On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

Smit, J.; van der Velde, J.; Huang, J.; Trauschke, V.; Henrikus, S.; Chen, S.; Eleftheriadis, N.; Warszawik, E.; Herrmann, A.; Cordes, T.

doi:10.1101/371443

bioRxiv · 10.1101/371443

On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

Smit, J.·van der Velde, J.·Huang, J.·Trauschke, V.·Henrikus, S.·Chen, S.·Eleftheriadis, N.·Warszawik, E.·Herrmann, A.·Cordes, T.

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Abstract

While buffer cocktails remain the gold-standard 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 various combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other rather than being additive or even synergistic. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. In this context we identified a new function of intramolecular photostabilizers, i.e., protection of fluorophores from reversible off-switching events caused by solution-additives, which were 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.

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Smit, J., van der Velde, J., Huang, J., Trauschke, V., Henrikus, S., Chen, S., Eleftheriadis, N., Warszawik, E., Herrmann, A., Cordes, T.. 2018-07-18. On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores. https://doi.org/10.1101/371443

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