Nanowaveguide-illuminated fluorescence correlation spectroscopy for single molecule studies.

Area of Science:

• Biophysics
• Nanophotonics
• Spectroscopy

Background:

• Conventional Fluorescence Correlation Spectroscopy (FCS) is limited by its femtoliter sample volume, restricting studies of high-concentration (μM) molecules on cell membranes.
• Biological processes on cell membranes often involve molecules at concentrations exceeding the capabilities of standard FCS, necessitating smaller measurement volumes and nanoscale confinement.

Purpose of the Study:

• To develop a novel approach using nanowire waveguides to achieve zeptoliter-scale measurement volumes for FCS.
• To investigate the concentration and dynamics of molecules, particularly proteins, on cell membranes at high densities.

Main Methods:

• Utilized finite element method simulations to obtain illumination profiles of dielectric nanowaveguides.
• Performed Monte Carlo simulations of fluorescence fluctuations for both 3D diffusion and 2D membrane-bound fluorophore movement.
• Developed analytical functions to fit simulation data and determine effective illumination sizes.

Main Results:

• Achieved an effective measurement volume down to the zeptoliter range using a nanowire waveguide.
• Demonstrated lateral dimensions of approximately 50 nm and longitudinal confinement of about 20 nm.
• Quantified effective illumination sizes of ~150 zl for 3D diffusion and 4 × 10⁻³ µm² for 2D membrane scenarios.

Conclusions:

• The nanowaveguide-illuminated FCS technique offers deep-subwavelength confinement suitable for high-resolution studies.
• This method is well-suited for analyzing the concentration and dynamics of densely distributed proteins on cell membranes.
• The advancement enables unprecedented insights into membrane-bound molecular behavior.