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Abstract:
Photoswitchable fluorophores─proteins and synthetic dyes─whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene (DAE) and cucurbit[7]uril (CB7) (denoted as DAE@CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host–guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φfl) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE@CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N-hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE@CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3@CB7 and DAE-NHS@CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70–90 nm optical resolution.
