Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

Abstract Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves structures (~100 nm), dynamics (~2 ms), molecular brightness from the exact same data set. Simultaneous super-resolution of details leads to an improved precision in estimating diffusion coefficient actin binding polypeptide Lifeact corrects structural artefacts. Multi-parametric analysis epidermal growth factor receptor (EGFR) suggests domain partitioning EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering inter-EGFR interactions but largely independent EGFR-actin interactions. These results demonstrate pixel-wise cross-correlation parameters obtained different techniques on set enables robust physicochemical parameter estimation provides biological knowledge cannot be sequential measurements.