Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

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.     

Masked Rhodamine Dyes of Five Principal Colors Revealed by Photolysis of a 2‐Diazo‐1‐Indanone Caging Group: Synthesis,Photophysics, and Light Microscopy Applications

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 (?)=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 (?=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.     

Designing Sub‐2 nm Organosilica Nanohybrids for Far‐Field Super‐Resolution Imaging

Stimulated emission depletion (STED) microscopy enables ultrastructural imaging of biological samples with high spatiotemporal resolution. STED nanoprobes based on fluorescent organosilica nanohybrids featuring sub‐2 nm size and near‐unity quantum yield are presented. The spin–orbit coupling (SOC) of heavy‐atom‐rich organic fluorophores is mitigated through a silane‐molecule‐mediated condensation/dehalogenation process, resulting in bright fluorescent organosilica nanohybrids with multiple emitters in one hybrid nanodot. When harnessed as STED nanoprobes, these fluorescent nanohybrids show intense photoluminescence, high biocompatibility, and long‐term photostability. Taking advantage of the low‐power excitation (0.5 μW), prolonged singlet‐state lifetime, and negligible depletion‐induced re‐excitation, these STED nanohybrids present high depletion efficiency (>96 %), extremely low saturation intensity (0.54 mW, ca. 0.188 MW cm^?2), and ultra‐high lateral resolution (ca. λ_em/28).     

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

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.     

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

The development of stimulated emission depletion (STED) microscopy represented a major breakthrough in cellular and molecular biology. However, the intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes, which significantly limits the performance and practical utility of STED microscopy. We herein developed a photoresistant fluorescent dye C‐Naphox as a practical tool for STED imaging. With excitation using either a λ=405 or 488 nm laser in protic solvents, C‐Naphox exhibited an intense red/orange fluorescence (quantum yield Φ_F>0.7) with a large Stokes shift (circa 5900 cm^?1). Even after irradiation with a Xe lamp (300 W, λ_ex=460 nm, full width at half maximum (FWHM)=11 nm) for 12 hours, 99.5 % of C‐Naphox remained intact. The high photoresistance of C‐Naphox allowed repeated STED imaging of HeLa cells. Even after recording 50 STED images, 83 % of the initial fluorescence intensity persisted.     

Water‐Soluble Red‐Emitting Distyryl‐Borondipyrromethene (BODIPY) Dyes for Biolabeling

A series of water‐soluble red‐emitting distyryl‐borondipyrromethene (BODIPY) dyes were designed and synthesized by using three complementary approaches aimed at introducing water‐solubilizing groups on opposite faces of the fluorescent core to reduce or completely suppress self‐aggregation. An additional carboxylic acid functional group was introduced at the pseudo‐meso position of the BODIPY scaffold for conjugation to amine‐containing biomolecules/biopolymers. The optical properties of these dyes were evaluated under simulated physiological conditions (i.e., phosphate‐buffered saline (PBS), pH 7.5) or in pure water. The emission wavelength (λ_max) of these labels was found in the 640–660 nm range with quantum yields from modest to unprecedentedly high values (4 to 38 %). The bioconjugation of these distyryl‐BODIPY dyes with bovine serum albumin (BSA) and the monoclonal antibody (mAb) 12A5 was successfully performed under mild aqueous conditions.     

Red‐Emitting Rhodamines with Hydroxylated,Sulfonated, and Phosphorylated Dye Residues and Their Use in Fluorescence Nanoscopy

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.     

“Reduced” Coumarin Dyes with an O‐Phosphorylated 2,2‐Dimethyl‐4‐(hydroxymethyl)‐1,2,3,4‐tetrahydroquinoline Fragment: Synthesis,Spectra, and STED Microscopy

Large Stokes‐shift coumarin dyes with an O‐phosphorylated 4‐(hydroxymethyl)‐2,2‐dimethyl‐1,2,3,4‐tetrahydroquinoline fragment emitting in the blue, green, and red regions of the visible spectrum were synthesized. For this purpose, N‐substituted and O‐protected 1,2‐dihydro‐7‐hydroxy‐2,2,4‐trimethylquinoline was oxidized with SeO₂ to the corresponding α,β‐unsaturated aldehyde and then reduced with NaBH₄ in a “one‐pot” fashion to yield N‐substituted and 7‐O‐protected 4‐(hydroxymethyl)‐7‐hydroxy‐2,2‐dimethyl‐1,2,3,4‐tetrahydroquinoline as a common precursor to all the coumarin dyes reported here. The photophysical properties of the new dyes (“reduced coumarins”) and 1,2‐dihydroquinoline analogues (formal precursors) with a trisubstituted C=C bond were compared. The “reduced coumarins” were found to be more photoresistant and brighter than their 1,2‐dihydroquinoline counterparts. Free carboxylate analogues, as well as their antibody conjugates (obtained from N‐hydroxysuccinimidyl esters) were also prepared. All studied conjugates with secondary antibodies afforded high specificity and were suitable for fluorescence microscopy. The red‐emitting coumarin dye bearing a betaine fragment at the C‐3‐position showed excellent performance in stimulation emission depletion (STED) microscopy.     

