<scp>RoundMi</scp> : A quantitative method to analyze mitochondrial morphology in mitotic cells

Mitochondrial morphology is a key readout of cellular physiology and pathophysiology, yet its quantitative analysis in mitotic cells remains technically demanding due to their rounded, three-dimensional architecture. Volumetric imaging approaches, while comprehensive, require extensive Z-stack acquisition, high computational resources, and specialized image analysis expertise, collectively limiting throughput and accessibility. Here, we present RoundMi, a streamlined workflow for rapid, quantitative analysis of mitochondrial morphology in mitotic cells using single focal plane imaging. RoundMi integrates automated preprocessing via the Nellie plugin in Napari with downstream segmentation and quantification in MitoSkel. Focal plane selection is guided by DNA staining and mitochondrial signal to capture representative morphological features while minimizing acquisition time and phototoxicity. We validated RoundMi in mouse embryonic fibroblasts (MEFs) and HeLa cells, demonstrating robust detection of established morphological differences between wild-type and DRP1-deficient cells in both interphase and mitosis. Benchmarking against volumetric methods, including deconvolution and maximum intensity projection, confirmed that single-plane analysis provides a reliable proxy for mitochondrial morphology while avoiding projection-induced artifacts and substantially reducing computational demand. RoundMi is applicable across multiple cell types and compatible with live-cell imaging, offering a versatile, high-throughput solution for mitochondrial morphology analysis in dividing cells.