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[Display omitted] • A responsive probe EHDI for ratiometric fluorescence analysis of bisulfite (HSO 3 -) and sulfite (SO 3 2-) is developed. • The probe EHDI exhibits blue shifts in both the UV–Vis and emission spectra upon responding to HSO 3 -. • The probe EHDI features high sensitivity and selectivity for HSO 3 - detection in buffer and imaging in live cells. • Detection of HSO 3 - in beef samples by probe EHDI is validated by HPLC. The development of reliable probe technology for the detection of bisulfite (HSO 3 -) in situ in food and biological samples is contributing significantly to food quality and safety assurance as well as community health. In this work, a responsive probe, EHDI, is developed for ratiometric fluorescence detection of HSO 3 - in aqueous solution, meat samples, and living cells. The probe is designed based on the HSO 3 - triggered 1,4-addition of electron deficit C = C bond of EHDI. As a result of this specific 1,4-addition, the π-conjugation system was destructed, resulting in blue shifts of the emission from 687 to 440 nm and absorption from 577 to 355 nm. The probe has good water solubility, high sensitivity and selectivity, allowing it to be used for imaging of HSO 3 - internalization and production endogenously. The capability of probe EHDI for HSO 3 - was then validated by traditional HPLC technology, enabling accurately detect HSO 3 - in beef samples. The successful development of this probe thus offers a new tool for investigating HSO 3 - in situ in food and biological conditions. [ABSTRACT FROM AUTHOR]

The precise release and characterization of nitroxyl (HNO) gas signaling molecule remain a challenge due to its short lifetime to date. To solve this issue, an azobenzene-based HNO donor (Azo-D1) was proposed as a colorimetric and fluorometric chemosensor for HNO releasing, to release both HNO and an azobenzene fluorescent reporter together. Specifically, the Azo-D1 has an HNO release half-life of ∼68 min under physiological conditions. The characteristic color change from the original orange to the yellow color indicated the decomposition of the donor molecule. In addition, the stoichiometry release of HNO was qualitatively and quantitatively verified through the classical phosphine compound trap. As compared with the donor molecule by itself, the decomposed product demonstrates a maximum fluorescence emission at 424 nm, where the increase of fluorescence intensity by 6.8 times can be applied to infer the real-time concentration of HNO. Moreover, cellular imaging can also be achieved using this Azo-D1 HNO donor through photoexcitation at 405 and 488 nm, where the real-time monitoring of HNO release was achieved without consuming the HNO source. Finally, the Azo-D1 HNO donor would open a new platform in the exploration of the biochemistry and the biology of HNO. [Display omitted] • The first azobenzene-based HNO donor (Azo-D1) was developed. • Azo-D1 can release HNO (t 1/2 –68 min) under physiological conditions. • HNO release can be monitored by colorimetric and fluorometric changes. • Cell imagines were successfully obtained using Azo-D1 donor. [ABSTRACT FROM AUTHOR]

• The review explores the world of functional chromophores synthesized via MCRs. • MCR-synthesized functional chromophores are reshaping imaging research. • Bioimaging probes synthesized by MCRs with tunable properties. This review aims to provide a comprehensive overview of recent advancements and applications of fluorescence imaging probes synthesized via MCRs (multicomponent reactions). These probes, also known as functional chromophores, belong to a currently investigated class of fluorophores that are presently being successfully applied in bioimaging experiments, especially in various living cell lineages. We describe some of the MCRs that have been employed in the synthesis of these probes and explore their applications in biological imaging, with an emphasis on cellular imaging. The review also discusses the challenges and future perspectives in the field, particularly considering the potential impact of MCR-based fluorescence imaging probes on advancing this field of research in the coming years. Considering that this area of research is relatively new and nearly a decade has passed since the first publication, this review also provides a historical perspective on this class of fluorophores, highlighting the pioneering works published between 2011 and 2016. [ABSTRACT FROM AUTHOR]

