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Related Experiment Videos

The photon counting histogram in fluorescence fluctuation spectroscopy.

Y Chen1, J D Müller, P T So

  • 1Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. yan@lfd.physics.uiuc.edu

Biophysical Journal
|July 2, 1999
PubMed
Summary

Photon counting histogram (PCH) analysis offers a novel method for analyzing fluorescence fluctuation data, extracting molecule counts and photon counts per molecule. This technique reveals super-Poissonian behavior, providing deeper insights beyond traditional fluorescence correlation spectroscopy (FCS).

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Physical Chemistry

Background:

  • Fluorescence correlation spectroscopy (FCS) typically analyzes fluorescence signals to determine particle numbers and diffusion coefficients.
  • Existing methods rely on autocorrelation functions, which may not fully capture complex fluorescence fluctuation data.

Purpose of the Study:

  • To introduce and validate photon counting histogram (PCH) analysis as a novel tool for fluorescence fluctuation data.
  • To extract key parameters like photon counts per molecule and average molecule numbers within the observation volume.
  • To theoretically model and experimentally verify PCH behavior, including its super-Poissonian characteristics.

Main Methods:

  • Derivation of a theoretical expression for PCH based on Mandel's formula and two-photon excitation intensity distribution.

Related Experiment Videos

  • Analysis of PCH for single and multiple molecular species, including convolution of distributions.
  • Explicit treatment of the influence of point spread functions (PSFs), specifically Gaussian and Gaussian-Lorentzian profiles, on photon counting statistics.

    • Main Results:

    • PCH analysis successfully extracts photon counts per molecule and average molecule numbers.
    • Observed super-Poissonian behavior in PCH for few molecules in the excitation volume, attributed to fluorescence intensity fluctuations.
    • Experimental PCH data from two-photon excitation closely matched theoretical calculations for a Gaussian-Lorentzian beam profile.

    Conclusions:

    • Photon counting histogram (PCH) analysis is a powerful, complementary technique to FCS for quantitative analysis of fluorescence fluctuations.
    • PCH provides detailed information on molecular properties and numbers, influenced by excitation profiles and particle dynamics.
    • The study validates PCH as a robust method for characterizing fluorescence fluctuations in biophysical and chemical systems.