Phosphorylated 3‐Heteroarylcoumarins and Their Use in Fluorescence Microscopy and Nanoscopy

Photostable and bright fluorescent dyes with large Stokes shifts are widely used as markers in far‐field optical microscopy, but the variety of useful dyes is limited. The present study introduces new 3‐heteroaryl coumarins decorated with a primary phosphate group (OP(O)(OH)₂) attached to C‐4 in 2,2,4‐trimethyl‐1,2‐dihydroquinoline fragment fused with the coumarin fluorophore. The general synthetic route is based on the Suzuki reaction of 3‐bromocoumarines with hetarylboronic acids followed by oxidation of the methyl group at the C?C bond with SeO₂ (to an aldehyde), reduction with NaBH₄ (to an alcohol), and conversion into a primary phosphate. The 4 position in the coumarin system may be unsubstituted or bear a methyl group. Phosphorylated coumarins were found to have high fluorescence quantum yields in the free state and after conjugation with proteins (in aqueous buffers). In super‐resolution light microscopy with stimulated emission depletion (STED), the new coumarin dyes provide an optical resolution of 40–60 nm with a low background signal. Due to their large Stokes shifts and high photostability, phosphorylated coumarins enable to combine multilabel imaging (using one detector and several excitation sources) with diffraction unlimited optical resolution.     

Polar Red‐Emitting Rhodamine Dyes with Reactive Groups: Synthesis,Photophysical Properties,and Two‐Color STED Nanoscopy Applications

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.     

Designing Sub-2 nm Organosilica Nanohybrids for Far-Field Super-Resolution Imaging

Stimulated emission depletion (STED) microscopy enables ultrastructural imaging of biological samples with high spatiotemporal resolution. STED nanoprobes based on fluorescent organosilica nanohybrids featuring sub-2 nm size and near-unity quantum yield are presented. The spin–orbit coupling (SOC) of heavy-atom-rich organic fluorophores is mitigated through a silane-molecule-mediated condensation/dehalogenation process, resulting in bright fluorescent organosilica nanohybrids with multiple emitters in one hybrid nanodot. When harnessed as STED nanoprobes, these fluorescent nanohybrids show intense photoluminescence, high biocompatibility, and long-term photostability. Taking advantage of the low-power excitation (0.5 μW), prolonged singlet-state lifetime, and negligible depletion-induced re-excitation, these STED nanohybrids present high depletion efficiency (>96 %), extremely low saturation intensity (0.54 mW, ca. 0.188 MW cm⁻²), and ultra-high lateral resolution (ca. λ_em/28).     

4‐Trifluoromethyl‐Substituted Coumarins with Large Stokes Shifts: Synthesis,Bioconjugates, and Their Use in Super‐Resolution Fluorescence Microscopy

Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light‐emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7‐dialkylamino‐4‐trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3‐chloro‐4‐trifluoromethylcoumarins and available (hetero)aryl‐ or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2‐trifluoroacetyl‐5‐dialkylaminophenols with available (hetero)aryl‐ or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3‐(hetero)aryl or 3‐[2‐(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C‐4 of the 2,2,4‐trimethyl‐1,2‐dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413–480 and 527–668 nm, respectively. The phosphorylated dye, 9 ,CH?CH‐2‐py,H, with the 1‐(3‐carboxypropyl)‐4‐hydroxymethyl‐2,2‐dimethyl‐1,2‐dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3‐[2‐(2‐pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super‐resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two‐color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.     

New Fluorinated Rhodamines for Optical Microscopy and Nanoscopy

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.     

In Situ Visualization of Block Copolymer Self‐Assembly in Organic Media by Super‐Resolution Fluorescence Microscopy

Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media.     

DMAP‐BODIPY Alkynes: A Convenient Tool for Labeling Biomolecules for Bimodal PET–Optical Imaging

Several new boron dipyrromethene/N,N‐dimethylaminopyridine (BODIPY‐DMAP) assemblies were synthesized as precursors for bimodal imaging probes (optical imaging, OI/positron emission tomography, PET). The photophysical properties of the new compounds were also studied. The first proof‐of‐concept was obtained with the preparation of several new BODIPY‐labeled bombesins and evaluation of the affinity for bombesin receptors by using a competition binding assay. Fluorination reactions were investigated on DMAP‐BODIPY precursors as well as on DMAP‐BODIPY‐labeled bombesins. Chemical modifications on the BODIPY core were also performed to obtain luminescent dyes emitting in the therapeutic window (650–900 nm), suitable for in vivo imaging, making these compounds promising precursors for PET/optical dual‐modality imaging agents.     

