Optical properties of zero mode waveguides

Single molecule fluorescence techniques have contributed considerably to our understanding of a variety of biological systems. Unfortunately, single molecule techniques are fundamentally limited by the concentration of fluorescent species and volume being observed. Nanofabricated structures have emerged as ideal tools for volume confinement in fluorescence spectroscopy. This has allowed the extension of single molecule techniques to high concentration regimes. Zero Mode Waveguides, nanometer scale holes in a thin metal film produce observation volumes in the attoliter to zeptoliter range. These structures have been used to measure protein kinetics in the micro molar concentration range as well as for observations of polymerase incorporating single nucleotide bases. We have investigated the optical properties of these structures using a combination of fluorescence spectroscopy, scanning electron microscopy and optical transmission measurements in an effort to accurately model the optical properties of these structures. These measurements have allowed a detailed characterization of the optical behavior of sub-wavelength apertures in a thin metal film.