Imaging mitochondrial membrane potential via concentration-dependent fluorescence lifetime changes

Abstract Mitochondria are central to cellular metabolism. Various fluorescence tools have been developed for imaging the mitochondrial environment. Yet, new reporters and imaging methods for directly reading the mitochondrial status are needed for high spatial-temporal resolution imaging. Here, we introduce PK Mito Deep Red (PKMDR), a low-phototoxicity mitochondrial probe for time-lapse imaging, whose fluorescence lifetime serves as a sensitive indicator of mitochondrial membrane potential (Δψ m ). The positively charged PKMDR accumulates within mitochondria under a higher Δψ m , leading to concentration-induced quenching and a measurable decrease in fluorescence lifetime. Since mitochondrial respiration primarily regulates Δψ m , PKMDR’s fluorescence lifetime effectively reports on the status of oxidative phosphorylation. Using PKMDR with fluorescence lifetime imaging microscopy (FLIM), we visualize heterogeneous Δψ m across individual cells, organoids, and tissues over time. This method reliably reveals the heterogeneity between metabolically active peripheral mitochondria and relatively inactive perinuclear mitochondria in various cell types. Overall, PKMDR-FLIM is a robust tool for directly visualizing Δψ m with high spatiotemporal resolution.