Total internal reflection fluorescence spectroscopy for probing porphyrin fluorescence in cell membranes

摘要

Abstract: The application of total internal reflection fluorescence spectroscopy for probing fluorescence of protoporphyrin selectively in cell membranes is described. Penetration depths of the evanescent field were calculated for a wavelength of 543 nm. Penetration depths varied between 75 nm and 190 nm, depending on the incident angle of the light. In contrast to fluorescence spectra obtained by epi- illumination, spectra obtained by total internal reflection fluorescence spectroscopy were characterized by a very low autofluorescence background. This indicates that only protoporphyrin located in the plasma membrane or in close vicinity to the plasma membrane was excited. Furthermore total internal reflection fluorescence spectroscopy setup was used for the determination of photobleaching and polarized fluorescence measurements. Illumination of cells incubated with protoporphyrin resulted in a biexponential photobleaching with a rapidly and a slowly bleaching portion. During the whole period of light exposure a degree of polarization P equals minus 0.22 was determined. !15