19 June 2003 Fluorescence liftime imaging (FLIM) using ps-pulsed diode lasers in laser scanning microscopes
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A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting of an 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 time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). The time-resolved fluorescence characteristics of 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated before and during PDT with subcellular resolution. For cells which were incubated with 5-ALA, a component with a fluorescence lifetime of about 7 ns was correlated with a structured fluorescence, which probably coincides with mitochondria, whereas a shorter lifetime was found in the cytoplasm. In the case of 5-ALAhe the lifetime of PPIX was longer, which could be due to different localization. During PDT the component with the longer lifetime completely vanished, whereas the shorter liftime was retained. It seems that FLIM is a valuable method to selectively identify and localize the photodynamically active photosensitizer.
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Angelika C. Ruck, Angelika C. Ruck, Frank Dolp, Frank Dolp, Claudia Happ, Claudia Happ, Rudolf Steiner, Rudolf Steiner, Michael Beil, Michael Beil, "Fluorescence liftime imaging (FLIM) using ps-pulsed diode lasers in laser scanning microscopes", Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); doi: 10.1117/12.477828; https://doi.org/10.1117/12.477828

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