New Microscopic Techniques to Investigate Intracellular Localization and Reactions of Photosensitizers: Laser Scanning Fluorescence Lifetime Imaging (LS-FLIM)

Abstract

Summary This work describes the time-resolved fluorescence characteristics of 5-ALA induced PPIX in living cells. The intracellular fluorescence lifetime was determined from time-gated spectra as well as single photon counting, using a ps pulsed diode laser for fluorescence excitation. The diode laser which emits pulses at 398 nm with 70 ps duration was coupled to a confocal laser scanning microscope. For time resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in “spot scan” mode. In addition, a novel developed time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. A fluorescence lifetime of 7.4 ns was found for PPIX, a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. Short pulsed diode lasers coupled to a laser scanning microscope, equipped with appropriate detection units allows therefore time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.