Conventional Fluorophores Research Articles

One-step nanoscale expansion microscopy reveals individual protein shapes.

The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

Fluorescence of BODIPY dyes in gas phase at near-ambient conditions

Molecular fluorescence is a phenomenon that is usually observed in condensed phase. It is strongly affected by molecular interactions. The study of fluorescence spectra in gas-phase can provide a nearly ideal model for the evaluation of the intrinsic properties of fluorophores. Unfortunately, most conventional fluorophores are not volatile enough to allow for the study of their fluorescence in gas phase. Here we report very bright gas phase fluorescence of simple BODIPY dyes that can be readily observed at atmospheric pressure using conventional fluorescence instrumentation. To our knowledge, this is the first example of visible-range gas-phase fluorescence at near-ambient conditions. Evaporation of the dye in vacuum allowed us to demonstrate organic molecular electroluminescence in gas discharge excited by the electric field produced by a Tesla coil.

Early Detection and Noninvasive Staging of Kidney Dysfunction by a PEGylated Conventional Fluorophore via GFR-Sensitive Renal Transport.

Noninvasive fluorescence imaging of renal function is a valuable technique for understanding kidney disease progression and the development of renal medicine. This technique requires sensitive imaging probes for reporting renal dysfunction accurately at early stage. Herein, a molecularly engineered imaging probe (800CW-PEG45-COOH) was synthesized by simply PEGylating conventional near-infrared fluorophore IRDye800CW with NH2-PEG45-COOH (molecular weight ∼2100 Da) for early detection and staging of renal dysfunction through noninvasive real-time kidney imaging. 800CW-PEG45-COOH not only cleared through the kidney efficiently (>90% injection dosage at 24 h postinjection) but was also found to be freely filtered by glomeruli without renal tubular reabsorption and secretion. Despite this simple construction strategy, the transport of 800CW-PEG45-COOH within the kidneys was extremely sensitive to the alteration of the glomerular filtration rate (GFR), which enabled it to detect renal dysfunction much earlier than commonly used serum biomarkers and stage kidney function impairments (mild vs severe dysfunction) via imaging-based kidney clearance kinetics. This work not only provides a promising optical imaging probe for the noninvasive evaluation of kidney function but also highlights the utility of PEGylation in enhancing the performance of conventional organic dyes in biomedical applications.

Tuning the emission of anti-rigidochromic fluorophore by alternating the polymer matrices for advanced data encryption

Anti-rigidochoromic fluorophores with bathochromic emission in rigidifying polymer matrix offer advantages such as high fluorescent contrast, broadly tunable emissions, and versatile stimuli-responsive behaviors. In this work, we systematically investigated the impact of polymer chemical structure on the emission of the anti-rigidochromic fluorophore. By precisely tuning intermolecular polar-π and π-π interactions between the polymer matrix and fluorophore, a range of emission colors could be achieved, spanning from deep blue (450 nm) to the yellow-orange region (600 nm). Notably, the emission of polymer/fluorophore composites can be precisely adjusted over temperature. Leveraging the distinctive features, we showcase advanced fluorescence encryption using the non-emissive polymers as an information carrier and the fluorophore as the decryption key, which efficiently addresses issues of conventional fluorophores such as photo-bleaching and low adhesive force. This work establishes the structure-fluorescence relationship for the anti-rigidochromic system and extends its application for advanced data encryption with a high level of security.

Development and Application of Cationic Nile Blue Probes in Live-Cell Super-Resolution Imaging and Specific Targeting to Mitochondria.

