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

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.
• 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.