The major drawback of pulsed two-photon excitation schemes in fluorescence correlation spectroscopy compared to one-photon applications is the existence of comparably long dark periods (12ns) for pulsed excitation, since the fluorescence lifetime is for most fluorophors only several ns. By splitting the laser beam and using different delay lines, we built a pulse doubler and quadrupler that was applied to FCS. The signal-to-noise ratio could be improved by a factor of 1.5 with proper alignment.
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