Fig. 2: Detailed singular and composite segmentation.

a, An example batch of tiles that spans 1–n stains from MICSSS undergoes new composite segmentation. The first column shows the original staining before segmentation, then the nuclear segmentation mask if each marker were independently segmented, then the nuclear segmentation from reconciling multiple nuclear stains, followed by the composite segmentation with nuclear boundaries extended by 3 pixels to simulate cytoplasm. The last column provides examples of the improvements that composite segmentation has over the singular segmentation. Scale bar, 30 µm. b, An example tile of MICSSS depicts MARQO composite segmentation across diverse cell densities; the zoomed-in images show how MARQO similarly segments in low-, medium- and high-density areas with a 1.6× linear magnification relative to the main image. Scale bar, 50 µm. c, Example tiles of MARQO-segmented cells compared with a pathologist’s manual identification of true cells for low-density (left) and middle- to high-density (right) regions. If a manually clicked point resides within the nuclear boundaries of a cell segmented by MARQO, that cell is considered ‘MARQO+, manual+’ (red). If no point resides within the nuclear boundaries of a cell segmented by MARQO, that cell is considered ‘MARQO+, manual−’ (blue). If a clicked point did not reside within any MARQO-segmented cell nuclear boundaries, an overlaid circle with a radius of 3 pixels (green) is plotted, simulating a ‘MARQO−, manual+’ cell. Scale bars, 20 µm. d, Total cell counts segmented are plotted for all tiles (n = 15) for our manual quantification (left) versus our MARQO automated quantification (right).