Herbert Schneckenburger^1,2, Reinhard Sailer¹,

Michael H. Gschwend¹, Wolfgang S.L. Strauss¹

Advanced methods and recent applications of laser microscopy and time-resolved fluorescence spectroscopy of cultivated cells are presented. Intrinsic fluorescence was studied after inhibition of the mitochondrial respiratory chain, deprivation of oxygen or during the process of phagocytosis. In all cases pronounced changes of fluorescence of the free coenzymes NADH or NADPH were observed. Non-radiative energy transfer from NADH to the mitochondrial marker rhodamine 123 (R123) was examined for selective detection of mitochondrial malfunction.

Photosensitizers, e.g. protoporphyrin IX (PPIX) or sulphonated meso-tetraphenyl porphyrins, could be detected selectively within the plasma membrane or within lysosomes using total internal reflection fluorescence spectroscopy (TIRFS) or time-resolved fluorescence spectroscopy. Phototoxicity of PPIX towards endothelial cells was related to plasma membrane-associated monomers with fast photobleaching kinetics. Advantages of steady-state and time-resolved fluorescence techniques are outlined.