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 scales of ECM dynamics are challenging to measure with current microscopic techniques. We have developed fluorescence correlation spectroscopy super-resolution optical fluctuation imaging or “fcsSOFI,” as a super-resolution optical signal processing technique to simultaneously characterize the nanometer dimensions of and diffusion dynamics within the porous ECM using correlation. Here, we have analytically defined the experimental parameters of frame rate, total number of frames, signal-to-background ratio, and diffuser concentration to accurately obtain ECM structure, diffusion dynamics, and diffuser numbers. We determine these parameters based on correlation mathematics and simulated data with a known ground truth, and demonstrate the accuracy with application to experimental data of diffusing polymer and proteins within collagen. Overall, fcsSOFI images the complex local, nanoscale, and physicochemical variations in the ECM.
Lydia Kisley (Sun,) studied this question.