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.