Fluorescent Molecules Research Articles

Seeing Beyond the Limits With Super-Resolution Microscopy

When I was young, I wanted to be an astronaut and I was a hard-core science fiction fan. I was always drawn to heroic characters that invented something new that resulted in an amazing breakthrough. When I started my scientific career, I did not want to do something small—I wanted to do something that would be really different and impactful. This is why I chose to work on one of the most difficult problems in the field of optical (light-using) microscopy—how to see objects that are smaller than the wavelength of visible light. This meant challenging a limit that was long believed to be unbreakable. In my research, I developed a method called photoactivated localization microscopy (PALM), which enabled us to break that limit with the help of glowing (fluorescent) molecules. Using the PALM method, and other methods based on glowing molecules, scientists can learn new things about living cells and single molecules and significantly advance our understanding of life.

A Photo‐ and Thermo‐Driven Azoarene‐Based Circularly Polarized Luminescence Molecular Switch in a Liquid Crystal Host

AbstractThe development of chiral optical active materials with switchable circularly polarized luminescence (CPL) signals remains a challenge. Here an azoarene‐based circularly polarized luminescence molecular switch, (S, R, S)‐switch 1 and (R, R, R)‐switch 2, are designed and prepared with an (R)‐binaphthyl azo group as a chiral photosensitive moiety and two (S)‐ or (R)‐binaphthyl fluorescent molecules with opposite or the same handedness as chiral fluorescent moieties. Both switches exhibit reversible trans/cis isomerization when irradiated by 365 nm UV light and 520 nm green light in solvent and liquid crystal (LC) media. In contrast with the control (R, R, R)‐switch 2, when switch 1 is doped into nematic LCs, polarization inversion and switching‐off of the CPL signals are achieved in the resultant helical superstructure upon irradiation with 365 nm UV and 520 nm green light, respectively. Meanwhile, the fluorescence intensity of the system is basically unchanged during this switching process. In particular, these variations of the CPL signals could be recovered after heating, realizing the true sense of CPL reversible switching. Taking advantage of the unique CPL switching, the proof‐of‐concept for “a dual‐optical information encryption system” based on the above CPL active material is demonstrated.

A Photo- and Thermo-Driven Azoarene-Based Circularly Polarized Luminescence Molecular Switch in a Liquid Crystal Host.

The development of chiral optical active materials with switchable circularly polarized luminescence (CPL) signals remains a challenge. Here an azoarene-based circularly polarized luminescence molecular switch, (S, R, S)-switch 1 and (R, R, R)-switch 2, are designed and prepared with an (R)-binaphthyl azo group as a chiral photosensitive moiety and two (S)- or (R)-binaphthyl fluorescent molecules with opposite or the same handedness as chiral fluorescent moieties. Both switches exhibit reversible trans/cis isomerization when irradiated by 365 nm UV light and 520 nm green light in solvent and liquid crystal (LC) media. In contrast with the control (R, R, R)-switch 2, when switch 1 is doped into nematic LCs, polarization inversion and switching-off of the CPL signals are achieved in the resultant helical superstructure upon irradiation with 365 nm UV and 520 nm green light, respectively. Meanwhile, the fluorescence intensity of the system is basically unchanged during this switching process. In particular, these variations of the CPL signals could be recovered after heating, realizing the true sense of CPL reversible switching. Taking advantage of the unique CPL switching, the proof-of-concept for "a dual-optical information encryption system" based on the above CPL active material is demonstrated.

Photo‐Responsive Phase‐Separating Fluorescent Molecules for Intracellular Protein Delivery

AbstractCellular membranes, including the plasma and endosome membranes, are barriers to outside proteins. Various vehicles have been devised to deliver proteins across the plasma membrane, but in many cases, the payload gets trapped in the endosome. Here we designed a photo‐responsive phase‐separating fluorescent molecule (PPFM) with a molecular weight of 666.8 daltons. The PPFM compound condensates as fluorescent droplets in the aqueous solution by liquid‐liquid phase separation (LLPS), which disintegrate upon photoirradiation with a 405 nm light‐emitting diode (LED) lamp within 20 min or a 405 nm laser within 3 min. The PPFM coacervates recruit a wide range of peptides and proteins and deliver them into mammalian cells. Photolysis disperses the payload from condensates into the cytosolic space. Altogether, a type of small molecules that are photo‐responsive and phase separating are discovered; their coacervates can serve as transmembrane vehicles for intracellular delivery of proteins, whereas photo illumination triggers the cytosolic distribution of the payload.

