Jump to main content
Jump to site search
Access to RSC content Close the message box

Continue to access RSC content when you are not at your institution. Follow our step-by-step guide.


Issue 41, 2015
Previous Article Next Article

Camera-based single-molecule FRET detection with improved time resolution

Author affiliations

Abstract

The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Förster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view decreases the number of molecules that can be monitored simultaneously. Here, we present a generalised excitation scheme termed stroboscopic alternating-laser excitation (sALEX) that significantly improves the time resolution without sacrificing highly parallelised detection in total internal reflection fluorescence (TIRF) microscopy. In addition, we adapt a technique known from diffusion-based confocal microscopy to analyse the complex shape of FRET efficiency histograms. We apply both sALEX and dynamic probability distribution analysis (dPDA) to resolve conformational dynamics of interconverting DNA hairpins in the millisecond time range.

Graphical abstract: Camera-based single-molecule FRET detection with improved time resolution

Back to tab navigation

Supplementary files

Article information


Submitted
15 Jul 2015
Accepted
27 Sep 2015
First published
28 Sep 2015

This article is Open Access

Phys. Chem. Chem. Phys., 2015,17, 27862-27872
Article type
Paper
Author version available

Camera-based single-molecule FRET detection with improved time resolution

S. Farooq and J. Hohlbein, Phys. Chem. Chem. Phys., 2015, 17, 27862
DOI: 10.1039/C5CP04137F

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

Reproduced material should be attributed as follows:

  • For reproduction of material from NJC:
    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
  • For reproduction of material from PCCP:
    [Original citation] - Published by the PCCP Owner Societies.
  • For reproduction of material from PPS:
    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
  • For reproduction of material from all other RSC journals:
    [Original citation] - Published by The Royal Society of Chemistry.

Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.


Social activity

Search articles by author

Spotlight

Advertisements