Spatially Resolving Size Effects on Diffusivity in Nanoporous Extracellular Matrix-like Materials with Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation Imaging.

It is well documented that the nanoscale structures within porous microenvironments greatly impact the diffusion dynamics of molecules. However, how the interaction between the environment and molecules influences the diffusion dynamics has not been thoroughly explored. Here, we show that fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) can be used to accurately measure the diffusion dynamics of molecules within varying matrices such as nanopatterned surfaces and porous agarose hydrogels. Our data demonstrate the robustness of fcsSOFI, where it is possible not only to quantify the diffusion speeds of molecules in heterogeneous media but also to recover the matrix structure with resolution on the order of 100 nm. Using dextran molecules of varying sizes, we show that the diffusion coefficient is sensitive to the change in the molecular hydrodynamic radius. fcsSOFI images further reveal that smaller dextran molecules can freely move through the small pores of the hydrogel and report the detailed porous structure and local diffusion heterogeneities not captured by the average diffusion coefficient. Conversely, bigger dextran molecules are confined and unable to freely move through the hydrogel, highlighting only the larger pore structures. These findings establish fcsSOFI as a powerful tool to characterize spatial and diffusion information of diverse macromolecules within biorelevant matrices.