[Display omitted] • FLHE , a eugenol derivative-based fluorescence turn-on probe for detecting Zn2+ was introduced. • FLHE demonstrates the CHEF effect, making it an excellent fluorescence "turn-on" probe for Zn2+ detection. • FLHE showed a high sensitivity for the Zn2+ detection (LOD = 31.8 nM) in live cells and environmental samples. • FLHE -based paper strip (FLHE-PS) assay allows Zn2+ detection (LOD = 63.2 nM) in aqueous solutions. • FLHE -based paper strip (FLHE-PS) assay allows quantification of water (LOD of 0.14%) in the organic solvent. Herein, a Eugenol-derived fluorescence 'turn-on' probe FLHE was synthesized by condensing 2-((3-(trifluoromethyl)phenyl)amino)benzohydrazide with 5-allyl-2-hydroxy-3-methoxybenzaldehyde. FLHE demonstrated very low fluorescence in the studied organic solvents of varying polarities. However, upon titration with Zn2+ in HEPES buffer (pH = 7.4, 50% ACN, v/v), FLHE showed 40-fold higher fluorescence signals indicating the formation of the FLHE–Zn2+ complex. The fluorescence turn-on phenomenon upon FLHE–Zn2+ complex formation results from a chelation-enhanced fluorescence (CHEF) effect. The FLHE–Zn2+ complexation demonstrated a stokes shift of 156 nm (λ ex = 350 nm, λ em = 506 nm) and an about 33-fold increase in the quantum yield (FLHE , Φ = 0.007; FLHE–Zn2+ complex, Φ = 0.23). The binding constant (K a) determined by the Benesi-Hildebrand plot for interaction between FLHE and Zn2+ was 5.33 × 103 M−1. FLHE demonstrated a LOD of 31.8 nM for detecting Zn2+ in the environmental samples without interference from other cations and anions. FLHE -based paper strip (FLHE-PS) assay was developed to quantify the Zn2+ ions in water and the water content of organic solvent. FLHE-PS allows the detection of Zn2+ in aqueous solutions with a LOD of 63.2 nM and quantifying water in acetonitrile with a LOD of 0.14%. These results indicate that the FLHE has high applicability for detecting Zn2+ in living cells and environmental samples and detecting the presence of water in the organic solvents. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new probe was designed, synthesized and characterized using NMR, mass and single crystal XRD studies. • The probe exhibited high selectivity and sensitivity towards cyanide in an aqueous solution. • A fluorescence turn-on process via deprotonation of phenolic –OH group was proposed. • The proposed sensing mechanism and optical properties were investigated using DFT and TD-DFT calculations. • The probe was utilized to detect cyanide in food materials and HeLa cells and to determine in natural water samples. A simple fluorescent probe (KS4) containing multiple reaction sites (phenolic –OH, imine and C = C bonds) is successfully synthesized and characterized using 1H NMR, 13C NMR, mass and single crystal XRD techniques. KS4 exhibits high selectivity towards CN– over a wide range of common anions in H 2 O:DMSO (1:1 v/v) leading to an amazing turn-on fluorescence at 505 nm via deprotonation of the phenolic –OH group. The limit of detection (1.3 µM) for CN– was much below the standard (1.9 µM) set by the World Health Organization (WHO). Stoichiometry of the interaction between KS4 and CN– was ascertained as 1:1 by the Job's plot method and the binding constant was determined to be 1.5x104 M−1. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) based theoretical insight has been appealed to understand the optical properties of KS4 before and after the addition of CN– ion. The probe shows respectable real-time applicability for qualitative detection of CN– in almond and cassava powder as well as quantification in real water samples with excellent recoveries (98.8 – 99.8%). In addition, KS4 is found to safe towards living HeLa cells and successfully applied to the detection of endogenous cyanide ions in HeLa cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • A "dual-locked" probe for detection of noradrenaline and HClO was synthesized. • The probe shows excellent selectivity for noradrenaline and HClO in cells. • The probe was successfully applied to accurately detect depressed cells. Due to the serious harm of depression to human health and quality of life, an accurate diagnosis of depression is warranted. For the complex etiology of depression, a single biomarker diagnostic method often leads to misdiagnosis. As noradrenaline and HClO are closely related to depression, a "dual-locked" fluorescence probe R-NE-HClO for diagnosing of depression through the simultaneous detection of noradrenaline and HClO was designed and synthesized. Fluorescence of R-NE-HClO can only be restored in the presence of both noradrenaline and HClO. The probe demonstrates excellent selectivity for noradrenaline and HClO and low cytotoxicity in cell imaging experiments. It is to be observed that we successfully applied the probe to accurately detect depressed cells which provides a possible tool for diagnosing depression. [ABSTRACT FROM AUTHOR]

