Gating & Analysis

Cytomaton supports four gate drawing tools plus boolean combinations:

Polygon gates — Press P or select the polygon tool. Click to place vertices around your population. A snap indicator appears when your cursor approaches the first vertex — click to close the polygon.

Rectangle gates — Press R and drag to draw a rectangle. Best for quick lymphocyte or singlet gates on FSC/SSC.

Ellipse gates — Press E and drag to draw an ellipse. The ellipse fits the start and end points as the major axis endpoints.

Quadrant gates — Press Q to place a quadrant crosshair that divides the plot into four quadrants.

Boolean gates — Combine existing gates with AND, OR, NOT operations via the boolean modal in the toolbar. Useful for exclusion strategies (e.g., "Live cells NOT doublets").

Select a parent gate in the gating tree before drawing a new gate. The new gate becomes a child of the selected gate — events are first filtered through the parent, then through the child. This is standard hierarchical gating as used in FlowJo and other tools.

Gates are auto-named with the parent path prefix (e.g., "Lymphocytes/CD4+"). Click any gate name in the tree to rename it inline.

Click a gate to select it. Drag any vertex to adjust boundaries — statistics update in real time as you drag.

To change a gate's color, click the color swatch next to its name in the gating tree. Gates can be enabled/disabled; disabling a parent gate propagates a warning to child gates.

Cytomaton provides native least-squares spectral unmixing for Cytek Aurora and Sony ID7000 data.

When a spectral file is detected, the gating canvas is blocked until unmixing is complete. The wizard guides you through autofluorescence reference selection. After unmixing, residuals are displayed in a heatmap labeled by fluorochrome name.

AI Unmixing QC analyzes residuals automatically and provides actionable flag messages with specific fluorochrome identification, cause, and corrective action.

FlowSOM-style clustering on gated populations with adjustable resolution (5×5 to 20×20 SOM grid).

Clusters get auto-generated phenotype summaries from the top 3 discriminating markers with a confidence indicator. A "View in Heatmap" bridge button links cluster results to the heatmap visualization.

Cluster overlay coloring is available on UMAP/tSNE/PCA embedding plots. Cluster statistics (Median FI per marker per cluster) are exportable via clipboard as TSV.

Note: FlowSOM uses the minisom SOM implementation. Cluster assignments may differ slightly from FlowJo or OMIQ.

Native dimensionality reduction on gated populations — no plugins required.

UMAP and tSNE produce 2D embeddings colored by gate membership or FlowSOM cluster. Back-gating lets you lasso a region on the embedding and create a gate on the parent scatter plot.

PCA axis labels show explained variance per component (e.g., "PC1 (34.2%)") with cumulative variance display. PCA is deterministic — same data always produces the same result.

A time estimate is shown during computation for all methods.

Expression-level heatmap (populations × markers) with Z-score normalization per marker as the default view.

Toggle between Z-score and raw Median FI. Hierarchical clustering on both axes uses optimal leaf ordering. Color scale options: Blue-White-Red (diverging), Viridis (sequential), and a deuteranopia-safe palette.

Works with both manually gated populations and FlowSOM clusters. Export as PNG, SVG, or PDF.

Compare up to 5 samples on a single histogram with distinct color-coded lines and line style variation (solid, dashed, dotted) for grayscale distinguishability.

Normalization toggle: Count, Percentage, or % of max (default for overlays, matching FlowJo convention). Legend shows event counts per sample.