Bright Fluorophores Research Articles

Affordable Small Molecules as Promising Fluorescent Labels for Biomolecules.

Fluorescent labels, commonly used in highly sensitive analytical techniques for detecting and tracking biomolecules in critical fields like cellular biology, medicine, medicinal chemistry, and environmental science, are currently too expensive for routine use in standard applications, with most exhibiting small Stokes shifts. This limitation underscores the potential of 4-diethylaminobenzaldehyde derivatives as a cost-effective alternative for developing new, bright fluorophores with larger Stokes shifts. In this work, using 4-diethylaminobenzaldehyde as starting material, we developed a simple, cost-effective, and efficient synthetic strategy to produce new affordable small molecules as effective fluorescent labels for biomolecules. Density functional theory and time-dependent density functional theory calculations were also conducted to gain insights into the observed photophysical properties.

Water-Soluble Lipophilic Near-Infrared Region II Fluorophores for High-Brightness Lipid Layer and Lipid Droplets Imaging Applications.

Fluorescence imaging in the second near-infrared region (NIR-II, 1000-1700nm) has garnered considerable attention for displaying the biological information of deep tissues. However, the lack of biocompatible contrast agents with bright NIR-II emission has hampered the precise clinical application of deep tissue imaging. Here, a lipophilic enhancement strategy employing donor-acceptor-donor (D-A-D) molecules, introducing long alkoxy chains and quaternary ammonium salts for the development of highly bright water-soluble NIR-II fluorophores (BBTD-2C-N), is described. Notably, liposome-encapsulated BBTD-2C-N nanoparticles (B-2C-N/DMPC) in aqueous solution exhibit a 1.8-fold increase in NIR-II fluorescence brightness compared to free BBTD-2C-N in methanol. Avoidance of the aggregation-caused quenching effect and enhanced NIR-II fluorescence are attributed to significantly attenuated π-π stacking interactions and maintained monodisperses in the hydrophobic liposome shell. Moreover, BBTD-2C-N demonstrates superior performance in visualizing lipid droplet-rich HeLa cells in vitro, as well as precise monitoring of adipose tissue and fatty liver in vivo. This study reveals a new avenue for the development of bright NIR-II fluorophores and precise in vivo imaging.

Bright and Fast Emission from Robust Supramolecular J-Aggregate Nanostructures through Silica-Encapsulation.

We introduce a two-step silica-encapsulation procedure to optimize both the optical efficiency and structural robustness of 5,5',6,6'-tetrachloro-1,1'-diethyl-3,3'-di(4-sulfobutyl)-benzimidazolocarbocyanine (TDBC), a two-dimensional sheet-like J-aggregate. We report a fluorescence quantum yield of ∼98%, the highest quantum yield recorded for any J-aggregate structure at room temperature, and a fast, emissive lifetime of 234 ps. Silica, as an encapsulating matrix, provides optical transparency, chemical inertness, and robustness to dilution, while rigidifying the J-aggregate structure. Our in situ encapsulation process preserves the excitonic structure in TDBC J-aggregates, maintaining their light absorption and emission properties. The homogeneous silica coating has an average thickness of 0.5-1 nm around J-aggregate sheets. Silica encapsulation permits extensive dilutions of J-aggregates without significant disintegration into monomers. The narrow absorbance and emission line widths exhibit further narrowing upon cooling to 79 K, which is consistent with J-type coupling in the encapsulated aggregates. This silica TDBC J-aggregate construct signifies (1) a bright, fast, and robust fluorophore system, (2) a platform for further manipulation of J-aggregates as building blocks for integration with other optical materials and structures, and (3) a system for fundamental studies of exciton delocalization, transport, and emission dynamics within a rigid matrix.

Highly Luminescent TCNQ in Melamine.

Optical properties of molecules change drastically as a result of interactions with surrounding environments as observed in solutions, clusters, and aggregates. Here, we make 7,7,8,8-tetracyanoquinodimethane (TCNQ) highly luminescent by encapsulating it in crystalline melamine. Colored single crystals are synthesized by slow evaporation of aqueous tetrahydrofuran solutions of melamine and TCNQ. Single-crystal X-ray diffraction reveals the lattice structure of pure melamine, meaning that the color is of impurities. Both mass spectrometry and UV-vis spectroscopy combined with density-functional theory calculations elucidate that the impurity species are neutral TCNQ and its oxidation product, dicyano-p-toluoyl cyanide anion (DCTC-), whose concentrations in a melamine crystal can be controlled by adjusting the molar ratio between melamine and TCNQ in the precursor solution. Fluorescence excitation-emission wavelength mappings on the precursor solutions illustrate dominant emissions from DCTC- while the emission from TCNQ is quenched by the resonance energy transfer to DCTC-. On the contrary, TCNQ in crystalline melamine is a bright fluorophore whose emission wavelength centered at 450 nm with internal quantum yields as high as 19% and slow fluorescence lifetimes of about 2 ns. The method of encapsulating molecules into transparent melamine would make many other molecules fluorescent in solids.

