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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 was further extended, the true grounds of this performance became clear. Small-molecule protective agents and/or auxiliary groups were attached at two different sites of the dye's scaffold. Thus, a rhodamine core, which is already quite photostable as it is, and an intramolecular stabilizer – a 4-nitrobenzyl or a 4-nitrobenzylthio group were combined to give potentially “everlasting dyes”. The fluorescence quantum yields (Φf) and the fluorescence lifetimes (τ) of the modified dyes were thoroughly measured with comparison to those of the parent dyes. The correlation of their STED performance with photostability and fluorescence color stability under illumination in water were explored. Unexpectedly, the anaerobic GSDIM (GOC) buffer proved unhelpful with respect to STED performance. It was demonstrated that, even dyes with a Φf of only 14–17% allow STED imaging with a sufficient photon budget and good signal-to-noise ratio. For the dyes with photostabilizing groups (PSG) the Φf values are 4–5 times lower than in the reference dyes, and lifetimes τ are also strongly reduced. Noteworthy are very high fluorescence color stability and constant or even increasing fluorescence signal under photobleaching in bulk aqueous solutions, which suggests a sacrificing role of the 4-nitrobenzyl-containing moieties. Straightforward and improved recipes for “last-minute” modifications and preparations of “self-healing” red-emitting fluorescent tags are described.

Hydroxylated rhodamines, carbopyronines, silico‐ and germanorhodamines with absorption maxima in the range of 530–640 nm were prepared and applied in specific labeling of living cells. The direct and high‐yielding entry to germa‐ and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila‐ and germafluoresceins, as well as ‐rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50–75 nm in one‐ and two‐color imaging of vimentin‐HaloTag fused protein and native tubulin. The established structure–property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo‐, silico‐, and germanorhodamines using simple additive schemes.

Nature Communications 7: Article number: 10144 (2016); Published 11 January 2016, Updated 24 July 2018 The original version of this Article omitted the following from the Acknowledgements: ‘This work was also financed by an ERC starting grant ‘SM-IMPORT’ (No. 638536 to T.C.)’. This has been corrected in both the PDF and HTML versions of the Article.

Aromatic nucleophilic substitution (SNAr) of fluorine in 9-(3′-carboxy-4′,5′,6′,7′-tetrafluorophenyl) groups of xanthene dyes constitutes a powerful tool in dye design. Thiols and amines regioselectively replace F-6′. This approach enables additional hydrophilic residues or functional groups required for bioconjugation to be introduced. By using this methodology, a “bright” and photostable dye for two-color superresolution microscopy was synthesized (with absorption and emission maxima at 604 and 627 nm, respectively). In the case of red-emitting rhodamine dyes with 3′-carboxy-4′,5′,7′-trifluorophenyl residues, two-dimensional NMR techniques and a chemical transformation were used to prove the precise position of the additional substituent – a carboxylic acid group linked through the S-atom at C-6′. Furthermore, simple 1H NMR spectra reliably permit the position of the additional carboxy substituent in the 3′-carboxyphenyl ring (at C-5′ or C-6′) to be established. Information on the exact position of this substituent is significant for the design of molecular probes and for the prediction of the properties of their bioconjugates.

Caged rhodamine dyes (Rhodamines NN) of five basic colors were synthesized and used as “hidden” markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to intense red light (λ∼750 nm; two-photon mode). All dyes possess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330–350 nm (molar extinction coefficient (ε)≈104 M−1 cm−1) with a band edge that extends to about λ=440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511–633 and 525–653 nm, respectively). The unmasked dyes are highly colored and fluorescent (ε= 3–8×104 M−1 cm−1 and fluorescence quantum yields (ϕ)=40–85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reactive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375–420 nm light or intense red light (λ=775 nm). Protein conjugates with optimal degrees of labeling (3–6) were prepared and uncaged with λ=405 nm light in aqueous buffer solutions (ϕ=20–38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10–40 % of the non-fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a “dark” non-emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified. Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Mitronova, Gyuzel Yu.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Boyarskiy, Vadim P.. St. Petersburg State University; Rusia Fil: Hebisch, Elke. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Geisler, Claudia. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Kolmakov, Kirill. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Wurm, Christian A.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Willig, Katrin I.. Max Planck Institute for Biophysical Chemistry ; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry ; Alemania

Nature Communications 7(1), 10144 (2016). doi:10.1038/ncomms10144, Published by Nature Publishing Group UK, [London]

