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Related Experiment Video

Updated: May 6, 2026

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures
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Photobleaching with a subnanosecond laser flash.

Y Yuan1, D Axelrod

  • 1Biophysics Research Division, University of Michigan, 48109, Ann Arbor, Michigan.

Journal of Fluorescence
|November 16, 2013
PubMed
Summary

Bright subnanosecond laser flashes cause significant bleaching in fluorescence recovery after photobleaching (FRAP) experiments. This study reveals the involvement of two-photon effects and reversible bleaching, crucial for polarized FRAP applications.

Area of Science:

  • Photophysics
  • Biophysical Techniques
  • Spectroscopy

Background:

  • Standard fluorescence recovery after photobleaching (FRAP) relies on irreversible bleaching from millisecond excitation pulses.
  • This method assumes low bleaching probability per excitation cycle.
  • Polarized FRAP (PFRAP) requires subnanosecond pulses for molecular rotational diffusion, posing challenges for bleaching efficiency and orientational pattern imprint.

Purpose of the Study:

  • To investigate bleaching depths as a function of subnanosecond pulse intensity.
  • To determine the mechanisms involved in short-pulse bleaching.
  • To assess the ability of polarized subnanosecond pulses to create orientational anisotropy.

Main Methods:

  • Measurement of bleaching depths induced by varying subnanosecond pulse intensities.

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  • Experimental setup involving a small labeled protein immobilized on fused silica.
  • Development of a theoretical molecular state model to explain observed bleaching phenomena.

    • Main Results:

    • Significant bleaching occurs with bright subnanosecond laser flashes.
    • Both two-photon effects and reversible bleaching contribute to the observed bleaching.
    • Polarized bleaching successfully induces an anisotropic orientational pattern in the unbleached fluorophore population.

    Conclusions:

    • Subnanosecond pulses are effective for inducing significant bleaching in FRAP and PFRAP.
    • Understanding reversible bleaching and two-photon effects is critical for optimizing short-pulse FRAP experiments.
    • The developed theoretical model provides a quantitative explanation for the intensity-dependent reversible bleaching observed.