2019, Smit, Jochem H., van der Velde, Jasper H. M., Huang, Jingyi, Trauschke, Vanessa, Henrikus, Sarah S., Chen, Si, Eleftheriadis, Nikolaos, Warszawik, Eliza M., Herrmann, Andreas, Cordes, Thorben

While buffer cocktails remain the most commonly used method for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with ‘self-healing’ or even functional properties such as photoswitching. In this contribution, we evaluated combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other. We find that although the rate of triplet-state quenching is additive, the photostability is limited by the faster pathway. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. Furthermore we show that intramolecular photostabilizers can protect fluorophores from reversible off-switching events caused by solution-additives, which was previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer–dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics in different biochemical environments.