Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

Organic fluorophores, which are popular labels for microscopy applications, intrinsically suffer from transient and irreversible excursions to dark-states. An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However, the working principles of this approach are not yet fully understood. In this contribution, we investigate the mechanism of intramolecular photostabilization in self-healing cyanines, in which photodamage is automatically repaired. Experimental evidence is provided to demonstrate that a single photostabilizer, that is, the vitamin E derivative Trolox, efficiently heals the cyanine fluorophore Cy5 in the absence of any photostabilizers in solution. A plausible mechanism is that Trolox interacts with the fluorophore through intramolecular quenching of triplet-related dark-states, which is a mechanism that appears to be common for both triplet-state quenchers (cyclooctatetraene) and redox-active compounds (Trolox, ascorbic acid, methylviologen). Additionally, the influence of solution-additives, such as cysteamine and procatechuic acid, on the self-healing process are studied. The results suggest the potential applicability of self-healing fluorophores in stochastic optical reconstruction microscopy (STORM) with optical super-resolution. The presented data contributes to an improved understanding of the mechanism involved in intramolecular photostabilization and has high relevance for the future development of self-healing fluorophores, including their applications in various research fields.

Fluorescence is a versatile tool for spectroscopic investigations and imaging of dynamic processes and structures across various scientific disciplines. The photophysical performance, that is, signal stability and signal duration, of the employed fluorophores is a major limiting factor. In this Letter, we propose a general concept to covalently link molecules, which are known for their positive effect in photostabilization, to form a combined photostabilizer with new properties. The direct linkage of two (or more) photostabilizers will allow one to obtain combined or synergetic effects in fluorophore stabilization and can simplify the preparation of imaging buffers that would otherwise require a mixture of photostabilizers for optimal performance. This concept was explored by synthesizing a molecule with a reducing and oxidizing moiety that is referred to as internal ROXS or "iROXS". Using single-molecule fluorescence microscopy, inter- and intramolecular healing of iROXS was observed, that is, strongly reduced blinking and increased photostability of the cyanine fluorophore Cy5. Moreover, it is shown that a covalently coupled photostabilizer can replace a mixture of molecules needed to make a functional photostabilizing ROXS buffer and might hence represent the new standard for defined and reproducible imaging conditions in single-molecule experiments. In self-healing fluorophores with intramolecular triplet-state quenching, an unprecedented photostability increase of >100-fold was obtained when using iROXS, which is even competitive with solution-based healing. Control experiments show that the oxidizing part of the iROXS molecule, an aromatic nitro group, dominates the healing process. The suggested synthetic concept and the proof-of-concept experiments represent the starting point for the quest to identify optimal combinations of linked photostabilizers for various fluorescence applications.

How photostabilizer molecules influence the photophysical properties of various organic fluorophores used for single-molecule and super-resolution imaging.

Covalent linkage of fluorophores and photostabilizers was recently revived as a strategy to make organic fluorophores "self-healing"via triplet-state quenching. Although Lüttke and co-workers pioneered this strategy already in the 1980s, the general design principles still remain elusive. In this contribution, we combine experiments and theory to understand what determines the photostabilization efficiency in dye-photostabilizer conjugates. Our results from single-molecule microscopy and molecular dynamics simulations of different Cy5-derivatives suggest that the distance and relative geometry between the fluorophore and photostabilizer are more important than the chemical nature of the photostabilizer, e.g. its redox potential, which is known to influence electron-transfer rates. We hypothesize that the efficiency of photostabilization scales directly with the contact rate of the fluorophore and photostabilizer. This study represents an important step in the understanding of the molecular mechanism of intramolecular photostabilization and can pave the way for further development of stable emitters for various applications.