The full spectrum tuning of fluorescent molecules via a one-pot multicomponent reaction

Fluorogenic probes for imaging enable visualization and analysis of difficult-to-reach cells and organelles. However, there are limited efficient examples of tuning these fluorescent molecules to higher wavelengths. This is vital since different tissues are sensitive to varying wavelength emissions. To address this need, we report the discovery, tuning, structure-photophysical property relationships (SPPR), and time-dependent DFT (TD-DFT) computations of 400–700+ nm fluorescent pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolines and substituted imidazo[1,2-a]pyridin-3-amines. The syntheses involve the trimethylsilylcyanide (TMSCN) modified Groebke-Blackburn-Bienaymé (GBB) multicomponent reaction as well as the TMSCN modified GBB combined with subsequent condensation of an aldehyde, and Aza-Friedel-Crafts-Intramolecular Cyclization-Oxidation all in one pot. The SPPR reveals that electron-withdrawing strength in the para-position of the aminopyridine starting material has direct control over the absorption and fluorescence emission wavelengths of these molecules. The TD-DFT computations show the changes in the natural transition orbitals (NTOs) with differing substitutions to the parent molecule that dictate the observed excitations, emissions, and fluorescence intensities. These findings give insights and directions for tuning the fluorescent properties of these motifs for various uses as probes and imaging agents.

Effect of cyano substitution in TADF molecules on luminescence properties: A theoretical study

Wise design strategies for efficient thermally activated delayed fluorescence (TADF) molecules are highly desirable. In this work, the effect of cyano-substitution effects on the molecular photophysical properties of five TADF molecules was investigated. The energy gap (△EST), natural transition orbitals (NTOs) properties, the Huang-Rhys factor and reorganization energies, rate constants for the intersystem crossing (kISC) and reverse intersystem crossing (kRISC) are analyzed based on density functional theory (DFT) and time-dependent density functional theory (TD-DFT) in combination with the thermal vibration correlation function (TVCF) method to measure method. CzTrzBp-CN-4 has the smallest △EST and larger spin-orbital coupling (SOC) in the film, leading to larger kRISC, suggesting that the cyano connections between the acceptor and other acceptors can effectively increase the twist angle and promote the kRISC, predicting that it may have TADF properties.

Single cell in vivo optogenetic stimulation by two-photon excitation fluorescence transfer

Optogenetic manipulation with single-cell resolution can be achieved by two-photon excitation. However, this frequently requires relatively high laser powers. Here, we developed a novel strategy that can improve the efficiency of current two-photon stimulation technologies by positioning fluorescent proteins or small fluorescent molecules with high two-photon cross-sections in the vicinity of opsins. This generates a highly localized source of endogenous single-photon illumination that can be tailored to match the optimal opsin absorbance. Through neuronal and vascular stimulation in the live mouse brain, we demonstrate the utility of this technique to achieve efficient opsin stimulation, without loss of cellular resolution. We also provide a theoretical framework for understanding the potential advantages and constrains of this methodology, with directions for future improvements. Altogether, this fluorescence transfer illumination method opens new possibilities for experiments difficult to implement in the live brain such as all-opticalneural interrogation and control of regional cerebral blood flow.

Single domain Camelid antibody fragments for molecular imaging and therapy of cancer.

Despite innovations in cancer therapeutics, cancer remains associated with high mortality and is one of biggest health challenges worldwide. Therefore, developing precise cancer imaging and effective treatments is an unmet clinical need. A relatively novel type of therapeutics are heavy chain variable domain antibody fragments (VHHs) derived from llamas. Here, we explored the suitability of VHHs for cancer imaging and therapy through reviewing the existing literature. We searched the MEDLINE, EMBASE and Cochrane databases and identified 32 papers on molecular imaging and 41 papers on therapy that were suitable for comprehensive reviewing. We found that VHHs harbor a higher specificity and affinity compared to mAbs, which contributes to high-quality imaging and less side-effects on healthy cells. The employment of VHHs in cancer imaging showed remarkably shorter times between administration and imaging. Studies showed that 18F and 99mTc are two optimal radionuclides for imaging with VHHs and that site-specific labelling is the optimal conjugation modality for VHHs with radionuclide or fluorescent molecules. We found different solutions for reducing kidney retention and immunogenicity of VHHs. VHHs as anticancer therapeutics have been tested in photodynamic therapy, targeted radionuclide therapy, immunotherapy and molecular targeted therapy. These studies showed that VHHs target unique antigen epitopes, which are distinct from the ones recognized by mAbs. This advantage means that VHHs may be more effective for targeted anticancer therapy and can be combined with mAbs. We found that high cellular internalization and specificity of VHHs contributes to the effectiveness and safety of VHHs as anticancer therapeutics. Two clinical trials have confirmed that VHHs are effective and safe for cancer imaging and therapy. Together, VHHs seem to harbor several advantages compared to mAbs and show potential for application in personalized treatment for cancer patients. VHH-based imaging and therapy are promising options for improving outcomes of cancer patients.

