Total Internal Reflection with Fluorescence Correlation Spectroscopy

Abstract

The combination of total internal reflection illumination with fluorescence correlation spectroscopy (TIR-FCS) allows one to examine in quantitative detail a variety of biophysical properties related to the motions and interactions of fluorescent molecules near the interface of a transparent planar surface and an adjacent solution. Several experimental and theoretical aspects of this combination will be discussed. TIR-FCS has allowed characterization of local diffusion coefficients and concentrations of fluorescently labeled antibodies in solution but very close to substrate-supported phospholipid bilayers. TIR-FCS has also been used to examine the interaction kinetics of fluorescently labeled mouse IgG specifically and reversibly associating with the mouse receptor Fc-gamma-RII, which was purified and reconstituted into substrate-supported planar membranes. This method also has the potential, through the use of a single fluorescent reporter, of providing information about the thermodynamics/kinetics of nonfluorescent molecules which participate in surface binding mechanisms; e.g., those that compete with fluorescent reporters for surface-immobilized receptors or those that interact on the surface with the receptors and reduce or enhance the interaction of the fluorescent reporters with the surface binding sites.