Pure and Applied Chemistry

Abstract: We present an overview on the applicability of fluorescence correlation spectroscopy (FCS) for the accurate determination of translational diffusion coefficients and thus, via the Stokes–Einstein relation, of molecular size. We consider several of the most common sources of optical aberrations and their impact on the outcome of conventional FCS measurements. We describe also a new variant of FCS, dual-focus FCS, which is robust against most of the considered aberrations, and we report reference values of diffusion coefficients for several fluorescent dyes across the visible spectrum.