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Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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)≈10(4)  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=3-8×10(4)  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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synthesis, reactivity, and photophysical properties 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 carbonates) are reported. All fluorophores contain an N-alkyl-1,2-dihydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary 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 conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two-color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting 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., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fluorescent dyes emitting red light are frequently used in conventional and super-resolution microscopy of biological samples, although the variety 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 performance in confocal and stimulated emission depletion (STED) microscopy. New dyes were prepared by means of reliable chemical transformations applied to a rhodamine scaffold with three variable positions. They feature polarity, water solubility, variable net charges, improved stabilities of N-hydroxysuccinimidyl (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 primary phosphate groups was explored as a valuable alternative to dyes with "classical" sulfonic acid groups. Due to the increased net charge of the phosphorylated dye (q=-4 at pH 8), it demonstrated a far better electrophoretic mobility compared with analogues with two sulfonic acid groups (q=-2). As an example, one fluorescent dye was designed to be especially convenient for practical use. It is characterized by sufficiently high chemical stability of the NHS ester, its simple isolation, handling, and solubility in aqueous buffers, as well as in organic solvents. 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 residue., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

New photostable rhodamine dyes represented by the compounds 1 a-r and 3-5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N',N-bis(2,2,2-trifluoroethyl) derivatives 1 e, 1 i, 1 j, 3-H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4' and 5'. Two fluorine atoms were introduced into the positions 2' and 7' of the 3',6'-diaminoxanthene fragment in compounds 1 a-d, 1 i-l and 1 m-r. The new rhodamine dyes may be excited with λ=488 or 514 nm light; most of them emit light at λ=512-554 nm (compounds 1 q and 1r at λ=576 and 589 nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98 %), relatively long excited-state lifetimes (>3 ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30 % SO3 in H2SO4 is compatible with the secondary amide bond (rhodamine-CON(Me)CH2CH2COOH) formed with MeNHCH2CH2COOCH3 to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very-high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live-cell-tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

The design, synthesis, and evaluation of new rhodamine spiroamides are described. These molecules have applications in optical nanoscopy based on random switching of the fluorescent single molecules. The new markers may be used in (co)localization studies of various objects and their (mutual) positions and shape can be determined with a precision of a few tens of nanometers. Multicolor staining, good photoactivation, a large number of emitted photons, and selective chemical binding with amino or thiol groups were achieved due to the presence of various functional groups on the rhodamine spiroamides. Rigidized sulfonated xanthene fragment fused with six-membered rings, N,N'-bis(2,2,2-trifluoroethyl) groups, and a combination of additional double bonds and sulfonic acid groups with simple aliphatic spiroamide residue provide multicolor properties and improve performance of the rhodamine spiroamides in photoactivation and bioconjugation reactions. Having both essential parts of the photoswitchable assembly-the switching and the fluorescent (reporter) groups-combined in one chemical entity make this approach attractive for further development. A series of rhodamine spiroamides is presented along with characterizations of their most relevant properties for application as fluorescent probes in single-molecule switching and localization microscopy. Optical images with resolutions on the nanometer scale illustrate the potential of the labels in the colocalization of biological objects and the two-photon activation technique with optical sectioning.

During the last decade far-field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous-wave laser beam. This bright marker enables strict control of on/off switching and provides single-molecule localization precision down to 15 nm in the focal plane. Two-photon induced nonlinear photoswitching of this marker with continuous-wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost-effective fluorescence nanoscopy with noninvasive optical sectioning.

Highly water soluble fluorescent dyes were synthesized and transformed into new amino reactive fluorescent labels for biological microscopy. To this end, rhodamine 8 (prepared from 7-hydroxy-1,2,3,4-tetrahydroquinoline (7) and phthalic anhydride in 85 % aq. H(3)PO(4)) was sulfonated with 30 % SO(3) in H(2)SO(4) and afforded the water soluble disulfonic acid 3 a (64 %). Amidation of the carboxy group in 3 a with 2-(methylamino)ethanol in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumPF(6) (-) (HATU) led to alcohol 3 b (66 %), which was transformed into the amino reactive mixed carbonate 3 d with di(N-succinimidyl)carbonate and Et(3)N. Reaction of the carboxy group in 3 a with MeNH(CH(2))(2)CO(2)Me and N,N,N',N'-tetramethyl-O-(N-succinimidyl)-uroniumBF(4) (-) (TSTU) yielded methyl ester 13. After saponification of the aliphatic carboxy group in 13, the compound was converted into NHS-ester 3 e (using HATU and Et(3)N). Heating of 7 with trimellitic anhydride in H(3)PO(4) gave a mixture of dicarboxylic acids 14 and 15 (1:1). Regioisomer 15 was isolated, sulfonated with 30 % SO(3) in H(2)SO(4), and disulfonic acid 3 f was used for the synthesis of the mono NHS-ester 3 g, in which the sterically unhindered carboxy group was selectively activated (with N-hydroxysuccinimide, HATU, and Et(3)N). The sulfonated rhodamines 3 b, c and f are soluble in water (up to 0.1 M), have excellent photostabilities and large fluorescence quantum yields. Subdiffraction resolution images of tubulin filaments of mammalian cells stained with these dyes illustrate their applicability as labels for stimulated emission depletion microscopy and other fluorescence techniques.

