Nanowaveguide-Illuminated Fluorescence Correlation Spectroscopy For Studying Molecular Dynamics On Cell Membranes

Fluorescence Correlation Spectroscopy (FCS) is a method for investigating particle concentration fluctuations in small volumes that are usually defined by a focused laser beam for excitation and a confocal pinhole for collection resulting in a size of about one femtoliter. Such sampling volumes are suitable for investigating dynamics of molecules in the nM concentration range. However, biological processes on cell membranes that involve molecules in the μM concentration range require sampling volumes well below the conventional FCS limit as well as nanoscale confinement in the longitudinal direction. In this study, we demonstrate that an effective measurement volume as small as one zeptoliter can be achieved via the introduction of a nanowire waveguide resulting in an illumination spot of about 50 nanometers in lateral dimensions and a longitudinal confinement of around 20 nanometers. Using illumination profiles obtained from finite element method simulations of dielectric nanowaveguides, we perform Monte Carlo simulations of fluorescence fluctuation for fluorophores that are confined in a membrane situated directly above the nanowaveguide exit surface with a background of fluorophores freely diffusing in the solution above the membrane. We have developed an analytical model to fit the simulation results and show that the signal is dominated by fluorophores on the membrane due to the extreme confinement of illumination in the longitudinal direction. These nanowaveguide devices are being fabricated and experiments are currently under way to verify the simulation results.