PT  - JOURNAL ARTICLE
AU  - Jagadish Sankaran
AU  - Harikrushnan Balasubramanian
AU  - Wai Hoh Tang
AU  - Xue Wen Ng
AU  - Adrian Röllin
AU  - Thorsten Wohland
TI  - SRRF ‘n’ TIRF - FCS: Simultaneous spatiotemporal super-resolution microscopy
AID  - 10.1101/2020.02.26.965905
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.02.26.965905
4099  - http://biorxiv.org/content/early/2020/03/27/2020.02.26.965905.short
4100  - http://biorxiv.org/content/early/2020/03/27/2020.02.26.965905.full
AB  - 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.