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Updated: Jun 4, 2026

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

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

Published on: November 23, 2021

Improved temporal resolution and linked hidden Markov modeling for switchable single-molecule FRET.

Stephan Uphoff¹, Kristofer Gryte, Geraint Evans

¹Department of Physics and Biological Physics Research Group, University of Oxford, Parks Road, Oxford OX1 3PU, UK.

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry

|February 1, 2011

Summary

Switchable Förster resonance energy transfer (smFRET) combines photoswitching with smFRET to sequentially probe multiple distances in single molecules. This technique offers improved temporal resolution for studying dynamic biological systems.

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Last Updated: Jun 4, 2026

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

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Published on: November 23, 2021

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

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High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Published on: July 25, 2014

Area of Science:

Biophysics
Single-molecule biophysics
Fluorescence microscopy

Background:

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for measuring distances within molecules.
Photoswitching allows reversible control over fluorophore brightness, enabling sequential measurements.

Purpose of the Study:

To introduce and validate switchable FRET, combining smFRET with photoswitching.
To enhance the temporal resolution and analytical capabilities for studying molecular dynamics.

Main Methods:

Utilized fast total internal reflection (TIRF) microscopy for high temporal resolution measurements.
Developed linked hidden Markov modeling (HMM) to analyze FRET and stoichiometry data.
Applied switchable FRET to immobilized molecules using both TIRF and confocal microscopy.

Main Results:

Achieved 10 ms temporal resolution for probing two FRET pairs within 2 seconds.
Demonstrated compatibility with both TIRF and confocal microscopy.
Linked HMM successfully identified transient states and their transitions in a 2D FRET-stoichiometry space.

Conclusions:

Switchable FRET provides enhanced temporal resolution for observing multiple distances in single molecules.
Linked HMM is an effective tool for analyzing complex switchable FRET data.
This technique advances molecular and structural biology research by enabling detailed studies of dynamic biological systems.