Abstract
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.
Item Type: | Journal article |
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EU Funded Grant Agreement Number: | 638536 |
EU Projects: | Horizon 2020 > ERC Grants > ERC Starting Grant > ERC Grant 638536: SM-IMPORT - Substrate import at work: single-molecule studies of ABC transporters |
Form of publication: | Publisher's Version |
Faculties: | Biology > Department Biology I |
Subjects: | 500 Science > 570 Life sciences; biology |
URN: | urn:nbn:de:bvb:19-epub-77656-4 |
Language: | English |
Item ID: | 77656 |
Date Deposited: | 26. Oct 2021, 12:55 |
Last Modified: | 03. Jan 2022, 18:22 |