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

We present diffusion measurements of the fluorescent dye Cy5 in aqueous solutions using conventional fluorescence correlation spectroscopy (FCS) and the recently introduced dual-focus fluorescence correlation spectroscopy (2fFCS). We study the sensitivity of both methods with respect to excitation intensity. Due to the light-driven transitions of Cy5 between fluorescent and non-fluorescent states, conventional FCS shows a strong dependence of the apparent diffusion coefficient on excitation intensity, whereas 2fFCS is virtually free from that artifact. Moreover, 2fFCS allows us to obtain the first precisely measured absolute value for the diffusion coefficient of Cy5.