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

• Published in: Bulletin of the Korean Chemical Society Vol. 43; no. 8; pp. 1069 - 1074
• Main Authors: Lee, Mina, Joo, Sihwa
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley‐VCH Verlag GmbH & Co. KGaA 01.08.2022; 대한화학회
• Subjects: gated‐STED; STED microscopy; sub‐nanosecond fiber laser; super‐resolution microscopy; time‐gating; 화학
• ISSN: 1229-5949; 0253-2964; 1229-5949
• DOI: 10.1002/bkcs.12583

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. Time‐gating was applied to an STED microscope system built with a sub‐nanosecond laser to improve the resolution. In contrast to previous studies that time‐gating would be useless for pulse STED, we found that it substantially increased the resolution and signal‐to‐background ratio of the STED images. This result suggests that time‐gating can address the reduction in resolution caused by sub‐nanosecond STED pulses.