Fluorescence nanoscopy, enabling single-digit nanometer resolution, revolutionizes our ability to visualize cellular structures and dynamics. Crucial to nanoscopy is the controlled switching of fluorophores between fluorescent ('on') and non-fluorescent ('off') states. Photoactivatable dyes improve these techniques by providing an irreversible light-triggered off-on transition, eliminating the need for specific buffers and high-intensity UV light. While rhodamine dyes are widely used due to their tunability, photostability, and brightness, current caging strategies involve photolabile protecting groups, limiting applications due to restrictions on substitution patterns, reduced water solubility, and toxic by-products. Existing caging strategies for rhodamines either involve photolabile protecting groups on nitrogen atoms or synthetic transformations of the lactone ring, both resulting in limitations. This study aims to address these limitations by developing a caging-group-free, compact, photoactivatable, and biocompatible fluorophore. The proposed approach utilizes photochemical reactions to 'lock' the fluorophores into a fluorescent state rather than 'unlocking' them from a caged state, potentially offering improved efficiency and reducing by-products.

The authors review existing photoactivatable or caged dyes and their limitations, focusing on rhodamine dyes and their caging strategies. They highlight the use of photolabile protecting groups (like nitroveratryloxycarbonyl or nitroso groups) and synthetic transformations of the lactone ring into cyclic α-diazoketones. They point out the drawbacks of these methods, such as restricted substitution patterns, reduced water solubility, toxic byproducts, varying uncaging efficiencies, and the formation of non-fluorescent side products. The literature also includes examples of photoactivation of silicon-pyronine analogues, but these suffer from susceptibility to nucleophilic addition products, limiting their applicability. This literature review sets the stage for the introduction of the novel caging-group-free approach.

The authors designed a new class of photoactivatable xanthone (PaX) dyes based on the photochemistry of diarylketones. The proposed mechanism involves a light-triggered cascade where a 3,6-diaminoxanthone core, functionalized with an intramolecular alkene radical trap, undergoes a photochemical reaction upon excitation to form a dihydropyran-fused pyronine dye. A series of Si-xanthones (1-7) were synthesized using an Ir-catalyzed ortho-selective C-H borylation followed by conversion to aryl bromide and Suzuki-Miyaura cross-coupling with various alkene substituents. Photoactivation kinetics were studied using UV irradiation in protic media. The formation of the 9-alkoxypyronine product was confirmed through LC-MS and NMR analyses. For bioconjugation, amino-reactive NHS ester (13), thiol-reactive maleimide (14), and actin-labeling phalloidin (15) derivatives were prepared. The photoactivation properties were characterized at various pH values and in the presence of thiols to assess their stability and biocompatibility. Live-cell compatibility and two-photon activation were also explored using various organelle-targeting moieties and self-labeling protein tags (HaloTag and SNAP-tag). Confocal, STED, PALM, and MINFLUX microscopy were used to image various cellular structures (microtubules, actin, nuclear pore complexes) in both fixed and living cells.

The synthesized PaX dyes exhibit rapid and complete conversion to highly fluorescent pyronine dyes upon irradiation, with no significant by-products. Photoactivation quantum yields ranged from 1 × 10⁻² to 6 × 10⁻². The rate of photoactivation was influenced by the alkene substituent. Si-bridged PaX dyes showed good live-cell compatibility and resistance to nucleophiles, with exceptional photostability for orange-emitting fluorophores. Successful super-resolution imaging was achieved using PaX dyes in STED, PALM, and MINFLUX microscopy. Live-cell imaging was demonstrated using organelle-targeted PaX dyes and self-labeling protein tag conjugates, showcasing two-photon activation using near-infrared light. The study also demonstrated the potential for channel duplexing using PaX dyes and established fluorophores, effectively doubling the imaging channels. Multiplexing of PaX labels was achieved through selective photoactivation using varying light doses. Finally, successful MINFLUX imaging of nuclear pore complexes was demonstrated.

The results demonstrate that the PaX dyes offer a superior alternative to traditional photoactivatable dyes, addressing limitations associated with caging groups. The caging-group-free design results in compact labels with improved water solubility and reduced toxicity. The efficient and clean photoactivation, coupled with good photostability and live-cell compatibility, makes them suitable for a wide range of microscopy techniques. The ability to tune photoactivation kinetics and spectral properties through substitution offers flexibility for various applications, including multiplexed imaging. The success in live-cell imaging and super-resolution microscopy highlights the potential for PaX dyes to advance biological research.

This study presents a general design strategy for caging-group-free, bright, and live-cell-compatible photoactivatable dyes. The PaX dyes are versatile, applicable to various microscopy techniques, and offer advantages over existing methods. Future research could focus on expanding the range of available PaX dyes, further optimizing photoactivation kinetics, and exploring applications in advanced nanoscopy techniques like MINSTED.

While the study demonstrates the versatility and efficacy of PaX dyes in various imaging techniques, potential limitations include the need for further optimization of the photoactivation parameters for specific applications and thorough in vivo testing for potential long-term toxicity.