Organic Fluorescent Dyes Research Articles

The use of nanocrystal quantum dot as fluorophore reporters in molecular beacon-based assays.

The utilization of molecular beacon (MB) biosensor probes to detect nucleic acid targets has received enormous interest within the scientific community. This interest has been stimulated by the operational qualities of MB-based probes with respect to their unique sensitivity and specificity. The design of MB biosensors entails not only optimizing the sequence of the loop to hybridize with the nucleic acid target or optimization of the length of the stem to tune the sensitivity but also the selection of the appropriate fluorophore reporter to generate the signal transduction read-out upon hybridization of the probe with the target sequence. Traditional organic fluorescent dyes are mostly used for signal reporting in MB assays but their optical properties in comparison to semiconductor fluorescent quantum dot (Qdot) nanocrystals are at a disadvantage. This review highlights the progress made in exploiting Qdot as fluorophore reporters in MB-based assays with the aim of instigating further development in the field of Qdot-MB technology. The development reported to date indicates that unparalleled fluorescence signal reporting in MB-based assays can be achieved using well-constructed Qdot fluorophores.

Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation.

The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time.

Live bio-imaging with fully bio-compatible organic fluorophores

We synthesized a new organic fluorescent dye named resveratrone glucoside from the photoreaction of naturally-occurring phytoalexin compound resveratrol glucoside (resveratrol-3-β-mono-d-glucoside), which is abundant in various plants such as berries, herbs, nuts and grapes. Just like its predecessor molecule resveratrone that was previously discovered by our group, resveratrone glucoside possesses excellent optical properties including a high fluorescence quantum yield, a large Stokes' shift, and a large two-photon absorption cross section. In addition to these highly desirable properties, both fluorescent molecules can also be used as ideal bio-compatible organic fluorophores since they have remarkably low cytotoxicity, which we verified through our cell morphological study, trypan blue exclusion assay, Western blot analysis and fluorescence imaging of various live biological specimens. In particular, we note that resveratrone glucoside is much more soluble in aqueous solution because of its glycosidic side chain and therefore highly suitable for in vivo imaging. We demonstrated that resveratrone and resveratrone glucoside can be used in one- and two-photon fluorescence microscopic imaging of E. coli, yeast (S. cerevisiae), and mammalian cell lines including HeLa and MCF10A cells as well as to the live imaging and real-time tracking of the zebrafish embryo development. Both organic fluorophores can be readily obtained from a simple photoreaction of commercially available, inexpensive samples.

Luminescent Downshifting by Photo‐Induced Sol‐Gel Hybrid Coatings: Accessing Multifunctionality on Flexible Organic Photovoltaics via Ambient Temperature Material Processing

A novel high‐durability multifunctional organic–inorganic hybrid coating material is presented in this work as luminescent down‐shifting (LDS) host matrix system for flexible organic photovoltaic (OPV) devices. Such new LDS coating is obtained by incorporating a convenient fluorescent organic dye in an appropriately functionalized fluoropolymeric resin that can be readily crosslinked by means of a dual‐cure mechanism with a single‐step ambient‐temperature photo‐induced sol–gel process. Due to its peculiar characteristics, the newly proposed system may be readily implemented in heat‐sensitive flexible devices. By carefully tuning the amount of organic fluorophore in the hybrid coating material, a maximum increase in power conversion efficiency exceeding 4% is achieved on devices incorporating the new LDS layer with respect to control systems. This represents the highest efficiency enhancement reported to date on flexible OPVs by means of a polymer‐based LDS layer. In addition, long‐term accelerated weathering tests (>550 h) highlight the excellent stability of LDS‐coated OPV devices, which can retain 80% of their initial performance, as opposed to the dramatic efficiency decay experienced by control uncoated devices. The approach presented here opens the way to the straightforward incorporation of versatile multifunctional light‐managing layers on flexible OPV systems for improved device efficiency and lifetime.

Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections.

Quantum dots are semiconductor fluorescent nanocrystals that exhibit excellent characteristics compared with more commonly used organic fluorescent dyes. For many years quantum dot conjugated products have been available in multiple forms for fluorescence imaging of tissue sections under the trademark name Qdot®. They have much increased brightness, narrow emission spectrum, large Stokes shift and photostability compared with conventional organic fluorescent dyes, which together make them the fluorophores of choice for demanding requirements. Vivid Qdots are recent replacements for original Qdots, modified to improve brightness, however this has affected the fluorescence stability in commonly used conditions for immunohistochemistry. We present here our investigation of the stability of original and Vivid Qdots in solution and in immunohistochemistry, highlight the potential pitfalls and propose a protocol for stable and reliable multiplex staining with current commercially available original and Vivid Qdots.

