Rapid and efficient quality control analysis of isolated mitochondria by interferometric light microscopy

Abstract Mitochondrial transplantation is a promising biotherapeutic strategy to restore cellular energy homeostasis in diseases associated with mitochondrial dysfunction. However, its clinical translation is hindered by the lack of rapid, reliable, and label‐free techniques for quantifying and characterizing freshly isolated mitochondria. Current methods are often time‐consuming, costly, or require pre‐labeled sample, limiting their suitability for routine quality control in mitochondrial research, engineering, and biotherapy. In this study, we employ interferometric light microscopy (ILM) via Videodrop technology to assess mitochondrial concentration and size with high precision and efficiency. Mitochondria were isolated from human mesenchymal stromal cells and comprehensively characterized, including through functional and structural marker expression, protein quantification, membrane integrity, and electron microscopy. ILM measurements showed strong correlation and robust accuracy compared to transmission electron microscopy and total protein assays. Our findings establish ILM as a label‐free, rapid, and scalable approach for mitochondrial quantification and sizing, paving the way for standardized quality control in mitochondrial biotherapy.