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Concentration effects in "single-molecule" spectroscopy.

Irina V Gopich1

  • 1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. irinag@niddk.nih.gov

The Journal of Physical Chemistry. B
|December 25, 2007
PubMed
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Even at low concentrations, multiple molecules can affect single-molecule Förster resonance energy transfer (FRET) measurements. This study provides a rigorous theory and a simple test to identify when these multiple-molecule effects become significant.

Area of Science:

  • Biophysics
  • Spectroscopy
  • Physical Chemistry

Background:

  • Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for studying molecular dynamics.
  • Accurate interpretation of smFRET data relies on the assumption of observing individual molecules.

Purpose of the Study:

  • To rigorously investigate the impact of multiple-molecule events on smFRET efficiency measurements.
  • To identify conditions under which the single-molecule approximation breaks down.

Main Methods:

  • Theoretical analysis of photon counting histograms and FRET efficiency distributions.
  • Consideration of molecular diffusion through a laser spot and fluorophore brightness.

Main Results:

Related Experiment Videos

  • Multiple-molecule effects are significant even at concentrations as low as 0.1 molecule per observation volume.
  • These effects are pronounced at high photon counts and large threshold values, potentially mimicking multiple conformational states.
  • The influence of multiple-molecule effects is strongly dependent on fluorophore brightness.

    • Conclusions:

    • The standard single-molecule description may be inadequate for smFRET measurements under certain conditions.
    • A simple experimental test is proposed to assess the validity of the single-molecule assumption.
    • Researchers should be cautious about misinterpreting multiple-molecule artifacts as biological heterogeneity.