Single molecule fluorescence spectroscopy - Approaches towards quantitative investigations of structure and dynamics in living cells

The investigation of the structure and dynamics of biomolecules and biomolecular assemblies in living cells is of current interest in molecular biology. Recent developments in single molecule fluorescence spectroscopy (SMFS) have opened ways for investigating the dynamics and stoichiometry of individual biomolecular complexes e.g., by application of single pair fluorescence resonance energy transfer (spFRET) with alternating laser excitation (ALEX), and by improved labels and labeling techniques. In the recent years, we have developed a set of techniques that allow the determination of the spatial distribution of single fluorescent molecules and their identification by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as the observation of the dynamics of individual molecules immobilized on surfaces. Based on SFLIM we currently focus on investigating the diffusion kinetics of biomolecules in living cells. By combining high-resolution confocal fluorescence nucroscopy of single molecules with fluorescence correlation spectroscopy (FCS) we seek to quantitate diffusion coefficients and concentrations of relevant fluorescently labeled biomolecules within living cells thereby visualizing the heterogencous distribution of local mobilities in the sample. The simultaneously acquired fluorescence intensity and lifetime images can further be used for additional single point measurements for obtaining i.e., information about the stoichiometry of' immobilized biomolecular complexes based on photon anti-bunching. In addition the simultaneous acquisition of multiple characteristic proper-ties by SFLIM, like spectral emission bands and fluorescence lifetime, offers the opportunity to discriminate different fluorescent probes and autofluorescence.