Selective Examination of Plasma Membrane–Associated Photosensitizers Using Total Internal Reflection Fluorescence Spectroscopy: Correlation between Photobleaching and Photodynamic Efficacy of Protoporphyrin IX

Fluorescence spectra, fluorescence decay kinetics, photobleaching kinetics and photodynamic efficacy of protoporphyrin IX (PP) were investigated in endothelial cells in vitro after different incubation times. Fluorescence spectra and photobleaching kinetics were determined during total internal reflection (TIR) illumination or epiillumination. Because penetration depth of the excitation light during TIR illumination was limited to about 100 nm, plasma membrane-associated PP was almost selectively examined. Spectra obtained by TIR fluorescence spectroscopy (FS) showed a very low background, where-as spectra obtained by epi-illumination exhibited considerable background by autofluorescence and scattered light. For photobleaching kinetics during TIR illumination after 1 h or 24 h incubation, a biexponential fluorescence decrease was observed with a rapidly and a slowly bleaching portion. After 1 h incubation, the rapidly bleaching portion was the predominant fraction, whereas after 24 h incubation comparable relative amounts of the rapidly and slowly bleaching portion were determined. The rapidly and slowly bleaching portion were assigned to PP monomers and aggregated species in close vicinity to the plasma membrane. Fluorescence decay measurements after epi-illumination support the decrease of PP monomers within the whole cell with increasing incubation time. In contrast to TIR illumination, photobleaching of PP during epi-illumination was characterized by slow monoexponential fluorescence decrease after 1 h or 24 h incubation. Photodynamic efficacy of PP using epi-illumination was found to depend strongly on incubation time. Considerable cell inactivation was determined for short incubation times (1 h or 3 h), whereas photodynamic efficacy was diminished for longer incubation times. Reduced photodynamic efficacy after long incubation times was assigned to the lower amount of photodynamically active monomers determined close to the plasma membrane as well as within the whole cell. In conclusion, TIRFS measurements are suggested to be an appropriate tool for the examination of the plasma membrane-associated photosensitizer fraction in living cells.