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

Abstract

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.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Self-healing dyes for super-resolution microscopy

“…It has to be stressed out that the time-point where signal-loss occurs (see Figure 5a, where the signal abruptly decreases to background level) might reflect either irreversible photobleaching or UV-reversible off-switching. In line with our previous work we refer to this as "apparent photobleaching lifetime" 44 , which reflects both pathways for fluorescence deactivation.…”

“…Related to this requirement, our group recently reported the effects of intramolecular photostabilizers on the action of solution-based additives such as Trolox, COT, TCEP and cysteamine (MEA). 44 In that paper, we evaluated the competition between inter-and intramolecular triplet-state quenching processes and showed that those are neither additive nor synergistic. Our findings revealed that intramolecular processes dominate the photophysical properties of different organic fluorophores for combinations of covalentlylinked and solution-based photostabilizers and importantly also photoswitching agents used for STORM.…”

“…Our findings revealed that intramolecular processes dominate the photophysical properties of different organic fluorophores for combinations of covalentlylinked and solution-based photostabilizers and importantly also photoswitching agents used for STORM. In that study 44 we identified a new function of intramolecular photostabilizers, i.e., protection of fluorophores from reversible photoswitching. Additionally, evidence was provided that the biochemical environment, here proximity of aromatic amino-acids such as tryptophan, significantly reduced the photostabilization efficiency of commonly used buffer cocktails.…”

“…Additionally, evidence was provided that the biochemical environment, here proximity of aromatic amino-acids such as tryptophan, significantly reduced the photostabilization efficiency of commonly used buffer cocktails. 44 Here, we detail the effects of the intramolecular photostabilizers TX, NPA and COT for Cy5 photoswitching kinetics and photon-yields, i.e., parameters relevant for STORM-type microscopy. We used an established approach where single-molecule TIRF-microscopy provides information on photobleaching lifetime, countrate, signal-to-noise ratio and total photon-count of individual fluorophores tethered to dsDNA on a microscope coverslide ( Figure 5A) 23,44 .…”

“…44 Here, we detail the effects of the intramolecular photostabilizers TX, NPA and COT for Cy5 photoswitching kinetics and photon-yields, i.e., parameters relevant for STORM-type microscopy. We used an established approach where single-molecule TIRF-microscopy provides information on photobleaching lifetime, countrate, signal-to-noise ratio and total photon-count of individual fluorophores tethered to dsDNA on a microscope coverslide ( Figure 5A) 23,44 . It has to be stressed out that the time-point where signal-loss occurs (see Figure 5a, where the signal abruptly decreases to background level) might reflect either irreversible photobleaching or UV-reversible off-switching.…”