Web-based Cellpose‑SAM segmentation enables quantification of cross-sectional area in formalin-fixed paraffin-embedded skeletal muscle sections stained with hematoxylin and eosin.

Cross‑sectional area (CSA) of skeletal myofibers is a primary morphometric indicator of atrophy and regeneration, yet it is most commonly quantified on cryosections after immunofluorescent labeling of basal lamina proteins, which requires specialized reagents and equipment and limits reuse of routine histology slides. Here we present a practical, fully web‑based workflow that leverages Cellpose‑SAM on a public browser interface to segment myofibers directly from formalin-fixed paraffin-embedded (FFPE) skeletal muscle sections stained with hematoxylin and eosin (H&E) or toluidine blue (TB), followed by downstream region‑of‑interest (ROI) curation and batch morphometry in Fiji/ImageJ. We validated this approach across whole muscles and tissue preparations in mice. In exemplar experiments on both FFPE and frozen sections of mouse soleus, CSA distributions from H&E and TB showed no significant differences. A sciatic nerve crush injury model exhibited a significant reduction in mean and median CSA in mouse soleus sections with a characteristic left-shift in distribution relative to sham controls, consistent with rapid denervation atrophy. This accessible pipeline lowers experimental barriers, enables analysis of archived H&E or TB slides, and supports reproducible, high-throughput morphometry for neuromuscular research.