Fluorescent dyes emitting red light are frequently used in conven- tional and super-resolution microscopy of biological samples, although the va- riety of the useful dyes is limited. We describe the synthesis of rhodamine- based fluorescent dyes with absorption and emission maxima in the range of 621-637 and 644-660 nm, respectively and demonstrate their high perform- ance in confocal and stimulated emis- sion depletion (STED) microscopy. New dyes were prepared by means of reliable chemical transformations ap- plied to a rhodamine scaffold with three variable positions. They feature polarity, water solubility, variable net charges, improved stabilities of N-hy- droxysuccinimidyl (NHS) esters, as well as large fluorescence quantum yields in dye solutions and antibody conjugates. The photophysical and imaging properties of dyes containing three different polar groups, namely primary phosphate, sulfonic acid (in two different positions), and hydroxyl were compared. A dye with two pri- mary phosphate groups was explored as a valuable alternative to dyes with "classical" sulfonic acid groups. Due to the increased net charge of the phos- phorylated dye (q =4 at pH 8), it demonstrated a far better electropho- retic mobility compared with analogues with two sulfonic acid groups (q =2). As an example, one fluorescent dye was designed to be especially conve- A for practical use. It is character- ized by sufficiently high chemical sta- bility of the NHS ester, its simple isola- tion, handling, and solubility in aque- ous buffers, as well as in organic sol- vents. All these features, accompanied by a zero net charge in conjugates, were accomplished by the introduction of hydrophilic groups of two types: two hydroxyl groups and one sulfonic acid

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group. Fil: Kolmakov, Kirill. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Wurm, Christian. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Sednev, Maksim V.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; Alemania Fil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemania

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.

Far-red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground-state depletion (GSDIM) super-resolution microscopy are presented. Fluorinated silicon–rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high-yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon–rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (

Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard., Synthetic organic fluorophores are powerful tools for bioimaging, but frequently display shortened observation times and signal fluctuations. Here, the authors report a general method to covalently label a biomolecule with a fluorophore and photostabilizer, reducing unwanted photophysical effects by intramolecular quenching of reactive fluorophore states.

The synthesis, reactivity, and photophysical prop- erties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the amino- reactive sites (NHS esters or mixed N-succinimidyl carbon- ates) are reported. All fluorophores contain an N-alkyl-1,2-di- hydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secon- dary carboxamide group. The absorption and emission maxima in water are in the range of 635-639 and 655- 659 nm, respectively. A vastly simplified approach to red- emitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed. As an example, a phosphorylated dye with an azide residue was prepared and was used in a click reaction with a strained alkyne bearing an N-hydroxysuccinimid (NHS) ester group. This method bypasses the undesired activation of phosphate groups, and gives an amphiphilic amino-reactive dye, the solubility and distribution of which between aqueous and organic phases can be controlled by varying the pH. The presence of two hydroxyl groups and a phenyl ring with two carboxyl residues in the dyes with another substitution pattern is sufficient for providing the hydrophilic properties. Selective formation of a mono-N-hydroxysuccinimidyl ester from 5-carboxy isomer of this rhodamine is reported. The fluorescence quantum yields varied from 58 to 92 % for free fluorophores, and amounted to 18-64 % for antibody conju- gates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two-color stimulated emis- sion depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Select- ing a pair of fluorophores with absorption/emission bands at 579/609 and 635/655 nm enabled two-color channels with low cross-talk and negligible background at approximately 40 nm resolution.

The cover picture shows a unique bird's eye view of the regular park in Strelna – one of the most desolate, yet stunning historical suburbs of St. Petersburg (Russia). Famous European architects of the 18th century, such as C.-B. Rastrelli, A. Le Blanc, G.-B. Chiprianni, P. Michetti, and L. Ruska, participated in the construction of “the Russian Versailles” – this is what the Russian Tsar Peter the Great planned this country residence to be. The northern facade of the Konstantinovsky Palace looks at the hazy and rainy Gulf of Finland. The regular cyclic pattern of the park lanes and shipping canals is reminiscent of the polycyclic structure of carbopyronine dyes decorated with hydrophilic groups. These fluorescent markers are described in the article by V. N. Belov, S. W. Hell et al. on p. 3593 ff. The authors report a detailed and strategically sound synthesis of the carbopyronine scaffold with great potential for dye design. Important photophysical properties and some nanoscopic applications of the new red emitting dyes are also described, and interesting future applications (e.g. as caged carbopyronines) are mentioned. Photo: V. N. Belov; the artwork of Mr. H. Sebesse (Max Planck Institute for Biophysical Chemistry, G?ttingen, Germany) is acknowledged.

Biological microscopy favors photostable fluorescent markers with large fluorescence quantum yields, low dark triplet state population, good biocompatibility and absorption and emission maxima in the near-infrared, where cellular autofluorescence is minimized. In the present study, carbopyronines absorbing around 640 nm and emitting at around 660 nm, with a low intersystem crossing rate (kisc ≈ 0.5×106 s-1) and excellent properties for cellular imaging were synthesized. A general synthetic route to carbopyronines with functional groups variable in the final steps of the synthesis or in the resulting fluorescent dye is presented. Possessing two 2-me-thoxyethyl groups, the parent dye is soluble in water and most organic solvents. Demethylation of the dye or its precursors is straightforward, clean, and furnishes compounds with one or two 2-hydroxyethyl groups, which can be used for further transformations. Modifications in the linker-containing carboxy group are also possible. A multistep synthesis of the dye starting from a simple precursor and utilizing a single temporary protective group is described. The presented approach may be further applied to the design of caged carbopyronines. © 2010 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim.