Fig. 1

Multispectral imaging and analysis. a Representative image of 7-color mIHC staining of a metastatic UM sample scanned with Mantra Quantitative Pathology Workstation at original magnification 20X. Color code is under the picture: CD4 marker is represented in yellow, CD8 in magenta, Granzyme B in green, FoxP3 in red, CD163 in white, melanoma mix in cyan, nuclei in blue. b Analysis workflow of acquired multispectral images: (b, left) tissue segmentation to differentiate tumor area (in red) and the surrounding stroma (in green); (b, middle) single cell segmentation (nuclei are in green, membrane in red); (b, right) cell phenotyping based on the detection of specific cell-surface or intracellular markers. c-d Representative image of cell-to-cell distance analyses: c the nearest neighbor analysis used for the determination of the mean cell distance between melanoma cells (light blue dots) and the nearest CD8+ cells (red dots); d count within analysis used for the calculation of the percentage of melanoma cells (light blue dots) within a specified μm radius from a CD8+ T lymphocyte (red dots)