21 February 2008 Recent advances in time-correlated single-photon counting
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We report about the time-resolved confocal fluorescence microscope MicroTime 200, which is completely based on TTTR format data acquisition and enables to perform very advanced FCS, FRET and FLIM analysis such as Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Two Focus FCS (2fFCS). FLCS is a fundamental improvement of standard FCS overcoming many of its inherent limitations. The basic idea of FLCS is a weighting of the detected photons based on the additional picosecond timing information (TCSPC start-stop time) when using pulsed laser excitation. 2fFCS goes even further, combining Pulsed Interleaved Excitation (PIE) with a time-gated FCS analysis. The basic implementation of 2fFCS uses two synchronized but interleaved pulsed lasers of the same wavelength but of different polarisation to generate two close by excitation foci in a pre-determined distance acting as a submicron ruler. In this case it it no longer necessary to have prior knowledge about the size and shape of the confocal volume. Maintaining the information about the photon´s origin, the dual focus data allows a precise calculation of absolute diffusion coefficients.
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Felix Koberling, Felix Koberling, Benedikt Krämer, Benedikt Krämer, Sebastian Tannert, Sebastian Tannert, Steffen Rüttinger, Steffen Rüttinger, Uwe Ortmann, Uwe Ortmann, Matthias Patting, Matthias Patting, Michael Wahl, Michael Wahl, Benjamin Ewers, Benjamin Ewers, Peter Kapusta, Peter Kapusta, Rainer Erdmann, Rainer Erdmann, "Recent advances in time-correlated single-photon counting", Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 686209 (21 February 2008); doi: 10.1117/12.761161; https://doi.org/10.1117/12.761161

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