Fluorescence correlation spectroscopy translational diffusion coefficients

In fluorescence correlation spectroscopy (FCS), the temporal fluctuations of the fluorescence intensity are recorded and analyzed in order to determine physical or chemical parameters such as translational diffusion coefficients, flow rates, chemical kinetic rate constants, rotational diffusion coefficients, molecular weights and aggregation. The principles of FCS for the determination of translational and rotational diffusion and chemical reactions were first described in the early 1970s. But it is only in the early 1990s that progress in instrumentation (confocal excitation, photon detection and correlation) generated renewed interest in FCS. 

Fluorescence correlation spectroscopy analyses the temporal fluctuations of the fluorescence intensity by means of an autocorrelation function from which translational and rotational diffusion coefficients, flow rates and rate constants of chemical processes of single molecules can be determined. For example, the dynamics of complex formation between /3-cyclodextrin as a host for guest molecules was investigated with singlemolecule sensitivity, which revealed that the formation of an encounter complex is followed by a unimolecular inclusion reaction as the rate-limiting step.263... 

Fluorescence correlation spectroscopy thus provides a way to study processes that change the translational diffusion coefficient, such as binding of a small, fluorescent ligand to a macromolecule. However, the spatial dependence of the light intensity in the focal region can be more complex than Eq. (5.83) assumes and this can add spurious components to the autocorrelation function [263]. 

At an early stage, it became apparent( 15-17) that interactions between diffusing macromolecules substantially modify what was then termed the translational diffusion constant. As had initially not been entirely transparent to the QELSS community, though it had been known elsewhere, there are in fact two translational diffusion coefficients, and (9,10). QELSS measures Dm, with complications at large q(9,10,16). If one can optically tag a few macromolecules, QELSS can also (as originally predicted(ll) and more recently confirmed(12) experimentally for fluorescence correlation spectroscopy) determine D. ... 

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