Fluorescence lifetime microscopy has become an important method of bioimaging, allowing not only to record intensity and spectral, but also lifetime information across an image. One of the most widely used methods of FLIM is based on Time-Correlated Single Photon Counting (TCSPC). In TCSPC, one determines this curve by exciting molecules with a periodic train of short laser pulses, and then measuring the time delay between the first recorded fluorescence photon after each exciting laser pulse. An important technical detail of TCSPC measurements is the fact that the delay times between excitation laser pulses and resulting fluorescence photons are always measured between a laser pulse and the first fluorescence photon which is detected after that pulse. At high count rates, this leads to so-called pile-up: ``early'' photons eclipse long-delay photons, resulting in heavily skewed TCSPC histograms. To avoid pile-up, a rule of thumb is to perform TCSPC measurements at photon count rates which are at least hundred times smaller than the laser-pulse excitation rate. The downside of this approach is that the fluorescence-photon count-rate is restricted to a value below one hundredth of the laser-pulse excitation-rate, reducing the overall speed with which a fluorescence signal can be measured. We present a new data evaluation method which provides pile-up corrected fluorescence decay estimates from TCSPC measurements at high count rates, and we demonstrate our method on FLIM of fluorescently labeled cells.
Joerg Enderlein, Daja Ruhlandt, Anna Chithik, René Ebrecht, Fred S. Wouters, and Ingo Gregor, "Dead-time correction for high-throughput fluorescence lifetime imaging microscopy
(Conference Presentation)," Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 971407 (Presented at SPIE BiOS: February 13, 2016; Published: 27 April 2016); https://doi.org/10.1117/12.2212167.4848767623001.
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