New Fluorescence Correlation Spectroscopy Enabling Direct Observation of Spatiotemporal Dependence of Diffusion Constants as an Evidence of Anomalous Transport in Extracellular Matrices

The potential of fluorescence correlation spectroscopy (FCS) is extended to enable the direct observation of anomalous subdiffusion (ASD) in inhomogeneous media that are of great importance particularly in many biological systems, such as membranes, cytoplasm, and extracellular matrices (ECMs). Because ASD can be confirmed by monitoring the spatiotemporal dependence of observable diffusion coefficients (Dobs), the size of the effective confocal volume (Veff) for FCS sampling (sampling volume) was continuously changed on a scale of 300–500 nm using a motorized variable beam expander through which an illuminating laser beam passes. This new method, namely, sampling-volume-controlled (SVC)-FCS, was applied to the analysis of hyaluronan (HA) aqueous solutions where the Dobs of light-emitting solute (Alexa 488) markedly changed, corresponding to the change in Veff (220–340 nm in the half-axis), because the network structure of HA of 7–33 nm (nanostructure) interferes with the material transport within it. The results indicate that moderate ASD may occur even in the presence of a small amount (∼0.1 wt %) of HA in ECM. Because the change in Dobs along with the traveling distance (the mean-square displacement) can be identified even in systems with no deformation of the autocorrelation function, this technique has a great potential for general applications to many biological systems in which ASD shows complex time and space dependences.