Rapid Synthesis of D‐A′‐π‐A Dyes through a One‐Pot Three‐Component Suzuki–Miyaura Coupling and an Evaluation of their Photovoltaic Properties for Use in Dye‐Sensitized Solar Cells

Twenty‐four D‐A′–π‐A dyes were rapidly synthesized through a one‐pot three‐component Suzuki–Miyaura coupling reaction, which was assisted by microwave irradiation. We measured the absorption spectra, electrochemical properties, and solar‐cell performance of all the synthesized dyes. The D5 πA4 dye contained our originally designed rigid and nonplanar donor and exerted the highest efficiency at 5.4 %. The short‐circuit current (J_sc) was the most important parameter for the conversion efficiency (η) in the case of the organic D‐A′‐π‐A dyes. Optimal ranges for the D‐A′‐π‐A dyes were observed for high values of J_sc/λ_max at λ=560–620 nm, an optical‐absorption edge of λ=690–790 nm, and E_HOMO and E_LUMO values of <1.14 and ?0.56 to ?0.76 V, respectively.     

Water‐Soluble Phospholo[3,2‐b]phosphole‐P,P′‐Dioxide‐Based Fluorescent Dyes with High Photostability

We recently reported that fluorescent dye PB430, which consisted of a 2‐phenyl‐substituted benzophosphole P‐oxide skeleton that was reinforced by a methylene bridge, showed pronounced photostability and, thus, high utility for applications in super‐resolution stimulated emission depletion (STED) microscopy. Herein, we replaced the methylene bridge with another P=O group to 1) investigate the role of the bridging moieties; and 2) further modulate the fluorescence properties of this skeleton. We synthesized a series of phospholo[3,2‐b]phosphole‐based dyes—trans‐PO‐PB430, cis‐PO‐PB430, and trans‐PO‐PB460—all of which showed sufficient water solubility. Moreover, trans‐PO‐PB430 and trans‐PO‐PB460 exhibited intense green and orange fluorescence, respectively, and a high photostability that was comparable to that of PB430. In contrast, cis‐PO‐PB430 underwent rapid photobleaching upon continuous photoirradiation, which demonstrated the importance of steric shielding of the polycyclic skeleton by the substituents on the bridging moieties. The fluorescence properties of these dyes were insensitive to concentration, pH value, and polarity changes of the environment in solution. In addition, even in the solid state, these dyes showed strong green to orange emissions. These results demonstrate the potential utility of trans‐PO‐PB430 and trans‐PO‐PB460 as highly photostable fluorescent dyes.     

Color Tuning of the Aggregation‐Induced Emission of Maleimide Dyes by Molecular Design and Morphology Control

Aggregation‐induced emission (AIE)‐active maleimide dyes, namely, 2‐p‐toluidino‐N‐p‐tolylmaleimide, 3‐phenyl‐2‐toluidino‐N‐p‐tolylmaleimide, 2‐p‐thiocresyl‐3‐p‐toluidino‐N‐p‐tolylmaleimide, and 2,3‐dithiocresyl‐N‐arylmaleimides, were synthesized by facile synthetic procedures. The dyes show intense emission in the solid state, and emission colors were controlled from green (λ_max=527 nm) to orange (λ_max=609 nm) by varying the substituents at the 2‐ and 3‐positions of the maleimide and the packing structures in the solid state. 2,3‐Disubstituted maleimide dyes effectively underwent redshifts of their emission wavelength. Furthermore, some of the dyes exhibited mechanochromism and polymorphism, and their emission properties were dramatically dependent on the morphology of the solid samples. The mechanisms of the emission behaviors were investigated by X‐ray diffraction. The substituent of the nitrogen atom of the maleimide ring affected the intermolecular interactions and short contacts, which were observed by single crystal X‐ray crystallography, to result in completely different emission properties.     

Stable,Wavelength‐Tunable Fluorescent Dyes in the NIR‐II Region for In Vivo High‐Contrast Bioimaging and Multiplexed Biosensing

Small‐molecule organic fluorophores, spectrally active in the 900–1700 nm region, with tunable wavelength and sensing properties are sought‐after for in vivo optical imaging and biosensing. A panel of fluorescent dyes ( CX ) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo‐ and photo‐stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high‐contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug‐induced hepatotoxicity by detection of OONO^?. This report presents a series of NIR‐II dyes with promising spectroscopic properties for high‐contrast bioimaging and multiplexed biosensing.     

Stable,Wavelength‐Tunable Fluorescent Dyes in the NIR‐II Region for In Vivo High‐Contrast Bioimaging and Multiplexed Biosensing

Small‐molecule organic fluorophores, spectrally active in the 900–1700 nm region, with tunable wavelength and sensing properties are sought‐after for in vivo optical imaging and biosensing. A panel of fluorescent dyes ( CX ) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo‐ and photo‐stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high‐contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug‐induced hepatotoxicity by detection of OONO^?. This report presents a series of NIR‐II dyes with promising spectroscopic properties for high‐contrast bioimaging and multiplexed biosensing.