PMID: 8583282Dec 1, 1995Paper

Time-resolved microfluorometric study of the binding sites of lipophilic cationic pyrene probes in mitochondria of living HeLa cells

Journal of Photochemistry and Photobiology. B, Biology
D HüglinH W Zimmermann

Abstract

Lipophilic dye cations specifically bind to the mitochondria of living cells. Using fluorescent dyes, the mitochondria can easily be observed with a fluorescence microscope. Electron microscopy has shown that the dyes are bound to the inner mitochondrial membranes and the cristae. Using time-resolved fluorescence microscopy we have investigated, whether the dye molecules are preferentially accumulated at the strongly hydrophobic protein complexes of energy metabolism or at the lipids of the inner membrane system. In order to use our nanosecond-pulsed laser fluorometer we synthesized specially designed lipophilic pyrene cations with S1 lifetimes in the nanosecond domain, which specifically stain mitochondria in living HeLa cells. Model experiments with artificial membranes such as liposomes, proteoliposomes and also protein complexes have shown that the fluorescence is strongly quenched by oxygen if the pyrene probes are bound to lipids. Binding to proteins causes a much smaller quenching effect. In artificial systems, all decays were single exponential. This is in contrast with incubated HeLa cells, which showed double-exponential fluorescence decays. Comparing these with the artificial systems we came to the conclusion that in...Continue Reading

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