A semi-automated pipeline for morphological analysis of myonuclei along single muscle fibers

Manual quantitation of skeletal muscle myonuclear number, spatial orientation, and morphology is time-consuming and subject to error and bias. To overcome these limitations, we developed and validated a semi-automated, quantitative, and reproducible image-analysis pipeline. The workflow combines FIJI-based preprocessing with custom Python scripts to process immunohistological images of individual muscle fibers, enabling high-resolution and scalable quantification of nuclei. Analyses incorporate morphometric parameters including nuclear position, shape, and three-dimensional orientation, as well as centroid-to-skeleton distance and nearest-neighbor relationships to capture spatial patterns of myonuclear organization along the fiber. Outputs include per-fiber and biopsy-level summaries integrated with Imaris metrics. This semi-automated approach provides a robust and efficient platform for high-throughput analysis of myonuclear number and structural features across large single fiber datasets.