Inclusion of Ethyl Acetoacetate Bearing 7-Hydroxycoumarin Dye by β-Cyclodextrin and its Cooperative Assembly with Mercury(II) Ions: Spectroscopic and Molecular Modeling Studies.

The inclusion of the fluorescent organic dye, ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-oxopropanoate (1) by the host β-cyclodextrin (β-CD), and its response toward mercuric ions (Hg2+ ), was studied by UV/Vis, fluorescence, and 1 H NMR spectroscopic analyses, mass spectrometry and molecular modeling studies. 1 H NMR measurements together with molecular modeling studies for dye 1 demonstrate that it exhibits two tautomeric forms (keto and enol); however, when the dye is included into the β-CD cavity, the enol form predominates. Moreover, by using spectroscopic and spectrometry techniques, a 1:1 stoichiometry was determined for the complexes formed between dye 1 (enol form) and β-CD, with a binding constant (Kb1 =1.8×104 m-1 ) and for the dye 1 (keto form)-Hg2+ (Kb2 =2.3×103 m-1 ). Interestingly, in the presence of 1-β-CD complex and mercuric ions, a ternary supramolecular system (Hg-1-β-CD complex) was established, with a 1:1:1 stoichiometry and a Kb3 value of 4.3×103 m-1 , with the keto form of the dye being the only one present in this assembly. The three-component system provides a starting point for the development of novel and directed supramolecular assemblies.

Interaction between fluorescein isothiocyanate and carbon dots: Inner filter effect and fluorescence resonance energy transfer

Carbon dots (CDs) have been widely used for the preparation of multifunctional probes by conjugation with organic fluorescent dyes. However, the effect of organic fluorescent dyes on CDs still remains poorly understood. Herein, the effect of fluorescein isothiocyanate (FITC) on CDs was explored by spectroscopic techniques at pH5.1, 7.0 and 9.0. The fluorescent intensity of CDs was found to be quenched gradually after mixing directly with different concentrations of FITC, but the fluorescent lifetime of CDs remained unchanged. According to the results of UV–vis absorption spectra and fluorescent lifetime measurements, a pH-dependent inner filter effect (IFE) between CDs and FITC was proposed. However, the fluorescent lifetime of CDs deceased after their conjugation with FITC, implying the fluorescence resonance energy transfer (FRET) between CDs and FITC. This study has revealed two different effects of FITC on CDs with varying pH values and provided useful theoretical guidelines for further research on the interaction between other nanoparticles and fluorophores.

Rapid Imaging of Latent Fingerprints Using Biocompatible Fluorescent Silica Nanoparticles.

Fluorescent silica nanoparticles (FSNPs) are synthesized through the Stöber method by incorporating silane-modified organic dye molecules. The modified fluorescent organic dye molecule is able to be prepared by allylation and hydrosilylation reactions. The optical properties of as-prepared FSNPs are shown the similar optical properties of PR254A (allylated Pigment Red 254) and have outstanding photostability. The polyvinylpyrrolidone (PVP) is introduced onto the surface of FSNP to enhance the binding affinity of PVP-coated FSNP for latent fingerprints (LFPs) detection. The simple preparation and easy control of surface properties of FSNPs show potential as a fluorescent labeling material for enhanced latent fingerprint detection on hydrophilic and hydrophobic substrates in forensic science for individual identification.

Nile Red and Nile Blue: Applications and Syntheses of Structural Analogues.

Nile red and Nile blue are highly fluorescent and photostable organic dyes from the benzo[a]phenoxazine family. They have been used as histological stains for imaging lysosomes and lipids in vitro. The dyes' high quantum yields and solvent-dependent optical properties make them ideal scaffolds for the development of pH probes and local polarity indicators. Reviews of the literature in this area are scarce with only one review ever published in 2006. It has been 10 years since and the field has evolved. This review aims to expand upon topics covered by the previous reviewers and to report on recent advances in the literature. As authors, we hope to convey a sense of scope and to spark renewed interest in this useful niche of dye chemistry.

Filtration markers and determination methods for the assessment of kidney function

Estimation of renal function is crucial for a number of clinical situations. Endogenous markers such as creatinine, as well as several exogenous agents including inulin, 99mTc-DTPA and iothalamate are used for evaluation of kidney function. At present, new generation filtration markers and determination methods based on organic fluorescent dyes and fluorescent detection technique are developed. This review gives an overview of filtration markers and clearance methods for the assessment of kidney function. New filtration markers and transcutaneous measurement of renal function are described. Problems and issues relating to the reabsorption or secretion of each of markers and the applicability of methods are also discussed.

