An extended analysis method of time, polarization and color resolved fluorescence correlation spectroscopy (filtered
FCS) is introduced. It uses multiparameter fluorescence detection (MFD) [1-3] to separate pure fluorescence signal of
multiple species and scatter contributions. This method allows monitoring of simultaneous and independent diffusion of
several molecular species in one sample and makes possible accurate and quantitative analyses of fractions. The
proposed method is simple to implement experimentally, because it requires only single wavelength excitation and MFD
widely used in single molecule experiments. In comparison to recently introduced fluorescence lifetime correlation
spectroscopy (FLCS) [4-7] this method is able to distinguish species when they have very close or even the same
fluorescence lifetime using just differences in time resolved fluorescence anisotropy.
The time-correlated single photon counting (TCSPC) technique combined with clock oscillator set by the pulsed laser provides a precise measurement of the arrival time of the detected photons with picosecond resolution for a time-scale of hours. If TCSPC is combined with other experimental techniques such as optical spectroscopy and mechanical manipulation, it is possible to coincide the detected fluorescence signal with the changes of the sample properties. High temporal resolution achieved in TCSPC (down to ps) allows us to monitor fast mechanical processes in single molecules. Here we present recent developments in fluorescence correlation spectroscopy (FCS) as well as the combination of TCSPC with optical scanning microscopy and mechanical manipulation by means of an atomic-force microscope (AFM).