Advancements in Superresolution Correlation Analysis to Image Anomalous Diffusion in Crowded Environments

Abstract

Fluorescence correlation spectroscopy super-resolution optical fluctuation imaging or “fcsSOFI” is a super-resolution optical imaging technique that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures. By utilizing pixel-by-pixel second-order autocorrelation, maps of diffusion coefficients at subdiffraction-limited levels are achieved. fcsSOFI has previously imaged and quantified one- and two-dimensional Brownian diffusion within liquid crystals and porous hydrogels. Application of fcsSOFI beyond Brownian diffusion and in non-porous, crowded environments will be presented. We have expanded the fcsSOFI algorithm to quantify multicomponent and anomalous diffusion. The computational analysis speed has been improved with GPU-based curve fitting. The success of the algorithm is demonstrated with simulated data. Experimentally, fcsSOFI is applied to the diffusion of rheological sensors within crowded (bio)polymer condensates formed by liquid-liquid phase separation. fcsSOFI quantifies anomalous diffusion components that were challenging to observe by conventional single particle tracking techniques due to low signal-to-background ratios. fcsSOFI is therefore a viable, objective imaging analysis technique to understand dynamics within porous or crowded environments below the diffraction limit.