Fluorescence correlation spectroscopy analysis of diffusion in a laser gradient field: a numerical approach

Fluorescence correlation spectroscopy (FCS) is widely used in biological systems. When the laser is intense enough, such as in two-photon experiments, the trapping force due to the laser gradient field can change the diffusion behavior of the fluorescent particles and induce error in the FCS measurements. Previous studies on biased FCS are qualitative. In this article, a numerical approach is proposed to treat the problem quantitatively. By assumption of a "spherical symmetry", biased FCS curves can be calculated numerically and fitted to the experimental data to retrieve the unbiased particle number, diffusion time, and polarizability of the fluorescent particles as well as the strength of the gradient field. It has been proven using simulated FCS data that the discrepancy caused by the spherical symmetry approximation is independent of the gradient field strength; therefore it can be eliminated by a calibration.