Fluorescence Correlation Spectroscopy with Nanowire Waveguide Illumination for High Concentration Conditions

Abstract

Fluorescence correlation spectroscopy (FCS), a technique based on correlating the fluorescence signal from a small sample volume, has been commonly used to study biological processes such as photophysical phenomena, binding kinetics, and intracellular dynamics. In a conventional FCS setup, the sample volume is defined by a focused laser beam for excitation and a confocal pin hole for collection resulting in a size of about one femtoliter. Evanescent waves from total internal reflection have been used to obtain a smaller sample volume, as a result of the improved confinement along the longitudinal axis for the illumination. However, applying FCS under physiological conditions where the molecule concentration is typically in the µM range requires an even smaller sample volume. Here we propose to apply FCS with nanowire waveguide illumination with nanometer-sized lateral and longitudinal confinement. Nanowire waveguides, made of a dielectric nanowire with a diameter of ∼50 nm embedded inside a metal film, allow for the efficient transmission of visible light by exploiting surface waves traveling along the dielectric-metal boundary. Such waveguides can generate a lateral illumination area of about 50 nanometers in diameter. In addition, a longitudinal confinement of around 20 nanometers is achieved with the rapidly-attenuating near fields exiting from the waveguide. The strong confinement in both lateral and longitudinal directions thus lead to a sample volume on the order of one zeptoliter, allowing for FCS measurements in the µM concentration range. By combining Brownian dynamics simulations with the illumination profile obtained from finite element method simulations of a zinc oxide nanowire waveguide in a silver metal film, we numerically calculate the correlation function to demonstrate the use of this method in the study of molecular dynamics under high concentration conditions.