[Display omitted] Carbon dots (CDs) have been widely used in recent years because of their excellent water solubility and abundant surface functional groups. However, compared with quantum dots or biological probes, the quantum yield of CDs is lower, and the fluorescence mainly concentrated in the blue-green range, which significantly limits the biological applications of CDs. Heteroatoms doping is the most common method to improve the luminescence of CDs. In this work, nitrogen and sulfur co-doped luminescent CDs were successfully synthesized by microwave assisted method using glutathione (GSH) and p-phenylenediamine (PPD) as raw materials. It can emit bright green fluorescence in ethanol solution, and the maximum emission wavelength is 535 nm when excited at 374 nm, and the absolute quantum yield is as high as 63%. Iron ion (Fe3+) can interact with the functional groups on the surface of the CDs to form CDs/Fe3+, which is a non-fluorescence complex, and Fe3+ can be reduced to ferrous ion (Fe2+). In other words, the reaction mechanism of CDs and Fe3+ is a combination of dynamic quenching and static quenching. The fluorescence of CDs quenched by Fe3+ can be restored by thiol, because there is a stronger binding force between sulfhydryl (-SH) on the surface of thiol and Fe3+, which enables CDs to be released. In addition, the CDs has good biocompatibility and stability, indicating that it has excellent potential in bioimaging. This discovery will expand the application of CDs in the fields of biosensing and imaging. [ABSTRACT FROM AUTHOR]

[Display omitted] Carbon dots have garnered significant attention owing to their versatile and highly tunable optical properties; however, the origins and the underlying mechanism remains a subject of debate especially for dual fluorescent systems. Here, we have prepared carbon dots from glutathione and formamide precursors via a one-pot solvothermal synthesis. Steady state and dynamic techniques indicate that these dual fluorescent dots possess distinct emissive carbon-core and a molecular states, which are responsible for the blue and red optical signatures, respectively. To further glean information into the fluorescence mechanism, electrochemical analysis was used to measure the bandgaps of the two fluorescent states, while femtosecond transient absorption spectroscopy evidenced the two-state model based on the observed heterogeneity and bimodal spectral distribution. Our findings provide novel and fundamental insights on the optical properties of dual fluorescent dots, which can translate to more effective and targeted application development particularly in bioimaging, multiplexed sensing and photocatalysis. [ABSTRACT FROM AUTHOR]

