Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

Area of Science:

• Biochemistry
• Nanotechnology
• Optical Engineering

Background:

• Single molecule fluorescence enzymology is limited by background fluorescence, restricting fluorophore concentrations to low nanomolar ranges.
• Zero mode waveguides (ZMWs) confine excitation light to zeptoliter volumes, enabling single-molecule imaging at higher micromolar fluorophore concentrations.
• Current ZMW fabrication relies on expensive electron beam lithography, hindering widespread adoption.

Purpose of the Study:

• To present an accessible alternative fabrication method for zero mode waveguides (ZMWs).
• To enable single-molecule studies at physiologically relevant concentrations.
• To reduce the cost and complexity of ZMW production.

Main Methods:

• Utilized colloidal (nanosphere) lithography to create nanometer-scale masks for waveguide fabrication.
• Developed a parallel fabrication process for thousands of ZMWs.
• Employed common laboratory equipment and a thermal evaporator for metal deposition.

Main Results:

• Successfully fabricated thousands of aluminum or gold ZMWs in parallel.
• Achieved final waveguide diameters and depths of 100-200 nm.
• Demonstrated a cost-effective and accessible method for ZMW production.

Conclusions:

• Nanosphere lithography offers a practical and scalable approach to fabricating ZMWs.
• Increased accessibility of ZMWs can facilitate single-molecule studies at cellular concentrations.
• This method has the potential to advance biochemical research by enabling studies at higher rates and concentrations.