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Clustering is widely used in MSI to segment anatomical features and differentiate tissue types, but existing approaches are both CPU and memory-intensive, limiting their application to small, single datasets. We propose a new approach that uses a graph-based algorithm with a twophase sampling method that overcomes this limitation. We demonstrate the algorithm on a range ofsample types and show that it can segment anatomical features that are not identified using commonly employed algorithms in MSI, and we validate our results on synthetic MSI data. We show that the algorithm is robust to fluctuations in data quality by successfully clustering data with a designed-in variance using data acquired with varying laser fluence. Finally, we show that this method is capable of generating accurate segmentations of large MSI datasets acquired on the newest generation of MSI instruments, and evaluate these results by comparison with histopathology
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