Fluorescence correlation spectroscopy analysis of diffusion in a laser gradient field: a numerical approach

Area of Science:

• Biophysics
• Optical Spectroscopy
• Physical Chemistry

Background:

• Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying molecular dynamics in biological systems.
• High laser intensities, common in two-photon experiments, can exert gradient forces on fluorescent particles.
• These forces can alter particle diffusion, leading to inaccuracies in standard FCS measurements, a phenomenon known as biased FCS.

Purpose of the Study:

• To develop a quantitative numerical method for analyzing biased FCS data.
• To accurately determine unbiased parameters like particle number, diffusion time, and polarizability.
• To quantify the strength of the laser gradient field influencing particle behavior.

Main Methods:

• A numerical approach assuming spherical symmetry was employed to model biased FCS curves.
• The numerical model was used to fit experimental FCS data.
• Simulated FCS data was utilized to validate the accuracy and robustness of the method.

Main Results:

• The numerical method successfully retrieves unbiased particle number, diffusion time, and polarizability.
• The strength of the laser gradient field can be quantitatively determined.
• The discrepancy introduced by the spherical symmetry approximation was found to be independent of the gradient field strength.

Conclusions:

• The proposed numerical method provides a quantitative solution for correcting errors in FCS measurements caused by laser-induced forces.
• Accurate determination of particle properties and laser field strength is achievable even under biased conditions.
• Calibration can eliminate systematic errors arising from the spherical symmetry approximation, enhancing the reliability of FCS in intense laser regimes.