Segmentation involves two steps, finding local maxima and region-growing using the maxima as seed points . 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  and adjacency matrices, improving separation of sub-populations.