Mitochondria are essential organelles involved in various metabolic processes in eukaryotes. The imaging, targeting, and investigation of cell death mechanisms related to mitochondria have garnered significant interest. Small-molecule fluorescent probes have proven to be robust tools for utilizing light to advance the study of mitochondrial biology. In this study, we present the rational design of cationic Nile blue probes carrying a permanent positive charge for these purposes. The cationic Nile blue probes exhibit excellent mitochondrial permeability, unique solvatochromism, and resistance to oxidation. We observed weaker fluorescence in aqueous solutions compared to lipophilic solvents, thereby minimizing background fluorescence in the cytoplasm. Additionally, we achieved photoredox switching of the cationic Nile blue probes under mild conditions. This enabled us to demonstrate their application for the first time in single-molecule localization microscopy of mitochondria, allowing us to observe mitochondrial fission and fusion behaviors. Compared to conventional cyanine fluorophores, this class of dyes demonstrated prolonged resistance to photobleaching, likely due to their antioxidation properties. Furthermore, we extended the application of cationic Nile blue probes to the mitochondria-specific delivery of taxanes, facilitating the study of direct interactions between the drug and organelles. Our approach to triggering cell death without reliance on microtubule binding provides valuable insights into anticancer drug research and drug-resistance mechanisms.

Supra-Fluorophores: Ultrabright Fluorescent Supramolecular Assemblies Derived from Conventional Fluorophores in Water.

Fluorescent organic nanoparticles (NPs) with exceptional brightness hold significant promise for demanding fluorescence bioimaging applications. Although considerable efforts are invested in developing novel organic dyes with enhanced performance, augmenting the brightness of conventional fluorophores is still one of the biggest challenges to overcome. This study presents a supramolecular strategy for constructing ultrabright fluorescent nanoparticles in aqueous media (referred to as "Supra-fluorophores") derived from conventional fluorophores. To achieve this, this course has employed a cylindrical nanoparticle with a hydrophobic microdomain, assembled by a cyclic peptide-diblock copolymer conjugate in water, as a supramolecular scaffold. The noncovalent dispersion of fluorophore moieties within the hydrophobic microdomain of the scaffold effectively mitigates the undesired aggregation-caused quenching and fluorescence quenching by water, resulting in fluorescent NPs with high brightness. This strategy is applicable to a broad spectrum of fluorophore families, covering polyaromatic hydrocarbons, coumarins, boron-dipyrromethenes, cyanines, xanthenes, and squaraines. The resulting fluorescent NPs demonstrate high fluorescence quantum yield (>30%) and brightness per volume (as high as 12060 m-1 cm-1 nm-3). Moreover, high-performance NPs with emission in the NIR region are constructed, showcasing up to 20-fold increase in both brightness and photostability. This Supra-fluorophore strategy offers a versatile and effective method for transforming existing fluorophores into ultrabright fluorescent NPs in aqueous environments, for applications such as bioimaging.

Dynamically Securing the Data by 1O2 Sensitization of Fluorescent Composites with a High Latency and Uncrackable Features

Comprehensive SummaryDynamic fluorescent materials play a crucial role in secure inks for data encryption; however, they are still plagued by issues such as photodegradation, poor latency, and susceptibility to unauthorized access. Herein, we propose a photochemically modulated dynamic fluorescent encryption system based on 1O2 sensitization of fluorescent composites, comprising a 1O2‐sensitive fluorophore (F2) and non‐emissive polymers. After UV irradiation, in‐situ generated 1O2 from the polymer effectively binds with F2 to form endoperoxides (F2EPO), resulting in a significant redshift in emission, up to 150 nm. The 1O2 concentration is closely related to the irradiation time, rendering different fluorescent colors in a time‐gated fashion. Moreover, the emission of F2EPO can be regulated by polymer chemical structure, molecular weight, and crosslinking density. Relying on these merits, we develop a dynamic data encryption method with various non‐emissive polymers as the data storage media, UV light irradiation as the data encoder, and F2 as the data decoder. UV light irradiation of diverse polymer solutions generates 1O2 at different concentrations, effectively encoding the data, which remains invisible under both UV and natural lights. The addition of F2 to these irradiated polymer solutions produces different redshifted fluorescence, enabling secure data decryption. Attributing to the non‐emissive nature of the polymers, time‐gated readout fashion, excellent latency of 1O2, and subtle interactions between 1O2 and F2, this data encryption is nearly undecipherable. This work offers an advantage data encryption approach beyond the reach of conventional fluorophores.