Development of organic RPL dosimeters based on fluorescent products of the radiation-induced reaction of 2′,7′-dichlorofluorescein diacetate

In this study, we achieved a shift in the fluorescence wavelength of the organic molecules by X-ray irradiation and fabricated organic materials for radiation dosimetry, focusing on the radiation-induced radical oxidation of 2′,7′-dichlorofluorescein-diacetate (DCFHDA). We doped DCFHDA into processable and transparent polymers to develop a novel tissue-equivalent dosimeters with 3D measurement capabilities. Photoluminescence spectra of the organic materials were measured before and after X-ray irradiation, and their sensitivities to X-rays were compared based on the changes in their fluorescence intensity. Fluorescent molecules were produced from DCFHDA upon X-ray irradiation, and the material was sensitive to X-rays at doses of up to 6 kGy. Furthermore, trichloroacetic acid doping enhanced the dose sensitivity of the material by promoting the radical reaction, which indicates that it can be used as a sensitizer. These results indicate that the fluorescence wavelengths of DCFHDA switched to those of another fluorescent molecule, i.e., the achievement of radiophotoluminescence (RPL). We achieved an organic RPL (ORPL) and showed the possibility of ORPL that can be utilized in dosimetry in radiotherapy.

Exploring the Synergies of Single-Molecule Fluorescence and 2D Materials Coupled by DNA.

The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection of single fluorescent molecules offer a set of advantages, for instance, high sensitivity and specificity, that allow the drawing of conclusions with unprecedented precision. Herein, it is argued how the study of 2D materials benefits from fluorescence-based single-molecule modalities, and vice versa. A special focus is placed on DNA, serving as a versatile adaptor when anchoring single dye molecules to 2D materials. The existing literature on the fruitful combination of the two fields is reviewed, and an outlook on the additional synergies that can be created between them provided.

Deep learning-enhanced single-molecule spectrum imaging

Fluorescence is widely used in biological imaging and biosensing. Rich information can be revealed from the fluorescence spectrum of fluorescent molecules, such as pH, viscosity and polarity of the molecule’s environment, and distance between two FRET molecules. However, constructing the fluorescence spectrum of a single fluorescent molecule typically requires a significant number of photons, which can suffer from photobleaching and, therefore, limit its potential applications. Here, we propose a deep learning-enhanced single-molecule spectrum imaging method (SpecGAN) for improving the single-molecule spectrum imaging efficiency. In SpecGAN, the photon flux required to extract a single-molecule fluorescence spectrum can be reduced by 100 times, which enables two orders of magnitude higher temporal resolution compared to the conventional single-molecule spectrometer. The concept of SpecGAN was validated through numerical simulation and single Nile Red molecule spectrum imaging on support lipid bilayers (SLBs). With SpecGAN, the super-resolution spectrum image of the COS-7 membrane can be reconstructed with merely 12 000 frames of single-molecule localization images, which is almost half of the previously reported frame count for spectrally resolved super-resolution imaging. The low photon flux requirement and high temporal resolution of SpecGAN make it a promising tool for investigating the molecular spectrum dynamics related to biological functions or biomolecule interactions.

Absorption bandwidth broadening of photon upconversion solid-solution organic crystals by co-dissolution of multiple sensitizers

Photon upconversion (UC) enhances the utilization efficiency of solar energy. However, UC materials generally exhibit a narrow optical absorption bandwidth of the sensitizing molecules that create excited triplet states, including the case of solid-state UC materials. Here, we demonstrate the concept of simultaneous dissolution of multiple sensitizers into crystals of fluorescent molecules to broaden the absorption bandwidth, by using an example of combining two sensitizing porphyrins. Consequently, we appreciably enhanced the UC emission intensity under simulated sunlight. In the air, the developed solid-solution crystals exhibited excellent photostability over 50 h and a low excitation threshold of <1-Sun intensity.

