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

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

doi:10.1039/c8cp05063e

Cited by 33 publications

(60 citation statements)

References 67 publications

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“…To assess possible applications and the impact of our labeling strategy, we next focused on characterization of self‐healing linker‐dye conjugates. We decided to focus on these (and not those of the metal‐sensing dyes) since we can benchmark the quantitative performance of the linker‐dye conjugates better in the context of our previous work [9b,c, 29a, 33] and that of others [18, 34] . We further reasoned that successful self‐healing linker‐dye conjugates would be able to fill an important gap in the field, since only very few commercial solutions are available for self‐healing dyes.…”

Section: Resultsmentioning

confidence: 99%

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

et al. 2022

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Many life‐science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super‐resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of ‘linker’ compounds, that bridge biotarget, fluorophore and a functional moiety via well‐established labeling protocols. Linker molecules were synthesized via the Ugi four‐component reaction (Ugi‐4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation‐ and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon‐rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol‐maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self‐healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self‐healing linker–fluorophore conjugates and demonstrated their applications in super‐resolution imaging and single‐molecule spectroscopy.

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Section: Resultsmentioning

confidence: 99%

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

et al. 2022

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“…[166] Self-healing dyes have advantages in SRM applications in terms of brightness and photostability (Figure 3B). [76,165] Nitrophenylalanine (NPA)-conjugated Atto647N dye and nitrophenylacetic acid-conjugated Star635P were prepared for STED imaging. Substantially larger fractions of self-healing dye molecules survived after STED imaging, and their average brightness was enhanced.…”

Section: Self-healing Dyesmentioning

confidence: 99%

“…Self‐healing dyes are synthetic organic fluorophores with covalently attached photostabilizing groups (PSG), such as COT, 4‐nitrobenzyl alcohol (NBA), and 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐caboxylic acid (Trolox) ( Figure 3 A ). [ 161 , 162 , 163 , 164 , 165 , 166 ] In buffer‐based systems, diffusion‐mediated reduction‐oxidation reactions restore two vulnerable states of fluorophores. Different pairs of reductants and oxidants are generally required for efficient photostabilization.…”

Section: Conventional Photostabilizing Strategies Applied In Super‐re...mentioning

confidence: 99%

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

2022

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Photobleaching is the permanent loss of fluorescence after extended exposure to light and is a major limiting factor in super‐resolution microscopy (SRM) that restricts spatiotemporal resolution and observation time. Strategies for preventing or overcoming photobleaching in SRM are reviewed developing new probes and chemical environments. Photostabilization strategies are introduced first, which are borrowed from conventional fluorescence microscopy, that are employed in SRM. SRM‐specific strategies are then highlighted that exploit the on–off transitions of fluorescence, which is the key mechanism for achieving super‐resolution, which are becoming new routes to address photobleaching in SRM. Off states can serve as a shelter from excitation by light or an exit to release a damaged probe and replace it with a fresh one. Such efforts in overcoming the photobleaching limits are anticipated to enhance resolution to molecular scales and to extend the observation time to physiological lifespans.

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“…44 Since there are no other fluorescent tags with these properties, the impact of FPs for 45 biological research cannot be overemphasized 1,[3][4][5] . Despite their importance, the 46 photophysical properties of FPs, i.e., brightness, count-rate and photostability 6-8 , are 47 relatively poor compared to synthetic organic fluorophores 9 or quantum dots [10][11] . 48 Extensive research has been done over the past decades to improve the photophysical 49 properties of FPs 12 .…”

Section: Introduction 41mentioning

confidence: 99%

Characterization of fluorescent proteins with intramolecular photostabilization

Henrikus

Tassis

Zhang

et al. 2020

Preprint

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Genetically encodable fluorescent proteins have revolutionized biological imaging in 21 vivo and in vitro. Since there are no other natural fluorescent tags with comparable features, 22 the impact of fluorescent proteins for biological research cannot be overemphasized. Despite 23 their importance, their photophysical properties, i.e., brightness, count-rate and photostability, 24 are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular 25 photostabilizers were recently rediscovered as an effective approach to improve photophysical 26properties. The approach uses direct conjugation of photostablizing compounds such as triplet-27 state quenchers or redox-active substances to an organic fluorophore, thereby creating high 28 local concentrations of photostabilizer. Here, we introduce an experimental strategy to screen 29 for the effects of covalently-linked photostabilizers on fluorescent proteins. We recombinantly 30 produced a double cysteine mutant (A206C/L221C) of -GFP for attachment of photostabilizer-31 maleimides on the ß-barrel in close proximity to the chromophore. Whereas labelling with 32 photostabilizers such as Trolox, Nitrophenyl, and Cyclooctatetraene, which are often used for 33 organic fluorophores, had no effect on -GFP-photostability, a substantial increase of 34 photostability was found upon conjugation of -GFP to an azobenzene derivative. Although the 35 mechanism of the photostabilizing effects remains to be elucidated, we speculate that the 36 higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the 37 near-UV and blue spectral range. Our study paves the way towards the development and 38 design of a second generation of fluorescent proteins with photostabilizers placed directly in 39 the protein barrel by methods such as unnatural amino acid incorporation.

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

Abstract: How photostabilizer molecules influence the photophysical properties of various organic fluorophores used for single-molecule and super-resolution imaging.

Cited by 33 publications

References 67 publications

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

Characterization of fluorescent proteins with intramolecular photostabilization

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