Mechanically gated data display via synergizing the mechanochromism of chiral photonic elastomers and optically responsive molecules

Mechanochromic fluorophores and elastomers gain great potential in dynamic data encryption and anti-counterfeiting due to their instant responses, excellent handleability, high-throughput data-verification, and multi-dimensional security features. However, the reported mechanochromic fluorophores generally require solvent fuming or thermal annealing to revert their responses, sacrificing the handleability and lengthening the authentication time. For the mechanochromic elastomers, the data input requires precisely control the dose of the crosslinkers or phase tuning agents, making the high-throughput data-processing rather challenging. Here we developed a mechanically gated data display by synergizing the mechanochromism of chiral photonic elastomers and optically responsive molecules. Specifically, the reflectance or emission of the dyes/fluorophores could be selectively reflected or transmitted by the elastomers with desired photonic bandgaps, leading to data displays of given color/fluorescence. Mechanically stretching the elastomers can readily tuning their photonic bandgaps over a wide range, thereby notably alternating the corresponding reflection and emission wavelength, and the outcome of displayed data. Relaxing the elastomers rapidly restores the initial data. Moreover, the displayed data varies notably under L- and R-circular polarizer filter, respectively, offering good programmability and multi-dimensional security features. This work represents a unique data protection approach and enables properties unattainable by conventional mechanochromic fluorophores and elastomers.

Plasmonic Gold Nanostar-Based Probes with Distance-Dependent Plasmon-Enhanced Fluorescence for Ultrasensitive DNA Methyltransferase Assay.

The ultrasensitive DNA methyltransferase (Dam MTase) assay is of high significance for biomedical research and clinical diagnosis because of its profound effect on gene regulation. However, detection sensitivity is still limited by shortcomings, including photobleaching and weak signal intensities of conventional fluorophores at low concentrations. Plasmonic nanostructures with ultrastrong electromagnetic fields and fluorescence enhancement capability that can overcome these intrinsic defects hold great potential for ultrasensitive bioanalysis. Herein, a silica-coated gold nanostars (Au NSTs@SiO2)-based plasmon-enhanced fluorescence (PEF) probe with 20 "hot spots" was developed for ultrasensitive detection of Dam MTase. Here, the Dam Mtase assay was achieved by detecting the byproduct PPi of the rolling circle amplification reaction. It is worth noting that, benefiting from the excellent fluorescence enhancement capability of Au NSTs originating from their 20 "hot spots", the detection limit of Dam Mtase was reduced by nearly 105 times. Moreover, the proposed Au NST-based PEF probe enabled versatile evaluation of Dam MTase inhibitors as well as endogenous Dam MTase detection in GW5100 and JM110 Escherichia coli cell lysates, demonstrating its potential in biomedical analysis.

In Situ Optical Detection of Amines at a Parts-per-Quadrillion Level by Severing the Through-Space Conjugated Supramolecular Domino.

Conventional fluorophores suffer from low sensitivity and selectivity in amine detection due to the inherent limitations in their "one-to-one" stoichiometric sensing mechanism. Herein, we propose a "one-to-many" chain reaction-like sensing mechanism by creating a domino chain consisting of one fluorescent molecule (e.g., PTF1) and up to 40 nonemissive polymer chains (pPFPA) comprising over thousand repeating units (PFPA). PTF1 (the domino trigger) interacts with adjacent PFPA units (the following blocks) through polar-π interactions and initiates the domino effect, creating effective through-space conjugation along pPFPA chains and generating amplified yellow fluorescent signals through charge transfer between PTF1 and pPFPA. Amine exposure causes rapid dismantling of the fluorophore-pPFPA-based domino chain and significantly reduces the amplified emissions, thus providing an ultrasensitive method for detecting amines. Relying on the above merits, we achieve a limit of detection of 177 ppq (or 1.67 × 10-12 M) for triethylamine, which is nearly 4 orders lower than that of previous methods. Additionally, the distinct reactivity of pPFPA toward different amines allows for the discrimination of primary, secondary, and tertiary amines. This study presents a "domino effect" sensing mechanism that has not yet been reported and provides a general approach for chemical detection that is beyond the reach of conventional methods.