Droplet-free and enzyme-free digital immunoassay based on fluorescent microspheres for protein detection

Digital immunoassays with high sensitivity offer the early disease diagnosis and therapeutic management of various diseases. By using fluorescent microspheres and magnetic microparticles (MMPs), a droplet-free and enzyme-free digital immunoassay method was proposed for protein detection. The utilization of fluorescent microspheres offers several advantages. Firstly, they are anchored onto the MMPs in digital detection, thereby eliminating the need for droplets. This innovative feature streamlines the assay process and enhances its efficiency. Secondly, the fluorescent microspheres were embedded with a lot of fluorescent molecules, allowing the detection of single fluorescent nanospheres. Signal amplification was achieved by the embedding of fluorescent molecules within the microspheres, thus eliminating the need for enzyme-based amplification methods of the signal. This approach not only simplifies the assay but also reduces costs associated with enzyme procurement and utilization. To validate the efficacy of this digital immunoassay method, interleukin 6 (IL-6) was chosen as the target protein for detection. The results showed a significant linear relationship between IL-6 concentrations of 0.01–50 pg/mL (R2 = 0.9982), highlighting the robustness of the assay. Furthermore, the assay showed exceptional sensitivity, with a limit of detection (LOD) of 0.006 pg/mL. Thus, this digital immunoassay method using fluorescent microspheres is a simple and low-cost technique, holding great potential for the industrialization of digital immunoassays.

Double‐State Anticounterfeiting Labels Based on Light‐Emitting Polymer‐Stabilized Blue Phase

AbstractAnticounterfeit labels with multiple information‐carrying functions serve a critical role in boosting security and minimizing counterfeit‐related losses, whose development remains challenging. Here, a security label with the ability to encode information in reflective and fluorescent sates is fabricated from light‐emitting polymer‐stabilized blue phase (LE‐PSBP) liquid crystals (LCs). The LE‐PSBP with temperature‐dependent reflective color can be encoded with information by UV irradiation through photomasks. On the other hand, introduction of a fluorescent molecule, which can be easily photocyclized upon UV irradiation, enables the convenient encoding of fluorescent information without impacting the reflective information. As an illustration, a security label with distinct information in reflective and fluorescent states is demonstrated. Such innovative security labels have tremendous potential to provide a platform for multi‐information encoding to enhance the protection level in anticounterfeiting technology.

Blue-green fluorescence during hypersensitive cell death arises from phenylpropanoid deydrodimers.

Infection of Arabidopsis with avirulent Pseudomonas syringae and exposure to nitrogen dioxide (NO2) both trigger hypersensitive cell death (HCD) that is characterized by the emission of bright blue-green (BG) autofluorescence under UV illumination. The aim of our current work was to identify the BG fluorescent molecules and scrutinize their biosynthesis, localization, and functions during the HCD. Compared with wild-type (WT) plants, the phenylpropanoid-deficient mutant fah1 developed normal HCD except for the absence of BG fluorescence. Ultrahigh resolution metabolomics combined with mass difference network analysis revealed that WT but not fah1 plants rapidly accumulate dehydrodimers of sinapic acid, sinapoylmalate, 5-hydroxyferulic acid, and 5-hydroxyferuloylmalate during the HCD. FAH1-dependent BG fluorescence appeared exclusively within dying cells of the upper epidermis as detected by microscopy. Saponification released dehydrodimers from cell wall polymers of WT but not fah1 plants. Collectively, our data suggest that HCD induction leads to the formation of free BG fluorescent dehydrodimers from monomeric sinapates and 5-hydroxyferulates. The formed dehydrodimers move from upper epidermis cells into the apoplast where they esterify cell wall polymers. Possible functions of phenylpropanoid dehydrodimers are discussed.

Performance improved white full-organic light emitting diodes (WOLED) based on tricolor dual-layer of hyperfluorescence (HF)

Efficiency-improved white-light organic light emitting diodes (WOLED) was designed, which based on thermally activated delayed fluorescence (TADF) tricolor-hyperfluorescent dual-layers. Benefited from the efficient utilization of triplet excitons by effective reverse intersystem crossing (RISC), this WOLED features improved efficiency and long lifetime. In this work, blue OLED based on emitting layer of mCP:BD340 was developed and dual-emissive hyperfluorescent OLEDs based on mono-emitting layer of CBP:4CzIPN (green TADF emitter):DBP (red emissive fluorescent molecule) were investigated. Then, the hyperfluorescent WOLED based on the two emitting layers was designed. The good performances were benefited from the highly efficient hyperfluorescent system. This device architecture design demonstrated here represents a kind of full-organic, low-cost white-light OLED for power-efficient light source.

