Ab Initio modelling of fluorescence fluctuation spectroscopy

Fluorescence correlation spectroscopy (FCS) has developed into a widely used and very successful spectroscopic technique for detecting and quantifying minute concentrations of fluorescing dyes in solution, as well as for obtaining information about molecular parameters such as diffusion coefficients, photophysical transition rates, or chemical reaction kinetics. The standard evaluation method of FCS data is based on the simplified assumption that the convolution of the excitation intensity distribution and the light collection efficiency function is a three-dimensional Gaussian distribution. Although that assumption leads to satisfactory fits of experimental data, the adjustable parameters of this standard model have a rather unphysical meaning. In the present paper, an ab initio approach to modeling FCS curves is developed that is based on exact wave-optical calculations of fluorescence excitation and detection. Comparison with the standard model is made, and the ab initio calculated model curves are used for fitting experimental data and deriving absolute values of diffusion coefficient and concentration.