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Bioengineering

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Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Article DOI: 10.3791/63124-v • 08:26 min • November 23rd, 2021
November 23rd, 2021 •
1, 2, 2, 2, 1
1Institute of Applied Physics, TU Wien, 2Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna

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This article presents a method for spatiotemporal analysis of mobile, single-molecule Förster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Tags

Automated Analysis Two-dimensional Spatiotemporal Analysis Mobile Single-molecule FRET Probes Surface-bound Probes Immobilized Molecules Biomolecule Diffusion FRET Deficiency Time Traces Spatiotemporal Aspects Molecular Forces Conformational Dynamics Binding Kinetics Live Cell Experiments High-quality Single-molecule FRET Experiments Signal-to-noise Ratio Single-molecule Tracks Sample Measurement Excitation Donor Fluorophores Acceptor Fluorophores Camera Readout Photobleaching Analysis
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