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Microglia exhibit diverse morphologies reflecting environmental conditions, maturity, or functional states. Thus, morphological characterization provides important information to understand microglial roles and functions. Most recent morphological analysis relies on classifying cells based on morphological parameters. However, this classification may lack biological relevance, as microglial morphologies represent a continuum rather than distinct, separate groups, and do not correspond to mathematically defined clusters irrelevant of microglial cells function. Instead, we propose a new open-source tool, MorphoCellSorter, which assesses microglial morphology by automatically computing morphological criteria, using principal component analysis and Andrews plots to score cells. MorphoCellSorter properly ranked cells from various microglia datasets in mice and rats of different ages, from  in vivo ,  in vitro , and  ex vivo  models, that were acquired using diverse imaging techniques. This approach allowed for the discrimination of cell populations in various pathophysiological conditions. Finally, MorphoCellSorter offers a versatile, easy, and ready-to-use method to evaluate microglial morphological diversity that could easily be generalized to standardize practices across laboratories.

MorphoCellSorter is an Andrews plot-based sorting approach to rank microglia according to their morphological features