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Abstract:
In this review a short introduction to the concept and experimental realization of single molecule multiparameter fluorescence spectroscopy is presented. The approach of Multiparameter Fluorescence Detection (MFD) is essentially the time-resolved observation of all five intrinsic properties of a chromophore that can be probed in a fluorescence experiment, i.e. spectral properties of absorption and fluorescence, F(lambda(A), lambda), fluorescence quantum yield, Phi(F), fluorescence lifetime, tau, and anisotropy, r. This harbors the potential to combine Fluorescence Correlation Spectroscopy (FCS) and Time-Correlated Single Photon Counting (TCSPC) in a single experiment. Additionally, species selective analysis of subensembles and even direct studies on single molecule dynamics become accessible. The application of MFD to complex multichromophoric systems is discussed. Examples range from studies of Donor-Acceptor pairs showing intra- or intermolecular Fluorescence Resonance Energy Transfer (FRET) for revealing dynamical and statical structural informations of biologically relevant macromolecules to the development of vastly improved analytical tools for the accurate identification of target molecules in multicomponent systems as needed in todays and future High-Throughput Screening (HTS) efforts. Many theoretical models of interactions and chemical reactions have been described on the molecular level although the primary source for our knowledge on chemical structure and dynamics so far are studies on molecule ensembles. Such studies, however, did not yet provide an answer to the question whether all members of the ensemble have the same properties. Here, experiments on single molecules promise new and unexpected insights, because they eliminate ensemble averaging and provide direct information on heterogeneity and kinetics of the system. That way distributions and spectra of different species in specific states are directly accessible even in heterogeneous systems (Fig.1).