PH-Sensitive and Lysosome Targetable Photosensitizers Based on BODIPYs.

Photodynamic therapy (PDT) is an effective and U.S. Food and Drug Administration (FDA) approved treatment for cancer and other diseases. Photosensitizer is one of the three key components that harvest the energy of light at a certain wavelength. Compared to the conventional fluorophores used as photosensitizers, boron dipyrromethene (BODIPY) derivatives have grown fast in recent years due to their low dark toxicity, versatile tunable sites, and easiness of being paired with other treatments. In this paper, two pH-sensitive BODIPY-based photosensitizers (BDC and BDBrC) were synthesized by adding carbazole moieties onto the BODIPY cores (BD and BDBr) through condensation reactions. BDBrC has two Br atoms at the BODIPY core that promote singlet oxygen generation and further red-shift the absorption maximum peak. Both compounds showed sensitivity toward pH change and generated more singlet oxygen under acidic conditions. The cellular uptake and cell imaging experiments showed that BDBrC can selectively target the lysosome organelle. The further dark cell viability and light cytotoxicity indicate the light triggered PDT treatment can be accomplished with BDBrC.

A Flexible, Quantitative Plasmonic-Fluor Lateral Flow Assay for the Rapid Detection of Orthoebolavirus zairense and Orthoebolavirus sudanense.

Filoviruses comprise a family of single-stranded, negative-sense RNA viruses with a significant impact on human health. Given the risk for disease outbreaks, as highlighted by the recent outbreaks across Africa, there is an unmet need for flexible diagnostic technologies that can be deployed in resource-limited settings. Herein, we highlight the use of plasmonic-fluor lateral flow assays (PF-LFA) for the rapid, quantitative detection of an Ebolavirus-secreted glycoprotein, a marker for infection. Plasmonic fluors are a class of ultrabright reporter molecules that combine engineered nanorods with conventional fluorophores, resulting in improved analytical sensitivity. We have developed a PF-LFA for Orthoebolavirus zairense (EBOV) and Orthoebolavirus sudanense (SUDV) that provides estimated limits of detection as low as 0.446 and 0.641 ng/mL, respectively. Furthermore, our assay highlights a high degree of specificity between the two viral species while also maintaining a turnaround time as short as 30 min. To highlight the utility of our PF-LFA, we demonstrate the detection of EBOV infection in non-human primates. Our PF-LFA represents an enormous step forward in the development of a robust, field-deployable assay for filoviruses.

Coordination-Induced Conformational Control Enables Highly Luminescent Metallo-Cages.

In recent years, luminescent materials have received a great deal of attention due to their wide range of applications. However, exploring a simple solution to overcome the fluorescence quenching resulting from the aggregation of conventional organic fluorophores remains a valuable area of investigation. In this study, we successfully constructed two metallo-cages, namely, SA and SB, through coordination-driven self-assemblies of the triphenylamine (TPA)-based donor L with different diplatinum(II) acceptors LA and LB, respectively. These metallo-cages take advantage of their steric nature and curved conformation to more effectively limit the free rotation of the benzene ring and hinder π-π stacking in the solid state, which successfully inhibited fluorescence quenching and realizing highly efficient luminescent properties. Therefore, this work offers a new design strategy for preparing materials with excellent luminescent properties.

A General Strategy Toward pH‐Resistant Phenolic Fluorophores for High‐Fidelity Sensing and Bioimaging Applications

AbstractAryl alcohol‐type or phenolic fluorophores offer diverse opportunities for developing bioimaging agents and fluorescence probes. Due to the inherently acidic hydroxyl functionality, phenolic fluorophores provide pH‐dependent emission signals. Therefore, except for developing pH probes, the pH‐dependent nature of phenolic fluorophores should be considered in bioimaging applications but has been neglected. Here we show that a simple structural remedy converts conventional phenolic fluorophores into pH‐resistant derivatives, which also offer “medium‐resistant” emission properties. The structural modification involves a single‐step introduction of a hydrogen‐bonding acceptor such as morpholine nearby the phenolic hydroxyl group, which also leads to emission bathochromic shift, increased Stokes shift, enhanced photo‐stability and stronger emission for several dyes. The strategy greatly expands the current fluorophores’ repertoire for reliable bioimaging applications, as demonstrated here with ratiometric imaging of cells and tissues.

