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

Abstract

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.

Figure 1.

fcsSOFI is a powerful technique to simultaneously image the structure of a porous matrix at high-resolution as well as characterize the diffusion dynamics of molecules inside the matrix with high accuracy. (A) Schematic representation of total internal reflection fluorescence (TIRF) microscopy experiment set up for detecting molecular diffusion on a patterned nano surface on glass. (B) Intensity fluctuations recorded on individual pixels on a 2D camera. The fcsSOFI code is monitoring the intensity fluctuations measured at each pixel individually and cannot distinguish between the diffusion types (i.e., vertical vs horizontal). (C) Example autocorrelation curves from individual pixels. (D) Final fcsSOFI images depicting the combined spatial and diffusion information. Scale bar is 2 μm. (E) Cumulative distribution plot showing the diffusion coefficients in the selected area from the fitting of the autocorrelation curves.

Figure 2.

The viscosity-dependent diffusion of beads in aqueous solutions is accurately quantified via fcsSOFI. (A) Diffusion coefficient cumulative distribution function for 100 nm fluorescent beads in water and with increasing concentration of dextrose using fcsSOFI. (B) Average diffusion coefficients for beads in varying aqueous solutions. The diffusion coefficient linearly decreases with increasing dextrose concentration. Error bars represent the SD of the mean from at least three replicates.

Figure 3.

fcsSOFI improves image resolution while also collecting accurate diffusion data in nanopatterned surfaces. (A) Average image, (B) fcsSOFI analyzed image, (C) line sections, and (D) cumulative distribution of diffusion coefficients. Scale bars are 2 μm.

Figure 4.

fcsSOFI analysis for dextran diffusion in agarose hydrogel reveals size-dependent diffusion behavior in the hydrogel as well as the gel pore structure. (A–C) Changes in the fcsSOFI images for dextran diffusion in agarose hydrogel as a function of dextran size. Smaller dextran molecules of molecular weight 76 and 155 kDa diffuse throughout the entire gel (A,B), whereas larger dextran molecules have localized diffusion within specific regions of the gel (C). All samples contain 20% Ficoll. Scale bars are 2 μm. (D) Average cumulative distribution plots with standard deviation from three experiments of diffusion coefficients for dextran of different sizes diffusing in agarose hydrogels. Larger dextran molecules of molecular weight 2000 kDa have slower speed compared to smaller dextran molecules of molecular weight 76 and 155 kDa.