Authors: Zdeněk Petrášek HannJörg Eckert Klaus Kemnitz
Publish Date: 2009/06/17
Volume: 102, Issue: 2-3, Pages: 157-
Abstract
Fluorescence lifetime imaging microscopy FLIM is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope We present a scanningless implementation of FLIM based on a time and spacecorrelated single photon counting TSCSPC method employing a positionsensitive quadrant anode detector and widefield illumination The standard timecorrelated photon counting approach leads to picosecond temporal resolution making it possible to resolve complex fluorescence decays This allows parallel acquisition of timeresolved images of biological samples under minimally invasive lowexcitation conditions 10mW/cm2 In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope where focusing of the excitation beam into a tight spot is required Therefore widefield FLIM permits to study Photosystem II PS II in a way so far not possible with a laser scanning microscope The potential of the widefield TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll dcontaining cyanobacterium Acaryochloris marina
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