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 withinporousmicroenvironments greatly impact the diffusion dynamics of molecules.However, how the interaction between the environment and moleculesinfluences the diffusion dynamics has not been thoroughly explored.Here, we show that fluorescence correlation spectroscopy super-resolutionoptical fluctuation imaging (fcsSOFI) can be used to accurately measurethe diffusion dynamics of molecules within varying matrices such asnanopatterned surfaces and porous agarose hydrogels. Our data demonstratethe robustness of fcsSOFI, where it is possible not only to quantifythe diffusion speeds of molecules in heterogeneous media but alsoto recover the matrix structure with resolution on the order of 100nm. Using dextran molecules of varying sizes, we show that the diffusioncoefficient is sensitive to the change in the molecular hydrodynamicradius. fcsSOFI images further reveal that smaller dextran moleculescan freely move through the small pores of the hydrogel and reportthe detailed porous structure and local diffusion heterogeneitiesnot captured by the average diffusion coefficient. Conversely, biggerdextran molecules are confined and unable to freely move through thehydrogel, highlighting only the larger pore structures. These findingsestablish fcsSOFI as a powerful tool to characterize spatial and diffusioninformation of diverse macromolecules within biorelevant matrices.

Automated summary

In this research, fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) was used to accurately measure the diffusion dynamics of molecules in different matrices. The results demonstrated the robustness of fcsSOFI in quantifying molecular diffusion speeds in heterogeneous media and recovering matrix structures with high resolution. Using dextran molecules of varying sizes, it was shown that the diffusion coefficient is sensitive to changes in molecular hydrodynamic radius. fcsSOFI images further revealed the ability of small dextran molecules to freely move through the hydrogel pores and reported detailed porous structure and diffusion heterogeneities.