Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements

Area of Science:

• Physical Chemistry
• Biophysics
• Analytical Chemistry

Background:

• Accurate measurement of diffusion coefficients is crucial for understanding molecular dynamics.
• Standard fluorescence correlation spectroscopy (FCS) methods can be susceptible to optical and photophysical artifacts.
• Measuring diffusion at nanomolar concentrations requires high precision and sensitivity.

Purpose of the Study:

• To introduce a novel two-focus fluorescence correlation spectroscopy (FCS) method for precise absolute diffusion coefficient measurements.
• To improve upon standard FCS by incorporating an external ruler for accurate diffusion time determination.
• To validate the new method by comparing results with established techniques like gradient pulsed field NMR (GPF NMR).

Main Methods:

• Modification of a standard FCS setup to include two laterally shifted, overlapping laser foci at a fixed distance.
• Development and application of a new two-parameter model for fitting the molecule detection function (MDF).
• Measurement of the diffusion coefficient of Atto655 using the two-focus FCS technique.

Main Results:

• The new two-focus FCS method provides high-precision measurements of absolute diffusion coefficients at nanomolar concentrations.
• The developed two-parameter model accurately describes the molecule detection function (MDF).
• The diffusion coefficient of Atto655 measured by two-focus FCS (4.26 x 10^-6 cm²/s) closely matches the GPF NMR value (4.28 x 10^-6 cm²/s).

Conclusions:

• The two-focus FCS method is robust against common optical and photophysical artifacts inherent in standard FCS.
• This new technique offers a reliable and precise approach for determining diffusion coefficients.
• The method is suitable for studying molecular diffusion in various conditions with high accuracy.