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

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

DOI:
10.3791/63124-v

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08:26 min

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November 23, 2021

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, , , ,
1Institute of Applied Physics,TU Wien, 2Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology,Medical University of Vienna

Chapters

  • 00:05Introduction
  • 01:05Measurement of Samples
  • 01:38Localization, Tracking, and Fluorescence Intensity Analysis of Single Molecules
  • 04:03Analysis and Filtering of Single-molecule Data
  • 06:41Plotting of Results and Further Analysis
  • 07:17Results: Spatiotemporal Analysis of Mobile, smFRET-based Probes Using Widefield Fluorescence Microscopy
  • 07:45Conclusion

Summary

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Automatic Translation

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

Keywords: Single-molecule FRETMobile ProbesSpatiotemporal AnalysisFluorescenceImage AnalysisBiomolecular DynamicsLive Cell ExperimentsData ProcessingImage RegistrationLocalization

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