Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

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 spatial structures (similar to 100 nm), temporal dynamics (similar to 2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

Automated summary

This study presents an experimental strategy that optimizes spatial and temporal resolution simultaneously by implementing a GPU-supported, camera-based measurement strategy. The results demonstrate the importance of pixel-wise cross-correlation of parameters obtained from different techniques on the same data set for robust physicochemical parameter estimation and providing unique biological knowledge.