> For the complete documentation index, see [llms.txt](https://help.codex.bio/codex/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.codex.bio/codex/processor/technical-notes/segmentation-and-spillover.md).

# Segmentation & Spillover

### Cell Segmentation

Segmentation involves two steps, finding local maxima and region-growing using the maxima as seed points [\[1\]](https://en.wikipedia.org/wiki/Region_growing). Threshold values can be specified by the user to control the region growth. Cell volumes are grown in 3D to encompass all corresponding marker expression signal. Expression levels are integrated for each marker using all voxels within a cell.

### Spatial Spillover Compensation

Cells may have spillover fluorescence expression from adjacent cells that may hamper efforts to differentiate positive and negative expressions. Spillover compensation is used to reassign such expression back to the correct cells using a Gabriel graph [\[2\]](https://en.wikipedia.org/wiki/Gabriel_graph) and adjacency matrices, improving separation of sub-populations.

[Goltsev, Y., Samusik, N., Kennedy-Darling, J., Bhate, S., Hale, M., Vazquez, G., ... & Nolan, G. P. (2018). Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell, 174(4), 968-981.](https://www.sciencedirect.com/science/article/pii/S0092867418309048)


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