Improved fabrication of zero-mode waveguides for single-molecule detection

Metallic subwavelength apertures can be used in epi-illumination fluorescence to achieve focal volume confinement. Because of the near field components inherent to small metallic structures, observation volumes are formed that are much smaller than the conventional diffraction limited volume attainable by high numerical aperture far field optics (circa a femtoliter). Observation volumes in the range of 10−4fl have been reported previously. Such apertures can be used for single-molecule detection at relatively high concentrations (up to 20μM) of fluorophores. Here, we present a novel fabrication of metallic subwavelength apertures in the visible range. Using a new electron beam lithography process, uniform arrays of such apertures can be manufactured efficiently in large numbers with diameters in the range of 60–100nm. The apertures were characterized by scanning electron microscopy, optical microscopy, focused ion beam cross sections/transmission electron microscopy, and fluorescence correlation spectrosc...

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