Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

• Published in: Advanced science 2022-03, Vol.9 (9), p.e2101817-n/a
• Main Authors: Kwon, Jiwoong, Elgawish, Mohamed Saleh, Shim, Sang‐Hee
• Format: Article
• Language: English
• Subjects: Fluorescent Dyes - chemistry; fluorophore; Hypochlorous Acid; Light; Localization; Microscopy; Microscopy, Fluorescence; Photobleaching; photostability; photoswitching; Review; Reviews; super‐resolution fluorescence microscopy
• ISSN: 2198-3844; 2198-3844
• DOI: 10.1002/advs.202101817

Photobleaching is the permanent loss of fluorescence after extended exposure to light and is a major limiting factor in super‐resolution microscopy (SRM) that restricts spatiotemporal resolution and observation time. Strategies for preventing or overcoming photobleaching in SRM are reviewed developing new probes and chemical environments. Photostabilization strategies are introduced first, which are borrowed from conventional fluorescence microscopy, that are employed in SRM. SRM‐specific strategies are then highlighted that exploit the on–off transitions of fluorescence, which is the key mechanism for achieving super‐resolution, which are becoming new routes to address photobleaching in SRM. Off states can serve as a shelter from excitation by light or an exit to release a damaged probe and replace it with a fresh one. Such efforts in overcoming the photobleaching limits are anticipated to enhance resolution to molecular scales and to extend the observation time to physiological lifespans. In super‐resolution fluorescence microscopy, photobleaching not only leads into gradual loss of fluorophores but also greatly influences on spatiotemporal resolution. Both conventional photostabilization strategies applied in super‐resolution imaging and specific approaches repurposing fluorescence‐off states as shelters, are reviewed. Off states, originally devised for overcoming the diffraction limit, can shelter fluorophores from excitation or for replacing to a new one.