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Related Experiment Videos

Photon counting histogram: one-photon excitation.

Bo Huang1, Thomas D Perroud, Richard N Zare

  • 1Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry
|November 13, 2004
PubMed
Summary

Photon counting histogram (PCH) analysis, a fluorescence method, struggles with standard models. An improved PCH model corrects for out-of-focus light, enhancing species characterization in confocal microscopy.

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Area of Science:

  • Fluorescence spectroscopy
  • Biophysical chemistry
  • Analytical chemistry

Background:

  • Photon counting histogram (PCH) analysis is a fluorescence fluctuation technique.
  • It quantifies brightness and concentration of fluorescent species in solution.
  • Standard PCH models often use a 3D Gaussian observation volume.

Purpose of the Study:

  • To evaluate the adequacy of the standard 3D Gaussian PCH model for confocal microscopy.
  • To develop and validate an improved PCH model for enhanced fluorescence analysis.
  • To assess the impact of instrumental parameters on species resolvability.

Main Methods:

  • Utilized one-photon excitation in a confocal setup.
  • Applied photon counting histogram (PCH) analysis.

Related Experiment Videos

  • Developed a modified PCH model incorporating corrections for out-of-focus emission.

    • Main Results:

    • The standard 3D Gaussian PCH model showed inadequacy for experimental confocal data.
    • The improved PCH model, accounting for out-of-focus light, provided better data fitting.
    • The enhanced model successfully resolved different fluorescent species across various conditions.

    Conclusions:

    • A corrected observation volume profile is crucial for accurate PCH analysis in confocal setups.
    • The improved PCH model offers enhanced resolution for complex fluorescent samples.
    • This work facilitates the study of instrumental effects on fluorescence analysis.