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Issue 41, 2015
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Camera-based single-molecule FRET detection with improved time resolution

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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

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Publication details

The article was received on 15 Jul 2015, accepted on 27 Sep 2015 and first published on 28 Sep 2015


Article type: Paper
DOI: 10.1039/C5CP04137F
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Citation: Phys. Chem. Chem. Phys., 2015,17, 27862-27872
  • Open access: Creative Commons BY-NC license
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    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

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