SRRF ‘n’ TIRF - FCS: Simultaneous spatiotemporal super-resolution microscopy
Jagadish Sankaran, Harikrushnan Balasubramanian, Wai Hoh Tang, Xue Wen Ng, Adrian Röllin, Thorsten Wohland
Received Date: 27th March 20
Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either time or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~60 nm), temporal dynamics (≤ 2 ms), and molecular brightness from the exact same data set. We demonstrate the applicability and advantages of multi-parametric measurements to monitor the super-resolved structure and dynamics of two different biomolecules, the actin binding polypeptide LifeAct, and the epidermal growth factor receptor (EGFR). Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of LifeAct in dependence of the cellular actin network. Multi-parametric analysis 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 new biological knowledge that cannot be obtained from sequential measurements.
Read in full at bioRxiv.
This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.