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 (~100 nm), temporal dynamics (~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.

No abstract

No abstract

17Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually 18 exclusive experimental strategies optimizing either time or spatial resolution. To achieve both, 19we implement a GPU-supported, camera-based measurement strategy that highly resolves 20 spatial structures (~60 nm), temporal dynamics (≤ 2 ms), and molecular brightness from the 21 exact same data set. We demonstrate the applicability and advantages of multi-parametric 22 measurements to monitor the super-resolved structure and dynamics of two different 23 biomolecules, the actin binding polypeptide LifeAct, and the epidermal growth factor receptor 24 (EGFR). Simultaneous super-resolution of spatial and temporal details leads to an improved 25 precision in estimating the diffusion coefficient of LifeAct in dependence of the cellular actin 26 network. Multi-parametric analysis suggests that the domain partitioning of EGFR is primarily 27 determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-28 EGFR interactions but is largely independent of EGFR-actin interactions. These results 29 demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques 30 on the same data set enables robust physicochemical parameter estimation and provides new 31 biological knowledge that cannot be obtained from sequential measurements. 32 33 Key Words 34 35 Super-resolution, Imaging fluorescence correlation spectroscopy, Number and brightness 36 analysis, Super-resolution radial fluctuations, FCS diffusion law, Epidermal growth factor 37 receptor 38 egfr 39 Simultaneous spatiotemporal super-resolution microscopy

We describe a protocol for the preparation of live cell samples for fluorescence spectroscopy and computational super-resolution imaging. We detail here how to culture, transfect, and prepare the cells for fluorescence applications.