Unlocking the NIR-II AIEgen for High Brightness through Intramolecular Electrostatic Locking.

Fluorescent imaging and biosensing in the near-infrared-II (NIR-II) window holds great promise for non-invasive, radiation-free, and rapid-response clinical diagnosis. However, it's still challenging to develop bright NIR-II fluorophores. In this study, we report a new strategy to enhance the brightness of NIR-II aggregation-induced emission (AIE) fluorophores through intramolecular electrostatic locking. By introducing sulfur atoms into the side chains of the thiophene bridge in TSEH molecule, the molecular motion of the conjugated backbone can be locked through intramolecular interactions between the sulfur and nitrogen atoms. This leads to enhanced NIR-II fluorescent emission of TSEH in both solution and aggregation states. Notably, the encapsulated nanoparticles (NPs) of TSEH show enhanced brightness, which is 2.6-fold higher than TEH NPs with alkyl side chains. The in vivo experiments reveal the feasibility of TSEH NPs in vascular and tumor imaging with a high signal-to-background ratio and precise resection for tiny tumors. In addition, polystyrene nanospheres encapsulated with TSEH are utilized for antigen detection in lateral flow assays, showing a signal-to-noise ratio 1.9-fold higher than the TEH counterpart in detecting low-concentration antigens. This work highlights the potential for developing bright NIR-II fluorophores through intramolecular electrostatic locking and their potential applications in clinical diagnosis and biomedical research.

Tetraanionic Oligo-Dioxaborines: Strongly Absorbing Near-Infrared Dyes.

Polymethine dyes of tetraanionic nature comprising 1,3,2-dioxaborine rings in the polymethine chain and end-groups of different electron-accepting abilities have been synthesized. They can be considered as oligomeric polymethines, where a linear conjugated π-system passes through three 1,3,2-dioxaborine units and a number of tri- and dimethine π-bridges between two end-groups. The obtained dyes exhibit near-infrared absorption and fluorescence, with molar absorption coefficients reaching as high as 564000 M-1 cm-1 in DMF, rendering them among the strongest absorbers known. The novel compounds are bright NIR fluorophores, with fluorescence quantum yields up to 0.13 in DMF. A comparative analysis of the electronic structure of the obtained dyes with respective dianionic and trianionic oligomers was conducted through quantum chemical calculations.

Abstract 84: StarBright Dyes: Fluorophores with superior performance for multiplexing in flow cytometry and Western blotting

Abstract New StarBrightTM Dyes from Bio-Rad are bright fluorescent fluorophores ideal for many antibody-based applications, for example, western blotting and flow cytometry. Traditional fluorophores can be dim, have high cross laser excitation, or don’t work with all protocols, leading to poor data. StarBright Dyes have been designed to address these pain points. Western blotting secondary antibodies are available conjugated to StarBright Blue 700 and StarBright Blue 520 Dyes. Their brightness, the short exposure time required, and low background make them ideal for detection of a single target, or as shown here for multiplex detection of several proteins in combination with other common fluorophores. For flow cytometry, we have a full range of 32 StarBright Dyes excited by the most common laser lines: 355 nm, 405 nm, 488 nm, 561 nm, or 640 nm. They are available conjugated to highly cited clones against common immunophenotyping targets. Data shown here on the ZE5 Cell Analyser demonstrates how the features of StarBright Dyes can improve multiplexing immunophenotyping data. They have narrow excitation and emission spectral profiles, which reduce compensation and spread. This, combined with their exceptional brightness, enables high resolution of cell populations. They work in all buffers and fixatives, so including them in existing panels does not require a change in protocol. They can also be premixed in a master mix for over six months with no loss in performance. Premixes are ideal when using the same panel over an extended period, such as in clinical and longitudinal studies, to ensure reproducible results, also saving time and money. StarBright Dyes are ideal for multiplexing assays in both western blotting and flow cytometry. BIO-RAD and STARBRIGHT are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are the property of their respective owner. Citation Format: Sharon Sanderson, Oliver Rosenwasser, Michael Blundell. StarBright Dyes: Fluorophores with superior performance for multiplexing in flow cytometry and Western blotting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 84.

