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.

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis.

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography.