Fig. 6.

Hypserspectral imaging workflow. Firstly, the tissue was stained with an optimized multiplexed antibody panel. The staining was then assessed by a pathologist and regions of interest were identified for image acquisition. Hyperspectral images corresponding to five areas per tissue slide were then captured. Acquired images underwent spectral unmixing and mapping of unmixed stain concentrations. Images were then segmented using nuclear stain characteristics. Segmented images were further analyzed to determine stain positivity thresholds, and based on stain positivity and negativity, cells were classified into groups. Finally, the spatial relationships of all groups were interrogated and quantified