Single molecule confocal fluorescence lifetime correlation spectroscopy for accurate nanoparticle size determination

Area of Science:

• Spectroscopy
• Physical Chemistry
• Nanotechnology

Background:

• Confocal single molecule fluorescence lifetime correlation spectroscopy (FLCS) is a powerful technique for studying molecular dynamics.
• Accurate application of autocorrelation functions is crucial for reliable data interpretation in FLCS.
• Understanding the influence of experimental parameters is key to optimizing FLCS measurements.

Purpose of the Study:

• To establish an experimental procedure for confocal FLCS to determine optimal excitation power and concentration ranges.
• To validate the use of unmodified model autocorrelation functions for accurate single-molecule analysis.
• To investigate the impact of pinhole size on diffusion measurements and nanoparticle characterization.

Main Methods:

• Development of an experimental protocol for confocal FLCS.
• Analysis of autocorrelation functions under varying excitation power and molecular concentrations.
• Systematic evaluation of pinhole size effects on diffusion length (r) and diffusion time (τD).
• Application of the Stokes-Einstein (S-E) equation using determined parameters for nanoparticle hydrodynamic radius calculation.

Main Results:

• Defined the operational window for justified use of unmodified autocorrelation functions in FLCS.
• Demonstrated accurate measurement of diffusion length (r) and diffusion time (τD) for single molecules.
• Quantified the dependency of r and τD on pinhole size in the confocal FLCS setup.
• Identified the particle size range where the S-E equation accurately reflects the real radius of spherical nanoparticles.

Conclusions:

• The developed procedure enhances the reliability of confocal FLCS for molecular diffusion studies.
• This method provides a robust approach for accurate nanoparticle size determination using the S-E equation.
• The findings offer critical guidance for optimizing experimental conditions in FLCS and nanoparticle characterization.