A General Strategy Toward pH-Resistant Phenolic Fluorophores for High-Fidelity Sensing and Bioimaging Applications.

Aryl alcohol-type or phenolic fluorophores offer diverse opportunities for developing bioimaging agents and fluorescence probes. Due to the inherently acidic hydroxyl functionality, phenolic fluorophores provide pH-dependent emission signals. Therefore, except for developing pH probes, the pH-dependent nature of phenolic fluorophores should be considered in bioimaging applications but has been neglected. Here we show that a simple structural remedy converts conventional phenolic fluorophores into pH-resistant derivatives, which also offer "medium-resistant" emission properties. The structural modification involves a single-step introduction of a hydrogen-bonding acceptor such as morpholine nearby the phenolic hydroxyl group, which also leads to emission bathochromic shift, increased Stokes shift, enhanced photo-stability and stronger emission for several dyes. The strategy greatly expands the current fluorophores' repertoire for reliable bioimaging applications, as demonstrated here with ratiometric imaging of cells and tissues.

Non-aromatic single amino acid, lysine: A simple fluorescence sensor for selective detection of Cu2+ and Co2+ with colorimetric distinction by the virtue of its self-aggregation behaviour

Non-aromatic amino acids are mostly considered as non-emissive due to their lack of conventional fluorophores. However, at high concentrations, they can undergo conformational rigidification via self-aggregation and emit in the visible range. Herein, we have exploited that very property of non-aromatic amino acids as a fluorescence sensor. To the best of our knowledge, this is the first report demonstrating a single unmodified non-aromatic amino acid, Lysine (Lys) as a selective and sensitive fluorescence sensor for the detection of Cu2+ and Co2+ along with colorimetric distinction. An as-prepared aqueous solution of Lys (6.8 mM) served as a fluorescence sensor for Cu2+ and Co2+ with a detection limit of 420 nM and 460 nM, respectively in water. The instantaneous appearance of blue and pink color upon adding Cu2+ and Co2+, respectively to the Lys solution makes the system useful for their real-time analysis. The sensing of Cu2+ and Co2+ are also demonstrated through the fabrication of paper strips. Moreover, the sensor could detect Cu2+ and Co2+ in real water samples (lake water and tap water). The exceptionally low cost (<1 U.S. cent/10 mg Lys), reversibility of the sensor, and its ease of use uniquely qualify Lys as a remarkably simple fluorescent sensor.

The Role of Trypan Blue as a Conventional and Fluorescent Dye for the Diagnosis of Superficial Mycoses by Potassium Hydroxide (KOH) Testing.

Direct microscopic examination of samples using potassium hydroxide (KOH) is a fast, simple, and inexpensive method to confirm clinical suspicion of superficial mycosis. However, the lack of color contrast in this test makes it difficult to separate any fungal structures from artifacts. The sensitivity of the KOH mount technique may be enhanced using both fluorochromes and conventional stains that highlight the fungal structures when observed under fluorescence microscopy and bright-field, respectively. Here we study the possibility of using Trypan Blue (TB), an azo dye which is often used as a live/dead marker, in the diagnosis of superficial mycoses by KOH testing. TB at 0.01% displayed a fluorescent staining pattern similar to that of Calcofluor White (CFW), the conventional cell wall fluorophore. Furthermore, by adjusting the TB concentration to 0.1-0.3%, in addition to maintaining the fluorescent staining pattern, the fungal elements were stained in blue under bright-field microscopy. Thus, we demonstrate for the first time that TB has the unique property as a fungal stain that can be used in both bright-field and fluorescence microscopy for diagnosis of superficial mycoses by direct microscopic examination.

