3D Imaging of Extracellular Matrix Analogues via Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation Imaging (fcsSOFI)

The diffusion dynamics of biomolecules within porous microenvironments are greatly impacted by the nanoscale physiochemical structure of the matrix. Despite this understanding, much remains unknown about how the interplay between the matrix environment and biomolecules impacts their diffusion dynamics. In previous studies, we demonstrated the effectiveness of fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) in accurately assessing biomolecule diffusion dynamics within diverse extracellular matrix analogues, including nanopatterned surfaces and porous agarose hydrogels within a two-dimensional domain. In this work, we have taken a step further to extend this approach, employing fcsSOFI in conjunction with light sheet microscopy to reconstruct the three-dimensional structure of the matrix in various specific extracellular matrix analogues. Our findings underscore the robustness of fcsSOFI, as it allows for not only the quantification of the approximately 10 μm²/s diffusion rates of these molecules in complex media but also the super-resolution recovery of the matrix structure at a scale on the order of 10² nm. Moreover, we demonstrate how our technique allows us to start to understand how the matrix structure impacts the migration of biomolecules within the extracellular matrix.