Wide-field time-gated photoluminescence microscopy for fast ultrahigh-sensitivity imaging of photoluminescent probes.

Fluorescence microscopy is a fundamental technique for the life sciences, where biocompatible and photostable photoluminescence probes in combination with fast and sensitive imaging systems are continually transforming this field. A wide-field time-gated photoluminescence microscopy system customised for ultrasensitive imaging of unique nanoruby probes with long photoluminescence lifetime is described. The detection sensitivity derived from the long photoluminescence lifetime of the nanoruby makes it possible to discriminate signals from unwanted autofluorescence background and laser backscatter by employing a time-gated image acquisition mode. This mode enabled several-fold improvement of the photoluminescence imaging contrast of discrete nanorubies dispersed on a coverslip. It enabled recovery of the photoluminescence signal emanating from discrete nanorubies when covered by a layer of an organic fluorescent dye, which were otherwise invisible without the use of spectral filtering approaches. Time-gated imaging also facilitated high sensitivity detection of nanorubies in a biological environment of cultured cells. Finally, we monitor the binding kinetics of nanorubies to a functionalised substrate, which exemplified a real-time assay in biological fluids. 3D-pseudo colour images of nanorubies immersed in a highly fluorescent dye solution. Nanoruby photoluminescence is subdued by that of the dye in continuous excitation/imaging (left), however it can be recovered by time-gated imaging (right). At the bottom is schematic diagram of nanoruby assay in a biological fluid.

Dye-Doped Fluorescent Silica Nanoparticles for Live Cell and In Vivo Bioimaging

The need for novel design strategies for fluorescent nanomaterials to improve our understanding of biological activities at the molecular level is increasing rapidly. Dye-doped fluorescent silica nanoparticles (SiNPs) emerge with great potential for developing fluorescence imaging techniques as a novel and ideal platform for the monitoring of living cells and the whole body. Organic dye-containing fluorescent SiNPs exhibit many advantages: they have excellent biocompatibility, are non-toxic, highly hydrophilic, optically transparent, size-tunable and easily modified with various biomolecules. The outer silica shell matrix protects fluorophores from outside chemical reaction factors and provides a hydrophilic shell for the insoluble nanoparticles, which enhances the photo-stability and biocompatibility of the organic fluorescent dyes. Here, we give a summary of the synthesis, characteristics and applications of fluorescent SiNPs for non-invasive fluorescence bioimaging in live cells and in vivo. Additionally, the challenges and perspectives of SiNPs are also discussed. We prospect that the further development of these nanoparticles will lead to an exciting breakthrough in the understanding of biological processes.

Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion

Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels into the viral membrane and trapping fluorescent proteins in the virus interior enables the visualization of single virus fusion in living cells. Here, we implement a new approach to non-invasively label the viral membrane glycoproteins through metabolic incorporation of unnatural sugars followed by click-reaction with organic fluorescent dyes. This approach allows for efficient labeling of diverse viral fusion glycoproteins on the surface of HIV pseudoviruses. Incorporation of a content marker into surface-labeled viral particles enables sensitive detection of single virus fusion with live cells.

Sol–gel synthesis, spectral properties and stability of silica films doped by fluorescent dyes

Silicon dioxide thin films with organic fluorescent dyes with different amount of matrix layers were prepared by sol–gel spin coating and dripping technique, synthetic and subsequent treatment parameters were proposed for obtaining materials with high homogeneity and dye stability. The structural, morphological and optical properties of obtained materials have been studied by X-ray diffraction, atomic force microscopy and ultraviolet–visible spectroscopy. Immobilisation of the dye increases the degree of film amorphousness. Immobilisation greatly affects the spectral properties of coumarin 6 and BODIPY due to the dye protonation by acid catalyst, this effect is reversible in case of coumarin 6. Rhodamine 6G spectral characteristics are not influenced by immobilisation. In the hybrid materials monomeric form of the dyes retained at higher concentration compared with solutions. Dye immobilisation was found to increase dyes photostability.

Adamantyl side groups boosting the efficiency and thermal stability of organic solid-state fluorescent dyes

Ethyl-adamantyl side groups substituted onto para-bis(2-thienyl)phenylene, a solid-state fluorescent dye, were found to induce π–π interactions between the conjugated para-bis(2-thienyl)phenylene cores. At the same time, stacking between the central unit and the adamantyl-containing side group was observed. As a result, due to steric hindrance augmentation of the solid-state, luminescence was observed. Moreover, substitution of standard alkyl side chains with ethyl-adamantyl groups resulted in raising the melting point temperature from 55°C to ~250°C. Aside from possible further-functionalization of the para-bis(2-thienyl)phenylene conjugated unit, aforesaid approach can be applied in a wide range of molecules and polymers, when luminescent properties and thermal stability can be tune through the side-chain engineering of organic materials.