Water soluble silicon quantum dot based fluorescence immunoassay probe for C-reactive protein (CRP) detection

Silicon quantum dot (SiQD) based water soluble fluorescence immunoassay probe with aggregation induced emission features (AIE) was developed for the sensitive detection of C-reactive protein (CRP) biomarker. The solid state and solution phase photophysical properties of SiQD attributed from AIE effect was studied successfully. Later the ability of CRP antibody functionalized SiQD against the detection CRP antigen was evaluated systematically. The method relies on the decrease in fluorescence intensity of CRP antibody functionalized SiQD (CRPAb) upon the gradual addition of CRP antigen (CRPAg) via antigen-antibody interaction. The intensity of the antibody functionalized fluorophore quenched linearly by the addition of CRPAg within the concentration range of 50–150 nM and the limit of detection (LOD) was as sensitive as about 0.89 nM. Detection mechanism was assigned further by evaluating the interaction that exists between SiQD-CRPAb and CRPAg, which suggests that the effective complexation through a lock and key mechanism could be the plausible reason behind the fluorescence quenching of SiQD-CRPAb in presence of CRPAg. The solid state fluorescence features of SiQD arises from AIE phenomena which suggest that as a promising alternative to conventional QDs and organic fluorophores, integration of SiQD with lateral flow strips could result in powerful eco-friendly point-of-care device for in vitro diagnosis and the detection of other biomarkers.

Aggregation Induced Transition by Modulation of Substituent Groups on Rofecoxib Analogues and Multi‐Stimulus Response Properties

AbstractAggregation‐induced emission luminogens (AIEgens) have been widely investigated due to their promising applications in data storage, organic light‐emitting diodes, and deep tissue bioimaging as compared to conventional fluorophores. In this work, we have designed and synthesized two novel rofecoxib analogues functionalized with different terminal groups (−NH2, −Br) on the benzene ring B. Interestingly, the compound 2 a‐3 with −NH2 substituent shows aggregation‐caused quenching effect whereas the compound 1 b‐3 with −Br group displays aggregation‐induced emission (AIE) property which may be caused by the twisted conformation and restricted – stacking of 1 b‐3 according to the X‐ray single crystal diffraction analysis. These opposite results indicated that the terminal groups could have great influence on the photophysical properties of targets. Moreover, the compound 1 b‐3 exhibited multi‐stimuli response luminescence behaviors under various external stimuli including solvatochromism, mechanochromism and acidochromism. Furthermore, lipid droplets imaging investigation proved the compound 1 b‐3 was capable of achieving specific lipid droplets bioimaging. This work not only presents a better understanding of the structure‐fluorescent property relationship based on the scaffold of rofecoxib, but also provides a new avenue for spectral modulation and the development of new MSR materials.

Click chemistry in the design of AIEgens for biosensing and bioimaging

AbstractThe development of rapid, selective, and sensitive fluorescent sensors is essential for visualizing and quantifying biological molecules and processes in vitro, ex vitro, and in vivo, which is important for not only fundamental biological studies but the accurate diagnosis of diseases. The emerging field of activity‐based sensing (ABS), a sensing method that utilizes molecular reactivity for analyte detection possesses many advantages including high specificity, sensitivity and accuracy. The aggregation caused quenching phenomenon which occurs in most conventional fluorophores results in reduced labeling efficiency of the target analytes and low photobleaching resistance, therefore limiting the applications of the ABS strategy. In contrast, aggregation induced emission (AIE) active luminogens (AIEgens) provide exceptional molecular frameworks for ABS. Of the many reaction classes utilized in the AIEgen ABS approach, click chemistry has become increasing popular. In this review, the sensing concepts of the ABS approach with AIEgens and the principles of click chemistry are discussed, followed by a systematic summary of the application of specific click chemistry reactions in AIEgen ABS protocols for the detection of an array of target analytes. Furthermore, the utility of click chemistry in the construction of AIEgens for bioimaging will also be showcased throughout the review.