Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy
| Autor: | Shawn H. Pfeil, Kevin Y Chen, Ryan M. Jamiolkowski, Shane A. Fiorenza, Yale E. Goldman, Alyssa M. Tate |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Fluorophore
Materials science General Chemical Engineering Finite Element Analysis Article General Biochemistry Genetics and Molecular Biology law.invention chemistry.chemical_compound law Fluorescence Resonance Energy Transfer Colloids Lithography Fluorescent Dyes General Immunology and Microbiology business.industry General Neuroscience Colloidal crystal Single-molecule experiment Microspheres Single Molecule Imaging Nanostructures Nanolithography chemistry Microscopy Fluorescence Optoelectronics Nanosphere lithography Microtechnology Polystyrenes Gold business Crystallization Waveguide Porosity Electron-beam lithography Copper Aluminum |
| Zdroj: | J Vis Exp |
| ISSN: | 1940-087X |
| DOI: | 10.3791/61154-v |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|