Tandem High-Pressure Crystallography-Optical Spectroscopy Unpacks Noncovalent Interactions of Piezochromic Fluorescent Molecular Rotors.

To develop luminescent molecular materials with predictable and stimuli-responsive emission, it is necessary to correlate changes in their geometries, packing structures, and noncovalent interactions with the associated changes in their optical properties. Here, we demonstrate that high-pressure single-crystal X-ray diffraction can be combined with high-pressure UV-visible absorption and fluorescence emission spectroscopies to elucidate how subtle changes in structure influence optical outputs. A piezochromic aggregation-induced emitter, sym-heptaphenylcycloheptatriene (Ph7C7H), displays bathochromic shifts in its absorption and emission spectra at high pressure. Parallel X-ray measurements identify the pressure-induced changes in specific phenyl-phenyl interactions responsible for the piezochromism. Pairs of phenyl rings from neighboring molecules approach the geometry of a stable benzene dimer, while conformational changes alter intramolecular phenyl-phenyl interactions correlated with a relaxed excited state. This tandem crystallographic and spectroscopic analysis provides insights into how subtle structural changes relate to the photophysical properties of Ph7C7H and could be applied to a library of similar compounds to provide general structure-property relationships in fluorescent organic molecules with rotor-like geometries.

Plasma electrochemical synthesis of silicon quantum dots

Environmentally friendly and fast synthesis of silicon quantum dots (SiQDs) is realized with the assistance of plasma. The precursors used are N-(β-aminoethyl)-γ-aminopropyl trimethoxysilane (DAMO) and citric acid. When the excitation wavelength was 370 nm, the photoluminescence emission peak of the SiQDs appeared at 452.5 nm. The optimization of precursor concentration, reaction time and other parameters can effectively improve the quantum yield (QY) of SiQDs. The results show that the amidation and condensation of DAMO and citric acid plays an important role in the improvement of QY, as this means that more fluorescent molecules are produced and therefore QY is increased. This paper increases QY from 4.23% to 23.9%, providing a promising way to improve QY even more.

NIR-II organic small molecule probe for labeling lymph nodes and guiding tumor imaging

Organic small molecule fluorescent groups have injected new material support into the field of medical imaging due to their unique luminescence mechanism and easy tuning of structure. The great potential of NIR-II window imaging forces us to continuously optimize the structure of organic fluorophores to design better fluorescent molecules for fluorescence imaging-guided surgery. An ideal organic small molecule fluorescent group: it can penetrate into the inside of the organism, clearly present the internal structure and the edge contour of different tissues, so as to perfectly achieve internal imaging and accurately guide external surgery. In vivo, fluorescent groups do not damage normal tissues and organs. However, problems such as low quantum yield and poor biocompatibility greatly limit the clinical transformation of NIR-II fluorescent small molecules. To avoid the shortcomings of NIR-II fluorescent probes as much as possible and better realize image-guided surgery, in this experiment, the biplane donor unit was incorporated into the twisted D-π-A-π-D structure to expand the conjugated structure of the fluorescent group, which not only realized NIR-II emission, but also had high quantum yield and biosafety.

Engineering of cyanine-based nanoplatform with tunable response toward reactive species for ratiometric NIR-II fluorescent imaging in mice

High-quality second near-infrared (NIR-II) nanoprobes are of great significance for real-time bioimaging and medical diagnosis. Cyanine is an important class of fluorophores to construct activatable probes; however, there are still significant challenges hindering their biological applications, including weak fluorescence in aqueous solution, instability, and insufficient specificity. Herein, an integrated engineering strategy is conducted to develop the cyanine-based activatable NIR-II nanoplatforms with bright, stable emission and high specificity. Specifically, poly(styrene-co-maleic anhydride) (PSMA) is employed to encapsulate NIR-II fluorescent molecules (IR1048) to render the stable and bright NIR-II nanoparticles (PSMA@IR1048 NPs). By charge-modulated strategy, a series of cyanine-fluorophores are loaded on the surface of PSMA@IR1048 NPs and exhibit tunable response toward reactive species. Combing those two strategies, NIR-II ratiometric fluorescent nanoprobes (RNPs, including RNP1, RNP2, and RNP3) are constructed; among them, RNP2 displays hypochlorous acid (HClO) responsive performance and generates a higher NIR-II fluorescent ratio (FL2/FL1) signal. Such nanoprobe can reliably report the pathological HClO level in models of diabetic liver injury and lower limb ischemia–reperfusion (I/R) injury mice. Our study paves an engineering strategy to construct cyanine-based stable, bright, and specific NIR-II probes for bioimaging.