Quantitative Determination of Phototoxicity in Live Cell Super-Resolution Microscopy

In the past decades a number of novel fluorescence super-resolution microscopy techniques have emerged allowing to obtain images of cell structures and dynamics with unprecedented resolution by overcoming the diffraction limit. More recently, these approaches have been applied to imaging dynamics in living cells. Cell structures and molecules are in these applications labeled with cell permeable fluorescent organic dyes (e.g SiR, Oregon Green) or endogenously expressed fluorescent proteins. Since super-resolution microscopy tends to require higher laser radiation, phototoxicity and photodamage are potential limitations in live-cell fluorescence microscopy.Here, we present our recent systematic investigation of phototoxicity and photodamage in STED and FPALM/PALM/STORM super-resolution microscopy using a number of different cellular stress indicators, Ca2+ release (through measuring fluorescence levels of Fluoforte), Mitochondria depolarization (through measuring the decrease in fluorescence of JC-1) and reactive oxygen species formation (through monitoring the increase in fluorescence of MitoSOX, CMFDA and Enzo Total ROS/Superoxide indicator). We focus, in particular, on short to mid-term effects (up to 30 minutes), which is a typical time-scale of many cell-biological processes (membrane trafficking, etc.). We investigated, the influence of intensity, fluence, wavelength, dye location in the cell (e.g. ER, Golgi, mitochondria, cytoplasm using SNAP tags) and dependence on dye/tag labeling ratio.

ChemInform Abstract: Nanoparticles Made of π‐Conjugated Compounds Targeted for Chemical and Biological Applications

AbstractReview: 125 refs.

Fluorescence Lifetime Imaging of Cancer In Vivo.

Optical imaging of fluorescent reporters in animal models of cancer has become a common tool in oncologic research. Fluorescent reporters including fluorescent proteins, organic dyes, and inorganic photonic materials are used in fluorescence spectroscopy, microscopy, and whole body preclinical imaging. Fluorescence lifetime imaging provides additional, quantitative information beyond that of conventional fluorescence intensity signals, enabling signal multiplexing, background separation, and biological sensing unique to fluorescent materials.

Insight into a reversible energy transfer system.

Resonance energy transfer (RET) processes have wide applications; these processes involve a unidirectional energy transfer from a particular donor to a particular acceptor. Here, we report a plasmonic resonance energy transfer (PRET), which occurs from the surface of gold nanoparticles to fluorescent organic dyes, and coexists with a nanometal surface energy transfer (NSET) that operates in the reverse direction. The coexistence of both PRET and NSET in opposite directions means that the roles of both donor and acceptor can be interchanged, which could be identified by using spectrofluorometric measurements and light scattering dark field microscopic imaging. The experimental data could be further theoretically supported using Persson and Lang's model, the quasi-static approximation and finite-difference time-domain simulation. Moreover, disruption of the PRET process by altering the energy transfer pairs suggests that interactions occur inside the reversible energy transfer system, which manifest by increasing the fluorescence quenching efficiency of the NSET process.

Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nanographene Oxide and Applications in Three-Photon Fluorescence Bioimaging.

Organic fluorescent dyes with high quantum yield are widely applied in bioimaging and biosensing. However, most of them suffer from a severe effect called aggregation-caused quenching (ACQ), which means that their fluorescence is quenched at high molecular concentrations or in the aggregation state. Aggregation-induced emission (AIE) is a diametrically opposite phenomenon to ACQ, and luminogens with this feature can effectively solve this problem. Graphene oxide has been utilized as a quencher for many fluorescent dyes, based on which biosensing can be achieved. However, using graphene oxide as a surface modification agent of fluorescent nanoparticles is seldom reported. In this article, we used nanographene oxide (NGO) to encapsulate fluorescent nanoparticles, which consisted of a type of AIE dye named TPE-TPA-FN (TTF). NGO significantly improved the stability of nanoparticles in aqueous dispersion. In addition, this method could control the size of nanoparticles' flexibly as well as increase their emission efficiency. We then used the NGO-modified TTF nanoparticles to achieve three-photon fluorescence bioimaging. The architecture of ear blood vessels in mice and the distribution of nanoparticles in zebrafish could be observed clearly. Furthermore, we extended this method to other AIE luminogens and showed it was widely feasible.