Effect of time-gating on stimulated emission depletion microscopy using sub-nanosecond pulses

Stimulated emission depletion (STED) microscopy is a super-resolution microscopy technique that overcomes the diffraction limit using a donut-shaped beam switching off fluorescence through stimulated emission. Because the resolution of STED microscopy is directly affected by beam intensity, a light source for the donut beam is very important. Because of their output power, cost, and convenience, sub-nanosecond fiber lasers have become popular. However, their pulses (approximately 500 ps) are somewhat long, which can degrade the resolution. In this study, we used time-gating to improve the resolution of an STED microscope built with a sub-nanosecond fiber laser. Quantitative analysis of single DNA origami bead images revealed that time-gating increased the resolution and signal-to-background ratio by 15% and twofold, respectively. A similar effect was observed in cell imaging. This result suggests that time-gating would be beneficial for STED microscopes using slightly long pulses.