FIGURE 16.3 Principle of fluctuation correlation analysis (top). Representative data of fluctuating fluorescence signals induced by random motion of dye molecules through the detection volume (middle). Representative autocorrelation curve G(t) (bottom), describing the temporal decay function of fluctuations. The characteristic decay times for molecular residence times in the volume (td) and internal intensity fluctuations (tf) are indicated. (Adapted from Medina, M.A. et al., BioEssays, 24, 758, 2002.)

discussed and demonstrated [43-48]. FCS has been successfully applied for studying reaction kinetics of nucleic acids and proteins [45,46]. Based on fluctuations in the fluorescence yield of single-dye molecules, electron transfer, ion concentrations, and conformational changes of nucleic acid oligomers could be monitored [47,48]. The potential of FCS was further illustrated when substantial improvements in SNR were made by defining extremely small probe volumes using confocal and two-photon excitation. The potential of confocal FCS with high temporal resolution for rapid enzyme screening has been reported [49,50]. The tiny volume of confocal FCS in which the measurements are performed also makes it possible to evaluate molecular processes at the cell membrane. An increasing number of intracellular applications involving the study of molecular mobility of proteins and DNA in different locations inside cells have been developed using FCS [45,51,52]. Combined with TPE, FCS yields substantially improved signal quality in turbid samples such as deep cell layers in tissue [33]. At comparable signal levels, TPE minimizes photobleaching in spatially restrictive cellular compartments thereby preserving long-term signal stability. Since the definition of small volume is purely of an optical nature and no mechanical constraints are involved, FCS is relatively noninvasive and ideally suited for both in vitro and in vivo measurements. Potential artifacts that interfere with FCS measurements for intracellular applications include cellular autofluorescence, reduced signal quality due to light absorption and scattering, and dye depletion resulting from photobleaching in the restricted compartments inside cells.

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