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Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation
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Photobleaching pathways in single-molecule FRET experiments.

Xiangxu Kong1, Eyal Nir, Kambiz Hamadani

  • 1Department of Chemistry and Biochemistry, and California NanoSystems Institute, University of California-Los Angeles, Los Angeles, CA 90095, USA.

Journal of the American Chemical Society
|March 23, 2007
PubMed
Summary

Understanding acceptor photobleaching in single-molecule fluorescence resonance energy transfer (sm-FRET) is crucial. We found acceptor photobleaching is linked to FRET efficiency and donor excitation, suggesting pulsed donor excitation can improve sm-FRET experiments.

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Molecular Machines

Background:

  • Single-molecule fluorescence resonance energy transfer (sm-FRET) requires high photon flux for accurate biomolecular machine analysis.
  • High laser excitation intensity, while increasing photon flux, accelerates photobleaching, limiting experiment duration.
  • Antioxidant additives are commonly used to mitigate acceptor photobleaching.

Purpose of the Study:

  • To elucidate the primary photobleaching pathway of the acceptor in sm-FRET.
  • To identify factors influencing acceptor photobleaching rates.
  • To propose strategies for reducing acceptor photobleaching in sm-FRET measurements.

Main Methods:

  • Utilized advanced single-molecule and ensemble spectroscopy techniques.
  • Employed doubly labeled Acyl-CoA binding protein and double-stranded DNA as model systems.
  • Investigated photobleaching pathways under varying excitation conditions and FRET efficiencies.

Main Results:

  • Acceptor photobleaching rate positively correlates with FRET efficiency.
  • Picosecond-pulsed excitation enhances acceptor photobleaching compared to continuous-wave excitation.
  • Acceptor photobleaching intensity scales with donor excitation laser intensity, not acceptor laser intensity.

Conclusions:

  • The dominant acceptor photobleaching pathway involves absorption of a short-wavelength photon from the acceptor's first excited singlet state.
  • Donor photobleaching is generally not a limiting factor in sm-FRET.
  • Employing pulsed donor excitation is a potential strategy to minimize acceptor photobleaching in sm-FRET.