Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100-200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100-200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.