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Physical chemistry, chemical physics 21(7), 3721 - 3733 (2019). doi:10.1039/C8CP05063E, Published by RSC Publ., Cambridge

ChemBioChem 22(23), 3283-3291 (2021). doi:10.1002/cbic.202100276, Published by Wiley-VCH, Weinheim

In recent years, optical microscopy techniques have emerged that allow optical imaging at unprecedented resolution beyond the diffraction limit. These techniques exploit photostabilizing buffers to enable photoswitching and/or the enhancement of fluorophore brightness and stability. A major drawback with the use of photostabilizing buffers, however, is that they cannot be used in live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores, which undergo intramolecular photostabilization, in super-resolution microscopy examining both targeted (stimulated emission depletion (STED) microscopy) and stochastic readout (stochastic optical reconstruction microscopy (STORM)). The overall goal of the study was to identify dyes and conditions that lead to improved spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. As a result of previously shown superior performance, we identified an ATTO647N-photostabilizer conjugate as a potential candidate for STED microscopy. We have here characterized the photostability and resulting performance of this nitrophenylalanine (NPA) conjugate of ATTO647N on oligonucleotides in STED microscopy. We found that the superior photophysical performance resulted in optimal STED imaging and demonstrated that single-molecule fluorescent transients of individual fluorophores can be obtained with both the excitation-and STED-laser. In similar experiments, we also tested a nitrophenylacetic acid conjugate of STAR635P, another frequently used dye in STED microscopy, and present a characterization of its photophysical properties. Finally, we performed an analysis of the photoswitching kinetics of self-healing Cy5 dyes (containing trolox, cyclooctatetraene and NPA-based stabilizers) in the presence of Tris(2-carboxyethyl) phosphine and cysteamine, which are typically used in STORM microscopy. In line with previous work, we found that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.

Nature Communications 7: Article number: 10144 (2016); Published 11 January 2016, Updated 24 July 2018 The original version of this Article omitted the following from the Acknowledgements: ‘This work was also financed by an ERC starting grant ‘SM-IMPORT’ (No. 638536 to T.C.)’. This has been corrected in both the PDF and HTML versions of the Article.

In recent years optical microscopy techniques have emerged that allow optical imaging at unprecented resolution beyond the diffraction limit. Up to date, photostabilizing buffers are the method of choice to realize either photoswitching and/or to enhance the signal brightness and stability of the employed fluorescent probes. This strategy has, however, restricted applicability and is not suitable for live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores with intramolecular photostabilization in super-resolution microscopy with targeted (STED) and stochastic readout (STORM). The overall goal of the study was to improve the spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. Due to its past superior performance we identified ATTO647N-photostabilizer conjugates as suitable candidates for STED microscopy. We characterize the photostability and resulting performance of NPA-ATTO647N oligonucleotide conjugates in STED microscopy. We find that the superior photophysical performance results in optimal STED imaging and demonstrate the possibility to obtain single-molecule fluorescent transients of individual fluorophores while illuminating with both the excitation- and STED-laser. Secondly, we show an analysis of photoswitching kinetics of self-healing Cy5 dyes (comprising TX, COT and NPA stabilizers) in the presence of TCEP- and cysteamine, which are typically used in STORM microscopy. In line with previous work, we find that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.

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.

Nature Communications 7(1), 10144 (2016). doi:10.1038/ncomms10144, Published by Nature Publishing Group UK, [London]

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.

Reversible switching of conductance using redox triggered switching of a polymer-modified electrode is demonstrated. A bifunctional monomer comprising a central electroswitchable core and two bithiophene units enables formation of a film through anodic electropolymerization. The conductivity of the polymer can be switched electrochemically in a reversible manner by redox triggered opening and closing of the diarylethene unit. In the closed state, the conductivity of the modified electrode is higher than in the open state.