Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy

We have investigated spatial variations of the diffusion behavior of the green fluorescent protein mutant EGFP (F64L/S65T) and of the EGFP-beta-galactosidase fusion protein in living cells with fluorescence correlation spectroscopy. Our fluorescence correlation spectroscopy device, in connection with a precision x-y translation stage, provides submicron spatial resolution and a detection volume smaller than a femtoliter. The fluorescence fluctuations in cell lines expressing EGFP are caused by molecular diffusion as well as a possible internal and a pH-dependent external protonation process of the EGFP chromophore. The latter processes result in two apparent nonfluorescent states that have to be taken into account when evaluating the fluorescence correlation spectroscopy data. The diffusional contribution deviates from ideal behavior and depends on the position in the cell. The fluorescence correlation spectroscopy data can either be evaluated as a two component model with one fraction of the molecules undergoing free Brownian motion with a diffusion coefficient approximately five times smaller than in aqueous solution, and another fraction diffusing one or two orders of magnitude slower. This latter component is especially noticeable in the nuclei. Alternatively, we can fit the data to an anomalous diffusion model where the time dependence of the diffusion serves as a measure for the degree of obstruction, which is large especially in nuclei. Possible mechanisms for this long tail behavior include corralling, immobile obstacles, and binding with a broad distribution of binding affinities. The results are consistent with recent numerical models of the chromosome territory structure in the cell nucleus.