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Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution

T E Starr1, N L Thompson

  • 1Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290, USA.

Biophysical Journal
|February 27, 2001
PubMed
Summary
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Total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) quantifies surface binding kinetics and molecular concentrations. This study details the TIR-FCS autocorrelation function, incorporating surface kinetics and diffusion for enhanced interfacial analysis.

Area of Science:

  • Biophysics
  • Surface Science
  • Spectroscopy

Background:

  • Total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) measures surface interactions of fluorescent molecules.
  • Previous work established TIR-FCS autocorrelation functions without evanescent wave diffusion contributions.
  • Understanding these contributions is crucial for accurate interfacial measurements.

Purpose of the Study:

  • To describe the TIR-FCS autocorrelation function when surface kinetics and evanescent wave diffusion are both significant.
  • To provide a comprehensive theoretical framework for TIR-FCS analysis.
  • To enable more precise quantification of molecular behavior at interfaces.

Main Methods:

  • Development of theoretical expressions for the fluorescence fluctuation autocorrelation function.

Related Experiment Videos

  • Inclusion of parameters such as association/dissociation rates, surface site density, solution concentration, diffusion coefficient, and evanescent field depth.
  • Mathematical modeling of fluorescence fluctuations under combined kinetic and diffusion influences.

    • Main Results:

    • The TIR-FCS autocorrelation function is shown to depend on kinetic rate constants, surface density, solution concentration, diffusion, and evanescent field depth.
    • Both general and approximate mathematical expressions for the autocorrelation function are presented.
    • This work elucidates the interplay between surface binding and diffusion in TIR-FCS.

    Conclusions:

    • The presented expressions enhance the analytical capabilities of TIR-FCS for studying interfacial phenomena.
    • Accurate measurement of surface association/dissociation rates and molecular concentrations is achievable.
    • This theoretical advancement supports precise characterization of molecular dynamics at solution-substrate interfaces.