Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy

Dual-focus fluorescence correlation spectroscopy is a method for precisely measuring the diffusion coefficient of fluorescing molecules close to the infinite dilution limit in a reference-free and absolute manner. We apply the method to determine the diffusion coefficients of three fluorescent dyes across the visible spectrum. These values can be used as absolute reference standards for fluorescence correlation spectroscopy. In particular, it is found that the diffusion coefficient of the widely used reference dye Rhodamine 6G is by 37% larger than the value used in most publications on fluorescence correlation spectroscopy over the last three decades.