Everlasting rhodamine dyes and true deciding factors in their STED microscopy performance

Kolmakov, Kirill; Winter, Franziska R.; Sednev, Maksim V.; Ghosh, Subhabrata; Borisov, Sergey; Nizovtsev, Alexey V.

doi:10.1039/d0pp00304b

Cited by 6 publications

(45 citation statements)

References 58 publications

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“… a Relative quantum yields measured in PBS buffer at pH = 7.4, using oxazine 170 as a reference dye ( 9 ). b See Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A previous comparative study investigated the STED applications of red-emitting rhodamine dyes, demonstrating that fluorophore functionalization with charged polar functional groups (sulfate, phosphate) was crucial for the dye’s performance ( 9 ). However, although a large set of dyes was tested, positively charged compounds were not considered.…”

Section: Introductionmentioning

confidence: 99%

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Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging

Lucidi,

Capecchi,

Visaggio

et al. 2024

Microbiol Spectr

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Our understanding of bacteria is increasingly dependent on our ability to visualize cellular processes at the single-cell level with high spatial and temporal resolution. Advances in fluorescence microscopy are accompanied by an increasing demand for novel fluorophores that enable tagging specific bacterial components. In this context, new fluorescent probes emitting in the far-red (FR) are of particular interest as they reduce possible interference caused by sample autofluorescence and increase flexibility in multicolor imaging experiments. In this study, an extended set of previously reported and newly synthesized FR-emitting dyes has been characterized for their applicability in live single-cell imaging of the Gram-negative and Gram-positive prototype bacteria Escherichia coli and Bacillus subtilis . Toxicity tests demonstrated that these dyes do not interfere with the growth kinetics of both species, opening up the possibility of using them in live-cell imaging. Moreover, confocal laser-scanning microscopy imaging revealed that all the tested dyes can distinguish viable from dead bacterial cells. Among the newly synthesized fluorophores, the oxazine derivative KK 1905-NHS was particularly efficient in membrane staining and was effectively employed to monitor membrane biogenesis using a two-step labeling protocol on living cells. In addition, KK 1905-NHS was successfully used in super-resolution stimulated emission depletion microscopy. Overall, the new fluorophores presented in this study expand the microscopy toolbox, which is an asset for the investigation of fundamental bacterial processes. IMPORTANCE By harnessing the versatility of fluorescence microscopy and super-resolution imaging, bacteriologists explore critical aspects of bacterial physiology and resolve bacterial structures sized beyond the light diffraction limit. These techniques are based on fluorophores with profitable photochemical and tagging properties. The paucity of available far-red (FR)-emitting dyes for bacterial imaging strongly limits the multicolor choice of bacteriologists, hindering the possibility of labeling multiple structures in a single experiment. The set of FR fluorophores characterized in this study expands the palette of dyes useful for microbiologists, as they can be used for bacterial LIVE/DEAD staining and for tagging the membranes of viable Escherichia coli and Bacillus subtilis cells. The absence of toxicity makes these dyes suitable for live-cell imaging and allows monitoring of bacterial membrane biogenesis. Moreover, a newly synthesized FR-fluorophore can be employed for imaging bacterial membranes with stimulated emission depletion microscopy, a super-resolution technique capable of increasing the resolving power of conventional microscopes.

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“… a Relative quantum yields measured in PBS buffer at pH = 7.4, using oxazine 170 as a reference dye ( 9 ). b See Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging

Lucidi,

Capecchi,

Visaggio

et al. 2024

Microbiol Spectr

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“…First, it may be caused by aggregation of the close-by markers that may not only quench the emission but even prevent on-switching. Possible remedies are based on the incorporation of charged side groups (e.g., sulfonates) 64 or reducing the DOL values. The first option is already implemented in our DAEs having charged trimethylammonium groups, and the second one is inappropriate as it should result in dimmer images or longer recording times.…”

Section: Resultsmentioning

confidence: 99%

Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging

Kim¹,

Aktalay

Jensen³

et al. 2022

J. Am. Chem. Soc.

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Photoswitchable fluorophores—proteins and synthetic dyes—whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene ( DAE ) and cucurbit[7]uril (CB7) (denoted as DAE @CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host–guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φ fl ) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE @CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N -hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE @CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3 @CB7 and DAE-NHS @CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70–90 nm optical resolution.

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Section: Xanthene- Cyanine- Oxazine- and Bodipy-based Fluorophores Fo...mentioning

confidence: 99%

“…114 In this context, it is also important to highlight that the photostability and other fluorogenic properties of the STED-compatible red-emitting rhodamine dyes could be adequately modulated by attaching small-molecule intramolecular stabilizing agents and/or auxiliary groups at different sites of the rhodamine-based fluorescent probes. 115 Song et al reported the synthesis of some modified Sirhodamine (SiRMO) dyes, in which methoxybenzene units were incorporated into the xanthene moieties. 116 One of the newly synthesized Si-rhodamine dye (SiRMO-2) containing the 2,6dimethoxybenzene substituent, not only showed increased brightness but also possessed markedly improved stability owing to steric hindrance protection mediated by two methoxy groups on the ninth carbon atom of the xanthene (Fig.…”

Section: Bodipymentioning

confidence: 99%

Xanthene, cyanine, oxazine and BODIPY: the four pillars of the fluorophore empire for super-resolution bioimaging

Samanta,

Lai,

et al. 2023

Chem. Soc. Rev.

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Everlasting rhodamine dyes and true deciding factors in their STED microscopy performance

Abstract: The authors took an independent and closer look at the family of red-emitting rhodamine dyes known for a decade due to their excellent performance in STED microscopy. After the family...

Cited by 6 publications

References 58 publications

Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging

Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging

Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging

Xanthene, cyanine, oxazine and BODIPY: the four pillars of the fluorophore empire for super-resolution bioimaging

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