Strategic Construction of meso-Aryl-Substituted N,N-Carbonyl-Bridged Dipyrrinones as Small, Bright, and Tunable Fluorophores.

A series of novel N,N-carbonyl-bridged dipyrrinone fluorophores have been directly constructed from α-halogenated dipyrrinones, which are conveniently obtained from the acid-catalyzed hydrolysis of readily available α,α'-dihalodipyrrins. This novel methodology affords efficient modulation of the functional groups at both the meso- and α-positions of this fluorophore. These resultant dyes show tunable absorption and emission wavelengths, good molar absorption coefficients, relatively large Stokes shifts, and excellent fluorescence quantum yields up to 0.99, and have been successfully applied in both one- and two-photon fluorescence microscopy imaging in living cells.

Design of 2-Pyridone Fluorophores for Brighter Emissions at Longer Wavelengths.

The recent discovery of blue fluorophores with high quantum yields based on pyridone structures inspired the development of new low-molecular-weight fluorophores with bright emissions at tunable wavelengths, which are highly attractive for various applications. In this study, we propose a rational design strategy for 2-pyridone-based fluorophores with bright emissions at long wavelengths. With a detailed understanding of the positional substitution effects on each carbon atom of the 2-pyridone core, we developed a bright blue fluorophore (λabs =377 nm; λem =433 nm; ϵ=13,200 M-1 cm-1 ; ϕF =88 %) through C3 -aryl and C4 -ester substitutions followed by cyclization. Furthermore, by applying the intramolecular charge transfer (ICT) principle, we invented a bright green fluorophore through C3 - and C4 -diester and C6 -aryl substitutions. The ICT fluorophore based on the pyridone structure shows large molar absorptivity (ϵ=20,100 M-1 cm-1 ), longer emission wavelength (λem =539 nm), high emission quantum yield (ϕF =74 %), and large Stokes shift (Δv=5720 cm-1 ), which are comparable to those of practical fluorescent probes.

A rapid access to activity-based and mitochondria targeting rhodol-like chromenoxanthene dyes for selective bioimaging

Chromenoxanthenes are colorful dyes which do not suffer from any fluorescence turn-off induced by spirocyclic forms as usually observed for conventional rhodamine and fluorescein xanthene analogues. In that way, wide variety of applications in particular in bioimaging can be foreseen for those underexplored fluorophores. Herein, a one-pot synthesis of rhodol-like chromenoxanthene dyes is reported, which are further modified at the periphery of the core structure to tune the optical properties. Resulting neutral derivatives are bright fluorophores in the orange-red spectral window, while cationic counterparts are almost non-emissive. These optical properties are fully characterized and are also rationalized via first-principles calculations. In particular, the occurrence of twisted intramolecular charge transfers is shown to account for the dark-state quenching occurring in the charged compounds. This feature is harvested in the construction of an activity-based fluorescent probe for mitochondria imaging. Excellent organelle localization is achieved, while the emission signal is proportional to the hydrogen peroxide concentration in the mitochondria. Furthermore, the absence of cytotoxicity bestows rhodol-like chromenoxanthene dyes a bright future in cellular imaging applications.

Lipid Droplets Specific Fluorophore for Demarcation of Normal and Diseased Tissues.

Using high-fidelity, permeable, lipophilic, and bright fluorophores for imaging lipid droplets (LDs) in tissues holds immense potential in diagnosing conditions such as diabetic or alcoholic fatty liver disease. In this work, we utilized linear and Λ-shaped polarity-sensitive fluorescent probes for imaging LDs in both cellular and tissue environments, specifically in rats with diabetic and alcoholic fatty liver disease. The fluorescent probes possess several key characteristics, including high permeability, lipophilicity, and brightness, which make them well-suited for efficient LD imaging. Notably, the probes exhibit a substantial Stokes shift, with 143 nm for DCS and 201 nm for DCN with selective targeting of the lipid droplets. Our experimental investigations successfully differentiated morphological variations between diseased and normal tissues in three distinct tissue types: liver, adipose, and small intestine. They could help provide pointers for improved detection and understanding of LD-related pathologies.

