The use of photoswitchable fluorescent diarylethenes (fDAEs) as protein labels in fluorescence microscopy and nanoscopy has been limited by labeling inhomogeneity and the need for ultraviolet light for fluorescence activation (on-switching). To overcome these drawbacks, we prepared “turn-on mode” fDAEs featuring thienyl substituents, multiple polar residues, and a reactive maleimide group in the core structure. Conjugates with antibodies and nanobodies displayed complete on-switching and excitation with violet (405 nm) and yellow-green (<565 nm) light, respectively. Besides, they afforded high signal-to-noise ratios and low unspecific labeling in fluorescence imaging. Irradiation with visible light at 532 nm or 561 nm led to transient on-off switching (“blinking”) of the fDAEs of double-labeled nanobodies so that nanoscale superresolution images were readily attained through switching and localization of individual fluorophores.

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 (Φ fl ) 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.

Here we describe highly compact, click compatible, and photoactivatable dyes for super-resolution fluorescence microscopy (nanoscopy). By combining the photoactivatable xanthone (PaX) core with a tetrazine group, we achieve minimally sized and highly sensitive molecular dyads for the selective labeling of unnatural amino acids introduced by genetic code expansion. We exploit the excited state quenching properties of the tetrazine group to attenuate the photoactivation rates of the PaX, and further reduce the overall fluorescence emission of the photogenerated fluorophore, providing two mechanisms of selectivity to reduce the offtarget signal. Coupled with MINFLUX nanoscopy, we employ our dyads in the minimal-linkage-error imaging of vimentin filaments, demonstrating molecular-scale precision in fluorophore positioning.

New photoactivatable fluorescent dyes (rhodamine, carbo‐ and silicon‐rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2‐nitrobenzyloxycarbonyl unit with an alpha‐carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live‐cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live‐cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single‐digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.

A bright and photostable fluorescent dye with a disulfide (S−S) linker and maleimide group (Rho594‐S2‐mal), as cleavable and reactive sites, was synthesized and conjugated with anti‐GFP nanobodies (NB). The binding of EGFP (FRET donor) with anti‐GFP NB labeled with one or two Rho594‐S2‐mal residues was studied in vitro and in cellulo. The linker was cleaved with dithiothreitol recovering the donor (FP) signal. The bioconjugates (FP‐NB‐dye) were applied in FRET‐FLIM assays, confocal imaging, and superresolution STED microscopy.