The extracellular matrix (ECM) is a biophysical environment that plays an important role in physiological processes and disease development. The ECM is highly dynamic, with changes occurring as local, nanoscale, physicochemical variations in physical confinement and chemistry from the perspective of biological molecules. The length and time scale of ECM dynamics are challenging to measure with current microscopic techniques. Super-resolution fluorescence microscopy has the potential to probe local, nanoscale, physicochemical variations in the ECM. Here, I will share our development of super-resolution imaging and analysis methods and their application to study model nanoparticles and biomolecules within synthetic ECM hydrogels. This includes 1) fluorescence correlation spectroscopy super-resolution optical fluctuation imaging or “fcsSOFI,” a super-resolution optical signal processing technique that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures using correlation and 2) expansion microscopy using tensile force, a sample-based super-resolution method that physically expands stretchable hydrogels. Overall, super-resolution imaging is a powerful tool that can increase our understanding of extracellular environments at new spatiotemporal scales to reveal ECM processes at the molecular-level.