Comparison of optical saturation effects in conventional and dual-focus fluorescence correlation spectroscopy

The paper studies the excitation intensity dependence of diffusion measurements of the fluorescent dye Cy5 using conventional fluorescence correlation spectroscopy (FCS) and dual-focus fluorescence correlation spectroscopy (2fFCS). Due to the light-driven transitions of Cy5 between the fluorescent and non-fluorescent states, conventional FCS shows a strong dependency of the apparent diffusion coefficient on excitation intensity, whereas 2fFCS is virtually free from that artefact. Moreover, we provide an absolute value for the diffusion coefficient of Cy5.