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.

Date Published: 21 February 2008
PDF: 9 pages
Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 686209 (21 February 2008); doi: 10.1117/12.761161

Published in SPIE Proceedings Vol. 6862:
Single Molecule Spectroscopy and Imaging
Jörg Enderlein; Zygmunt K. Gryczynski; Rainer Erdmann, Editor(s)