1. Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy
- Author
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Thorsten Wohland, Xue Wen Ng, Adrian Röllin, Jagadish Sankaran, Wai Hoh Tang, and Harikrushnan Balasubramanian
- Subjects
Brightness ,Cell biology ,Science ,ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION ,Biophysics ,General Physics and Astronomy ,CHO Cells ,General Biochemistry, Genetics and Molecular Biology ,Article ,Fluorescence ,Diffusion ,Cricetulus ,Image processing ,Microscopy ,Fluorescence microscope ,Animals ,Humans ,Super-resolution microscopy ,Cluster analysis ,Image resolution ,Physics ,Multidisciplinary ,Estimation theory ,Cell Membrane ,General Chemistry ,Single-molecule experiment ,Actins ,Single Molecule Imaging ,Data set ,ErbB Receptors ,Spectrometry, Fluorescence ,Microscopy, Fluorescence ,Biological system - 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 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., Super-resolution microscopy and single molecule fluorescence spectroscopy require optimisation of the temporal or spatial resolution, which are usually mutually exclusive. Here the authors report a GPU-supported, camera-based strategy to achieve high spatial and temporal resolution from the same dataset.
- Published
- 2021