After nuclear segmentation identifies all cells, classification determines which are marker-positive:
- Intensity thresholding — measure mean signal in the marker channel within each nucleus; cells above threshold are positive
- Adaptive thresholding — threshold varies by brain region to account for background differences
- Machine learning classifiers — StrataQuest's classifier engine uses multiple features (intensity, texture, size, context) for more robust classification
The choice of method depends on signal quality and the degree of background variation across the section.