Disulfonated rhodamines are photostable and bright dyes widely used in life science and optical microscopy. However, di‐sulfonated dyes were considered cell impermeable and not applicable in living cells. We challenged this assumption with 5 most popular rhodamines (Rho) having two carboxylic acid residues, versatile sulfonation patterns and emitting green (AS488), yellow (Rho530), orange (Rho565) and red (Rho590 and STAR RED) light. The probes comprising one rhodamine entity and a HaloTagTM amine (O2) ligand (x) were prepared and applied for labeling of living, Vimentin‐Halo (VIM‐Halo) expressing U‐2 OS cells. Surprisingly, we observed specific and bright staining with simplest compounds Rho590‐x, Rho565‐x and Rho530‐x bearing two negative charges; they performed well also in stimulated emission depletion (STED) microscopy. Specific staining and red shifts in absorption and emission bands were observed with other probes having one negative charge; they were prepared by native chemical ligation and esterification. [ABSTRACT FROM AUTHOR]

Formylation of 2,6‐dichloro‐5‐R‐nicotinic acids at C‐4 followed by condensation with 3‐hydroxy‐N,N‐dimethylaniline gave analogs of the popular TAMRA fluorescent dye with a 2,6‐dichloro‐5‐R‐nicotinic acid residues (R=H, F). The following reaction with thioglycolic acid is selective, involves only one chlorine atom at the carbon between pyridine nitrogen and the carboxylic acid group and affords new rhodamine dyes absorbing at 564/ 573 nm and emitting at 584/ 597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with "small molecules" provided specific labeling (covalent and non‐covalent) of organelles as well as of components of the cytoskeleton in living cells and were combined with fluorescent probes prepared from 610CP and SiR dyes and applied in two‐color STED microscopy with a 775 nm STED laser. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μ m solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm. [ABSTRACT FROM AUTHOR]

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 (<600 Da). The use of the λ=800 nm STED beam instead of the commonly used one at λ=750-775 nm provides excellent imaging performance and suppresses re-excitation of SiRF and the oxazine dyes. The photophysical properties and immunofluorescence imaging performance of these new far-red emitting dyes (photobleaching, optical resolution, and switch-off behavior) are discussed in detail and compared with those of some well-established fluorophores with similar spectral properties. [ABSTRACT FROM AUTHOR]

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-9 H-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. [ABSTRACT FROM AUTHOR]

The synthesis, reactivity, and photophysical properties 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 carbonates) are reported. All fluorophores contain an N-alkyl-1,2-dihydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary 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 conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two-color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting 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. [ABSTRACT FROM AUTHOR]

Spatial resolution in far-field fluorescence microscopy is limited by diffraction to about 200 nm. With the aid of photoswitchable fluorophores, the diffraction barrier has been successfully overcome, allowing unprecedented resolution in the order of single biomolecules. The imaging process demands markers with strict and reliable control of the switching, to keep most of the markers in a non-emissive state most of the time and to bring a tiny number back to an emissive state, and detection at the single-molecule level. Herein, we describe the use of rhodamine spiroamides with unique photophysical properties as molecular probes for super-resolution techniques based on the localization of single emitters. This family of photochromic and fluorescent compounds fulfils the stringent requirements for such imaging methods; these compounds are robust and capable of enduring single-molecule detection in diverse environments. This has allowed meaningful images with resolution down to a few nanometres. Their design, synthesis and implementation is discussed along with imaging applications in material and life sciences. [ABSTRACT FROM AUTHOR]

Fluorescent dyes emitting red light are frequently used in conventional and super-resolution microscopy of biological samples, although the variety 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 performance in confocal and stimulated emission depletion (STED) microscopy. New dyes were prepared by means of reliable chemical transformations applied to a rhodamine scaffold with three variable positions. They feature polarity, water solubility, variable net charges, improved stabilities of N-hydroxysuccinimidyl (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 primary phosphate groups was explored as a valuable alternative to dyes with 'classical' sulfonic acid groups. Due to the increased net charge of the phosphorylated dye ( q=−4 at pH 8), it demonstrated a far better electrophoretic mobility compared with analogues with two sulfonic acid groups ( q=−2). As an example, one fluorescent dye was designed to be especially convenient for practical use. It is characterized by sufficiently high chemical stability of the NHS ester, its simple isolation, handling, and solubility in aqueous buffers, as well as in organic solvents. 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 residue. [ABSTRACT FROM AUTHOR]

All the beauty is revealed when sunlight shines on the statue of Polymnia, one of the nine Muses in ancient Greek mythology, masked with snow in winter and then liberated in all its splendor from the white cloak by bright sunshine. The parallel light ‐ induced transformation is depicted by the chemical formulae. Irradiation of colorless Rhodamines NN, masked fluorescent dyes, with UV or visible light irreversibly “releases” the hidden colors in all their depth and vibrant fluorescence. For more details, see the Full Paper by V. N. Belov, M. L. Bossi, S. W. Hell et al. on page 13162 ff. [ABSTRACT FROM AUTHOR]