Total internal reflection excitation used in combination with fluorescence correlation spectroscopy (TIR-FCS) is a method for characterizing the dynamic behavior and absolute concentrations of fluorescent molecules near or at the interface of a planar substrate and a solution. In this work, we demonstrate for the first time the use of TIR-FCS for examining the interaction kinetics of fluorescent ligands in solution which specifically and reversibly associate with receptors in substrate-supported planar membranes. Fluorescence fluctuation autocorrelation functions were obtained for a fluorescently labeled IgG reversibly associating with the mouse receptor FcgammaRII, which was purified and reconstituted into substrate-supported planar membranes. Data were obtained as a function of the IgG solution concentration, the Fc receptor surface density, the observation area size, and the incident intensity. Best fits of the autocorrelation functions to appropriate theoretical forms gave measures of the average surface density of bound IgG, the local solution concentration of IgG, the kinetic rate constant for surface dissociation, and the rate of diffusion through the depth of the evanescent field. The average number of observed fluorescent molecules, both in solution and bound to the surface, scaled with the solution concentration of IgG, observation area size, and Fc receptor surface density as expected. The dissociation rate constant and rate of diffusion through the evanescent field agree with previous results, and all measured parameters were independent of the incident intensity.

Total internal reflection with fluorescence correlation spectroscopy is a method for measuring the surface association/dissociation rate constants and absolute densities of fluorescent molecules at the interface of a planar substrate and solution. This method can also report the apparent diffusion coefficient and absolute concentration of fluorescent molecules very close to the surface. Theoretical expressions for the fluorescence fluctuation autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave, in solution, contribute to the fluorescence fluctuations have been published previously. In the work described here, the nature of the autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave contribute to the fluorescence fluctuations, and when fluorescent and nonfluorescent molecules compete for surface binding sites, is described. The autocorrelation function depends in general on the kinetic association and dissociation rate constants of the fluorescent and nonfluorescent molecules, the surface site density, the concentrations of fluorescent and nonfluorescent molecules in solution, the solution diffusion coefficients of the two chemical species, the depth of the evanescent field, and the size of the observed area on the surface. Both general and approximate expressions are presented.

The lateral diffusion coefficient of a ligand which repeatedly dissociates and rebinds to sites on a planar surface is described. An analytical expression showing the manner in which the diffusion coefficient depends on the propensity for rebinding after dissociation, as the molecule moves through the solution and across the surface, is derived. The diffusion coefficient is time dependent and ranges from the surface diffusion coefficient (at time zero when a tagged molecule is placed on the surface) to the solution diffusion coefficient (at time infinity). The time-dependent diffusion coefficient depends only on the intrinsic dissociation rate and a "rebinding parameter", which in turn depends on a group of constants including the intrinsic association and dissociation rates, the density of unoccupied surface binding sites, and the diffusion coefficient in solution.

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