Nanoaperture fabrication via colloidal lithography for single molecule fluorescence imaging

Abstract

15In single molecule fluorescence studies, background emission from labeled substrates often restricts 16 their concentrations to non-physiological nanomolar values. One approach to address this challenge is 17 the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and 18 optically confine the observation volume allowing much higher concentrations of fluorescent substrates. 19Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are 20 made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of 21 ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on 22 a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead 23interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by 24 partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in 25 toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving 26 behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By 27 using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology 28 avoiding the availability and expense of sophisticated clean-room environments and equipment. 29 30