Time-resolved fluorescence microscopy for probing mitochondrial metabolites

A novel set-up for time-gated (nanosecond) detection of fluorescence spectra and images of microscopic samples was recently developed. The apparatus was now used to measure the fluorescence of mitochondrial coenzymes (NADH, flavins) and a marker molecule (rhodamine 123) in endothelial cells from calf aorta. In these cultivated cells the electron transport chain was inhibited at various sites of the inner mitochondrial membrane. It could be shown that the fluorescence intensity of the free coenzyme NADH depended on the site of inhibition. In addition, an increased energy transfer from mitochondrial coenzymes (NADH, flavins) to rhodamine 123 molecules was observed, if the inhibition occurred in complex I (NADH- coenzyme Q reductase) or complex III (coenzyme QH2-cytochrome c reductase) of the respiratory chain. The diagnostic potential of these findings are discussed.