Spot fluorescence and perinuclear enrichment

Short Description

This protocol defines two ROIs based on nuclear fluorescence (DAPI). Nuclear ROIs are first extracted using the HK-mean algorithm on the DAPI channel. These nuclei are then morphologically dilated by x pixels to generate the perinuclear ROI, and further dilated by y pixels (y > x) to generate the extended ROI. Fluorescent spots are automatically detected within these ROIs, and spot size and fluorescence intensity are quantified. Signal enrichment in the perinuclear ROI relative to the extended ROI is then calculated using the following formula:

% perinuclear enrichment = [(Total spot fluorescence in perinuclear ROI)/(Total spot fluorescence in extended ROI)*(surface of extended ROI)/(surface of perinuclear ROI) – 1]*100