Label-free quantification of mitochondrial dynamics through mitosis using holotomographic microscopy

Live-cell visualization of mitochondria during mitosis is essential for understanding how energetic, redox, and biosynthetic homeostasis, as well as organelle inheritance, are maintained during cell division. Conventional fluorescence microscopy, while powerful, perturbs these processes through high-intensity illumination that induces reactive oxygen species (ROS), mitochondrial dysfunction, and signal loss over time. Here, we demonstrate that holotomographic microscopy (HTM), a quantitative phase-imaging technique based on refractive-index (RI) reconstruction using low-power illumination, enables label-free, subcellular-resolution volumetric imaging of mitochondrial network dynamics throughout the complete mitotic sequence. Using human umbilical vein endothelial cells (HUVECs), we captured all seven canonical mitotic phases over 6 h without exogenous dyes, validating mitochondrial identity through limited, low-intensity MitoTracker exposure. HTM revealed characteristic redistribution and compaction of mitochondrial networks from prophase through cytokinesis while preserving physiological mitotic timing and morphology. Three-dimensional RI reconstructions enabled continuous tracking of mitochondrial organization that is otherwise obscured in two-dimensional projections. By enabling prolonged, label-free volumetric imaging without detectable perturbation of mitotic progression or mitochondrial morphology, HTM establishes a quantitative platform for high-fidelity analysis of mitochondrial dynamics in dividing cells. Beyond methodological advance, this approach provides a framework for precision mapping of organelle behavior across diverse cell types and disease-relevant contexts.