Pseudo-spectral angle mapping for automated pixel-level analysis of highly multiplexed tissue image data

The rapid development of highly multiplexed microscopy systems has enabled the study of cells embedded within their native tissue, which is providing exciting insights into the spatial features of human disease [1]. However, computational methods for analyzing these high-content images are still emerging, and there is a need for more robust and generalizable tools for evaluating the cellular constituents and underlying stroma captured by high-plex imaging [2]. To address this need, we have adapted spectral angle mapping – an algorithm used widely in hyperspectral image analysis – to compress the channel dimension of high-plex immunofluorescence images. As many high-plex immunofluorescence imaging experiments probe unique sets of protein markers, existing cell and pixel classification models do not typically generalize well. Pseudospectral angle mapping (pSAM) uses reference pseudospectra – or pixel vectors – to assign each pixel in an image a similarity score to several cell class reference vectors, which are defined by each unique staining panel. Here, we demonstrate that the class maps provided by pSAM can directly provide insight into the prevalence of each class defined by reference pseudospectra. In a dataset of high-plex images of colon biopsies from patients with gut autoimmune conditions, sixteen pSAM class representation maps were combined with instance segmentation of cells to provide cell class predictions. Finally, pSAM detected a diverse set of structure and immune cells when applied to a novel dataset of kidney biopsies imaged with a 43-marker panel. In summary, pSAM provides a powerful and readily generalizable method for evaluating high-plex immunofluorescence image data.