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Related Experiment Videos

Total internal reflection with fluorescence correlation spectroscopy.

Nancy L Thompson1, Bridgett L Steele

  • 1Department of Chemistry, University of North Carolina at Chapel Hill, North Carolina 27599, USA. nlt@unc.edu

Nature Protocols
|April 21, 2007
PubMed
Summary
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Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) enables the study of molecular dynamics near interfaces. This protocol details constructing a TIR-FCS system for analyzing cellular membrane interactions.

Area of Science:

  • Biophysics
  • Analytical Chemistry
  • Cell Biology

Background:

  • Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is a powerful technique for investigating molecular events at interfaces.
  • It is particularly promising for studying dynamics near the basal membranes of living cells.
  • Understanding interfacial dynamics is crucial in various biological and chemical processes.

Purpose of the Study:

  • To provide a protocol for constructing and testing a Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) system.
  • To adapt TIR-FCS for use with either through-prism or through-objective internal reflection geometry.
  • To guide researchers in applying TIR-FCS to study interfacial molecular interactions.

Main Methods:

  • Detailed steps for building a TIR-FCS apparatus are presented.

Related Experiment Videos

  • The protocol covers system construction and testing procedures.
  • It includes adaptations for both through-prism and through-objective geometries.

    • Main Results:

    • The protocol facilitates the measurement of local fluorophore concentrations and mobilities at interfaces.
    • It enables the determination of kinetic rate constants for molecular association and dissociation.
    • The expected autocorrelation functions for different molecular behaviors (diffusion, binding) are discussed.

    Conclusions:

    • This protocol offers a practical guide for establishing a TIR-FCS system.
    • The technique allows for detailed analysis of molecular dynamics at cell membranes and other interfaces.
    • TIR-FCS is a versatile tool for quantitative studies of interfacial phenomena.