Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer

Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a subfraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria.