Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution...

Total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is a method for measuring the surface association/dissociation rates and absolute densities of fluorescent molecules at the interface of solution and a planar substrate. This method can also report the apparent diffusion coefficient and absolute concentration of fluorescent molecules very close to the surface. An expression for the fluorescence fluctuation autocorrelation function in the absence of contributions from diffusion through the evanescent wave, in solution, has been published previously (N. L. Thompson, T. P. Burghardt, and D. Axelrod. 1981, Biophys. J. 33:435-454). This work describes the nature of the TIR-FCS autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave contribute to the fluorescence fluctuations. The fluorescence fluctuation autocorrelation function depends in general on the kinetic association and dissociation rate constants, the surface site density, the concentration of fluorescent molecules in solution, the solution diffusion coefficient, and the depth of the evanescent field. Both general and approximate expressions are presented.