Harnessing polarity-dependent fluorescent probe for lipid droplet metabolism dissection and in vivo nonalcoholic fatty liver disease diagnosis

Nonalcoholic fatty liver disease (NAFLD) has emerged as a prevalent chronic liver condition worldwide. The dysregulated metabolism of lipid droplets (LDs) in cells is physiologically and pathologically linked to NAFLD. The development of fluorescent probes for imaging LD metabolism holds promise for noninvasive diagnosis and understanding of NAFLD. However, the development of such probes is challenging, and their in vivo diagnostic potential remains largely unexplored. Here, we present the synthesis of ZP-1, a bright fluorophore serving as a specific probe for LDs. ZP-1 possesses a donor-acceptor electronic structure with intramolecular charge transfer characteristics and shows polarity-dependent emission. In highly polar solvents, such as water, ZP-1 exhibits minimal fluorescence, while in low-polarity solvents, it displays strong fluorescence. This property enables high-specificity LD detection with excellent imaging contrast. Attributed to its high photostability and pH stability, ZP-1 effectively visualizes LD metabolism in live cells via real-time imaging, encompassing processes such as growth, migration, fusion, and fission. Moreover, we demonstrate in vivo diagnosis of NAFLD in a mouse model, providing rich information regarding LD morphology, numbers, volumes, and tissue structures. This work introduces ZP-1 as a valuable tool for NAFLD diagnosis and research, facilitating noninvasive assessment and improved understanding of the disease.

Improved photoluminescence of di, tri, and tetra-aminobenzene molecules hydrogen-bonded by polyethyleneimine and their application for metal ion sensing

Higher photoluminescence quantum yield (PLQY) is always a required property for fluorescent molecule dyes used as probes in aqueous environment because of non-radiative relaxation via O–H vibrations. 1,2,4-triaminobenzene (TriAB) dihydrochloride has been reported as a red-emitting fluorescent dye, although its photoluminescence (PL) intensity is very low. Here, a bright orange-emitting fluorophore was prepared by simply mixing TriAB and polyethyleneimine (PEI) in water at pH 7 and applied to metal ion sensing. The PLQY was increased from 3.1 % to 10.0 % by the addition of PEI into an aqueous TriAB solution. Our experiments by exchanging H2O for D2O and changing the pH from 7 to 1 revealed that the role of PEI in enhancing the PL intensity is attributed to the interaction between the amino groups of TriAB and PEI via hydrogen bonding, the prevention of contact between TriAB and water molecules, and thus the suppression of the non-radiative relaxation via O–H vibrations of H2O. We further demonstrated this role for 1,4-diaminobenzene and 1,2,4,5-tetraaminobenzene.

Optimization and validation of the fast blue BB (FBBB) screening test for the detection of delta-9-tetrahydrocannabinol (Δ9-THC) in oral fluid

The fast blue BB test (FBBB) colorimetric test has been previously validated for the differentiation between marijuana-type and hemp-type cannabis plants. Individuals under the influence of delta-9-tetrahydrocannabinol (Δ9-THC) may be impaired to perform activities such as driving and the rapid, on-site detection of recent use of Δ9-THC could help to prevent traffic accidents. The current study describes, for the first time, the optimization and validation of the FBBB color test from oral fluid (OF) for field use. The FBBB reaction with Δ9-THC forms a red chromophore and a bright red fluorophore that is visualized with a portable fluorescence reader under UV light. The test was optimized for a miniaturized reaction on a 6.3 mm diameter glass fiber filter spot. The limit of detection (LOD) was established as 0.5 µg/mL or 500 ng/mL of Δ9-THC in OF (5 ng of Δ9-THC on the spot) by fluorescence detection. Other figures of merit include linearity in the 0.5–10 µg/mL range, acceptable precision (9.6–28% RSD) and an accuracy of ± 23.2–56.2%. Results from interference studies using different OF collection devices and substances are reported. Interferences might also occur when minor cannabinoids (CBD, CBN, CBG, and Δ8-THC) and Δ9-THC metabolites (THC-COOH and THC-OH) are also present in the matrix. Pre-loaded FBBB reagent is stable on the substrate when stored below 4 °C for 15 days and the color and fluorescence persist on the spot for at least 30 days post reaction. Future studies include development of an OF extraction procedure coupled to a prototype device for field application.

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins.

Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a protein's behavior in situ. Some proteins can be analyzed by fusion to a protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here, we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in cells. Labeling is mediated by the formation of a high-affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific for one- or two-color imaging. Last, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin) in fixed cells, highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.

Coumarin-Based Fluorescent Inhibitors for Photocontrollable Bioactivation.

