Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

Abstract

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.

Small-molecule fluorogenic probes for mitochondrial nanoscale imaging

“…These fluorogenic probes have excellent photophysical properties including higher brightness ( εΦ ), better photostability, smaller molecular size, lower background fluorescence, and easier chemical modification than fluorescent proteins, quantum dots, and rare earth materials. 83,84 Furthermore, they have good biocompatibility and cell penetration to specifically penetrate the cell membrane. 85–88…”

“…These fluorogenic probes have excellent photophysical properties including higher brightness (eF), better photostability, smaller molecular size, lower background fluorescence, and easier chemical modification than fluorescent proteins, quantum dots, and rare earth materials. 83,84 Furthermore, they have good biocompatibility and cell penetration to specifically penetrate the cell membrane. [85][86][87][88] The protons in the mitochondrial matrix are pumped into the mitochondrial membrane space via the proton pump (PP) in the IMM, resulting in positive charges in the mitochondrial membrane space.…”

Superior Photoprotection of Cyanine Dyes with Thio-imidazole Amino Acids

“…This is critical to monitor processes that occur over long-time scales 1 and for acquiring robust statistical information in a single molecule experiment. 2 Signal stability, in turn, ensures that recorded signal fluctuations can be unequivocally assigned to e.g., associated conformational dynamics, 1 preventing systematic errors in data interpretation. At the crux of the low performance affecting a fluorophore survival time and its signal stability lies formation of a long-lived excited triplet state (red arrow in Fig 1a).…”

Superior Photoprotection of Cyanine Dyes with Thio-imidazole Amino Acids

“…Enhanced photostability will prolong the time window a system can be interrogated for and further extend the number of photons extracted before fading. This is critical to monitor processes that occur over long-time scales and for acquiring robust statistical information in a single-molecule experiment . Signal stability, in turn, ensures that recorded signal fluctuations can be unequivocally assigned to, e.g., associated conformational dynamics, preventing systematic errors in data interpretation.…”