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.1 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.2 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.3 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 imaging analysis technique to understand dynamics within porous or crowded environments.

Advancements in fluorescence correlation spectroscopy super resolution optical fluctuation imaging (fcsSOFI) to quantify anomalous diffusion in crowded environments

2019 Fall National Meeting

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