Improving zero-mode waveguide structure for enhancing signal-to-noise ratio of real-time single-molecule fluorescence imaging: A computational study

We investigated the signal-to-noise ratio ($S/N$) of real-time single-molecule fluorescence imaging (SMFI) using zero-mode waveguides (ZMWs). The excitation light and the fluorescence propagating from a molecule in the ZMW were analyzed by computational optics simulation. The dependence of the $S/N$ on the ZMW structure was investigated with the diameter and etching depth as the simulation parameters. We found that the SMFI using a conventional ZMW was near the critical level for detecting binding and dissociation events. We show that etching the glass surface of the ZMW by 60 nm enhances the $S/N$ six times the conventional nonetched ZMWs. The enhanced $S/N$ improves the temporal resolution of the SMFI at physiological concentrations.