# 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) --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://help.codex.bio/codex/processor/technical-notes/segmentation-and-spillover.md?ask= ``` The question should be specific, self-contained, and written in natural language. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.