ZMWs

Zero-mode waveguides (ZMWs) are nanoscale optical confinement structures used to observe single-molecule enzymatic activity in real time, most notably in Pacific Biosciences’ SMRT sequencing technology. They enable the direct visualization of DNA polymerase as it incorporates nucleotides labeled with fluorescent dyes.

A ZMW consists of a subwavelength hole or well in a metal film deposited on a glass or fused-silica substrate. The metal blocks propagating light at the excitation wavelength, so only an extremely small observation volume exists inside the bottom of the well. Typical wells have diameters on the order of 70–100 nanometers and depths of a few hundred nanometers, yielding a observation volume in the zeptoliter range. DNA templates are tethered to the glass surface, and a DNA polymerase enzyme localizes in the well to synthesize DNA using fluorescently labeled nucleotides supplied in the surrounding solution.

During sequencing, the polymerase incorporates nucleotides one by one, and the fluorescent label on each incorporated nucleotide emits a brief signal detectable by highly sensitive cameras. Because the illumination is confined to a tiny volume, high nucleotide concentrations can be used without excessive background, enabling real-time, single-molecule detection at the level of individual base incorporations. The approach supports long read lengths and direct assessment of base modifications.

Manufacturing of ZMW arrays yields millions of wells per chip, typically fabricated in a metal film on a transparent substrate. While powerful, ZMW technology requires specialized instrumentation, reagents, and data analysis workflows, and remains most closely associated with PacBio’s SMRT sequencing platform.