Improving the Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation (fcsSOFI) with Higher Order Cross-Correlation

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Project Summary We seek to understand how the physiochemical environment of the extracellular matrix affects the diffusion, structure, and folding of proteins. The extracellular matrix is spatially heterogeneous at nanoscales and can change over time. But current imaging methods are limited in spatial and temporal resolutions, while biochemical techniques are designed for model, pristine solutions that differ from the complexity of the extracellular matrix. Therefore, how proteins function locally in the extracellular matrix is an underexplored area in biophysics. We propose to understand how proteins function and interact in the complex extracellular matrix environment, and how the nanoscale variation in the chemical and physical composition of this environment can locally change protein dynamics. To achieve this, we will use a correlation-based super-resolution microscopy developed by our lab that can access the time- and spatial-scales of protein dynamics within the extracellular matrix. The goals of this proposal are to: 1) identify how the nanoscale chemical and steric properties of the extracellular matrix control protein diffusion, structure, and folding; 2) determine how cells tune the dynamics of proteins by changing the extracellular matrix; 3) develop instrumental and analysis methods that access the spatiotemporal regimes required to understand protein function in the extracellular environment. Achieving our goals will reveal the extracellular matrix and protein dynamics that drive biological regulation and will provide researchers with methods to understand and potentially control extracellular delivery of signaling proteins or therapeutics. Overall, an entirely new area of biology – the extracellular matrix - will be explored by super-resolution microscopy; the findings can be broadly applied to other biomolecules, cells, and tissues of relevance to fundamental biophysics, drug-delivery/therapeutics, and disease.