Adequate tumor margin delineation is crucial to maximize positive patient outcomes in molecular-guided surgery. Raman spectroscopy is highly specific in detecting tumor margins based on the differences in molecular composition between tumor and normal tissue; however, one major technical hurdle to its adoption is its slow acquisition speed. Previously, we described a "superpixel" acquisition approach that can expedite up to 10,000x compared to point-bypoint scanning while covering the entire surface area. We detected human basal cell carcinoma in Mohs surgical resection margins from eight patients and demonstrated superpixel acquisition had consistent diagnostic performance with point-by-point scanning. In this work, we further demonstrated examples of raster-scanned superpixel Raman classification images of positive and negative margins from three new patients. The performance of three superpixel sizes were evaluated, including 25×25μm2, 50×50μm2 and 100×100μm2. A previous established biophysical inverse model was applied to extract the biochemical composition of each superpixel, and a prior classification model was employed to generate the tumor heatmap. The classification result was then compared with the histopathological image. Our results show that superpixel Raman imaging can overcome the limitation of traditional Raman imaging in speed, allowing for rapid tumor margin assessment.
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