Activation of the IRE-1/XBP-1 pathway is related to many human diseases. Coumarin-based derivatives acting as both IRE-1 inhibitors and bright fluorophores are highly desirable to establish an integrated fluorescent inhibitor system. Here, we take insights into the aqueous stability of a photocaged IRE-1 inhibitor PC-D-F07 through a structure activity relationship. The substituent effects indicate that the electron-withdrawing -NO2 moiety in the photocage combined with the tricyclic coumarin fluorophore contribute to the structural stability of PC-D-F07. To optimize the photocage of PC-D-F07, we incorporate a 1-ethyl-2-nitrobenzyl or 2-nitrobenzyl photolabile moiety on the hydroxyl group of the IRE-1 inhibitor to generate RF-7 and RF-8. Upon photoactivation, both RF-7 and RF-8 present an increased fluorescence response, sequentially enabling the unlocking of the ortho-1,3-dioxane acetal for the release of active IRE-1 inhibitors. Moreover, RF-7 exhibits a high repolarization ratio of converting M2-type tumor-associated macrophages (M2-TAMs) to M1-type immune-responsive macrophages. This provides a novel prodrug strategy of modulating druggable fluorophore backbones to achieve spatiotemporally controllable drug release for precise cancer treatment.

Furan Donor for NIR-II Molecular Fluorophores with Enhanced Bioimaging Performance.

The second near-infrared (NIR-II, 1,000 to 1,700 nm) molecular fluorophores containing donor-acceptor-donor conjugated backbone have attracted substantial attention due to their outstanding advantages, such as stable emission and facilely tuned photophysical properties. However, it is still challenging for them to simultaneously achieve high brightness and red-shifted absorption and emission. Herein, furan is adopted as the D unit to construct NIR-II fluorophores, demonstrating red shift of absorption, enhanced absorption coefficient, and fluorescent quantum yield when compared with the generally used thiophene counterparts. The high brightness and desirable pharmacokinetics of the optimized fluorophore, IR-FFCHP, endows improved performance for angiography and tumor-targeting imaging. Furthermore, dual-NIR-II imaging of tumor and sentinel lymph nodes (LNs) has been achieved with IR-FFCHP and PbS/CdS quantum dots, enabling the in vivo imaging navigated LN surgery in tumor-bearing mice. This work demonstrates the potential of furan for constructing bright NIR-II fluorophores for biological imaging.

Dual‐Color Photoconvertible Fluorescent Probes Based on Directed Photooxidation Induced Conversion for Bioimaging

AbstractWe herein present a new concept to produce dual‐color photoconvertible probes based on a mechanism called Directed Photooxidation Induced Conversion (DPIC). As a support of this mechanism, styryl‐coumarins (SCs) bearing Aromatic Singlet Oxygen Reactive Moieties (ASORMs) like furan and pyrrole have been synthesized. SCs are bright fluorophores, which undergo a hypsochromic conversion upon visible light irradiation due to directed photooxidation of the ASORM that leads to the disruption of conjugation. SC‐P, a yellow emitting probe bearing a pyrrole moiety, converts to a stable blue emitting coumarin with a 68 nm shift allowing the photoconversion and tracking of lipid droplet in live cells. This new approach might pave the way to a new generation of photoconvertible dyes for advanced bioimaging applications.

Dual-Color Photoconvertible Fluorescent Probes Based on Directed Photooxidation Induced Conversion for Bioimaging.

We herein present a new concept to produce dual-color photoconvertible probes based on a mechanism called Directed Photooxidation Induced Conversion (DPIC). As a support of this mechanism, styryl-coumarins (SCs) bearing Aromatic Singlet Oxygen Reactive Moieties (ASORMs) like furan and pyrrole have been synthesized. SCs are bright fluorophores, which undergo a hypsochromic conversion upon visible light irradiation due to directed photooxidation of the ASORM that leads to the disruption of conjugation. SC-P, a yellow emitting probe bearing a pyrrole moiety, converts to a stable blue emitting coumarin with a 68 nm shift allowing the photoconversion and tracking of lipid droplet in live cells. This new approach might pave the way to a new generation of photoconvertible dyes for advanced bioimaging applications.

The Magic of Linking Rings: Discovery of a Unique Photoinduced Fluorescent Protein Crosslink.

Fluorosubstituted tryptophans serve as valuable probes for fluorescence and nuclear magnetic resonance (NMR) studies of proteins. Here, we describe an unusual photoreactivity introduced by replacing the single tryptophan in cyclophilin A with 7-fluoro-tryptophan. UV exposure at 282 nm defluorinates 7-fluoro-tryptophan and crosslinks it to a nearby phenylalanine, generating a bright fluorophore. The crosslink-containing fluorescent protein possesses a large quantum yield of ∼0.40 with a fluorescence lifetime of 2.38 ns. The chemical nature of the crosslink and the three-dimensional protein structure were determined by mass spectrometry and NMR spectroscopy. To the best of our knowledge, this is the first report of a Phe–Trp crosslink in a protein. Our finding may break new ground for developing novel fluorescence probes and for devising new strategies to exploit aromatic crosslinks in proteins.