next to the channel name. All other advanced analyses have similar labeling format.)
There are two plotting methods for dot plots colored by channel: Binned Pixels and Stacked Dots.
In Binned Pixels mode, collisions between events are binned into single pixels based on the chosen resolution of the plot. Each pixel then reports the median value of the events inside of it on the coloring channel selected for the plot.
The Stacked dot option arranges events on top of each other depending on the order in which they appear in the FCS file. The size of each event in pixels can be adjusted with the Dot size field. This functionality is useful for increasing visual quality of sparse data. No binning is performed with stacked dots, so data may be hidden by overlap in some cases. The Dot shape can be configured as well to be a circle, ring, or square. The ring shape (available only for sizes > 3) is hollow in the middle and can help visualize events that might be overlapping because of stacking. Changing dot shape has little impact unless larger plot resolutions and dot sizes are used.
(Comparison of dot plotting method and shape. All plots are 512-pixel resolution. All stacked dot images are dot size of 7.)
The Z-channel range is the minimum and maximum of the color scale that is applied to a dot plot colored by Z-channel, and shown in the color bar to the right of the plot. The color bar is the key for associating a color in the dot plot to a particular value for expression intensity. The minimum and maximum values of the color bar are determined according to the selected method:
- Global (all files): The ungated 2nd and 98th percentile values for the channel in question are calculated for all files displayed in the illustration. The smallest 2nd and largest 98th percentile values are used as the coloring range minimum and maximum for all files selected in the illustration on this channel.
- Local (per file): The ungated 2nd and 98th percentile values are calculated for the channel in question and assigned as coloring range minimum and maximum on a per-file basis.
- User-defined: The coloring range boundaries are set through user-defined values. One custom range can be defined per illustration and is not channel-specific.
The data values that define coloring behavior and range are calculated on data scaled according to the scaling method of the channel coloring the plot (and compensated, as applicable). However, the data values are untransformed for reporting in the color bar. The coloring follows a linear relationship through the transformed data, but not in the raw data. Therefore for non-linearly transformed scales (arcsinh, log) the raw values reported in the color bar do not grow linearly.
By default, a Global scale range is selected. To modify the channel range, navigate to the Plots menu and click on More plot settings. In the pop-up window look for the section named Z channel colored dot settings and select a different Z channel range from the dropdown menu.
(More plot settings for Z channel colored dot settings.)
The same color palettes available to other plot types in the Cytobank platform are available for dot plots colored by channel. The dropdown menu for palette selection shows a preview of each.
(Palette selection menu.)
The recommended color palette for most purposes is Spectrum. This should not be confused with Rainbow, which is similar but is more heavily represented in the greens. Examples of other palettes are shown below.
(viSNE maps colored by channel with different coloring schemes.)
When visualizing dot plots, you may want to combine different FCS files. This is often the case if you are trying to compare across groups and have multiple samples per group.
(Example tSNE-CUDA map colored by TNFa levels in the Z-axis. There are six FCS files, three individuals and two conditions.)
To virtually concatenate some samples, navigate to the Plots menu and select the Concatenate option that better fits your illustrations. Alternatively, navigate to the Layout menu, click on the arrow next to the Rows or Columns position and select Concatenated.
(Plots and Layout menus showing the access to the virtual concatenation tool.)
The resulting illustration will display the Rows or Columns concatenated.
(The samples of the three individuals are virtually concatenated to display to tSNE-CUDA maps colored by TNFa levels. On the left are the unstimulated samples and on the right the stimulated condition.)
To know more about the virtual concatenation tool please check this article.
*For Research Use Only. Not for use in diagnostic procedures.