Yersinia outer protein M (YopM) is one of the effector proteins and essential for virulence. YopM is delivered by the Yersinia type III secretion system (T3SS) into the host cell, where it shows immunosuppressive effect through interaction with host proteins. Therefore, protein-protein interactions of YopM is significant to understand its molecular mechanism. In this study, we aimed to explore protein-protein interactions of YopM with the two components of T3SS, namely LcrV and LcrG. We used bimolecular fluorescence complementation (BiFC) assay and monitored the reassembly of green fluorescence protein in Escherichia coli. As an indicator of the protein-protein interaction, we monitored the in vivo reconstitution of fluorescence by measuring fluorescence intensity and imaging the cells under fluorescence microscope. We showed, for the first time, that YopM interacts with LcrG, but not with LcrV. Here, we propose BiFC assay as a simple method to screen novel interaction partners of YopM. [Display omitted] • BiFC assay is used for the first time to explore the interaction partners of YopM. • LcrG is an interaction partner of YopM. • LcrV does not interact with YopM. • YopM-DDX3 and LcrV-LcrG interactions are confirmed by the BiFC assay. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) has been studied on the treatment of numerous diseases such as cancers, rheumatoid arthritis, and so on. However, real-time tracking and controlled release of CO gas for a safe and efficient therapy are till a challenging task. Although many CO-releasing molecules (CORMs) have been widely developed and used because of the release of CO by external stimuli, CORMs still have some disadvantages, such as fast CO release, potential toxicity and poor solubility. Herein, light-triggered CO-releasing hybrid material (UIO-66-NH 2 /CORM@FA-CS-5-FAM) based on UIO-66-NH 2 nanoparticles and [Mn(CO) 3 (CH 3 CN)(HPYDC)]·CH 3 CN (H 2 PYDC = 2, 5-pyridinedicarboxylic acid, abbreviated as CORM) coated with FA-CS-5-FAM was constructed. The composition characteristics of the hybrid material include: a chitosan (CS) increases the residence time of drugs on the application/absorption sites, folic acid (FA) targets the cancer cells and 5-carboxyfluorescein (5-FAM) is used as an imaging agent. Comparison with the free CORM, UIO-66-NH 2 /CORM@FA-CS-5-FAM nanocomposite exhibits a long-time CO release and possesses the excellent characteristics of targeted cancer cells and fluorescence imaging. Cell viability assays indicate that UIO-66-NH 2 /CORM@FA-CS-5-FAM shows good biocompatibility and anticancer activity. Light-triggered CO-releasing hybrid material (UIO-66-NH 2 /CORM@FA-CS-5-FAM) exhibits a long-time CO release and possesses the excellent characteristics of targeted cancer cells and fluorescence imaging. [Display omitted] • UIO-66-NH 2 /CORM@FA-CS-5-FAM nanocomposite exhibits a long-time CO release irradiated by UV and visible light. • The nanocomposite possesses the excellent characteristics of targeted cancer cells and fluorescence imaging. • The nanocomposite shows good biocompatibility and anticancer activity. [ABSTRACT FROM AUTHOR]

Lanthanide ion (Ln3+)-doped nanoscale hydroxyapatites (nHAp) with tunable luminescence have attracted increasing attention due to their potential applications as useful biomedical tools (e.g., imaging and clinical therapy). In this study, we reported that doping Terbium (III) ions (Tb3+) in self-activated luminescent nHAp via a facile hydrothermal reaction, using trisodium citrate (Cit3−), generates unique emission-tunable probes known as Cit/Tb-nHAp. The morphology, crystal phase, and luminescence properties of these Cit/Tb-nHAp probes are studied in detail. Moreover, the results demonstrate that the luminescence of self-activated nHAp originates from the carbon dots trapped within the nHAp crystals, in which partial energy transfer occurs from carbon dots (CDs) to Tb3+. The color tunability is successfully achieved by regulating the addition of Cit3−. Biocompatibility study indicates that when co-cultured with C6 glioma cells in vitro for 3 days, ≤800 ppm Cit/Tb-nHAp is not cytotoxic for C6 glioma cells. We also present in vitro data showing efficient cytoplasmic localization of transferrin conjugated Cit/Tb-nHAp into C6 glioma cells by fluorescence cell imaging. We have successfully engineered Cit/Tb-nHAp, a promising biocompatible agent for future in vitro and in vivo fluorescence bioimaging. [ABSTRACT FROM AUTHOR]

• Small molecule fluorescence probe. • Highly selective for Ni (II) (≥160-fold). • Compatible with living cells. • Simple and low-cost detection. In this paper, we unveil a new highly selective and sensitive mito-tracker (NiP) for Ni2+ detection. NiP itself held very weak fluorescence and exhibited a high selectivity (≥160-fold) toward Ni2+ over other metal ions, with a limit of detection of 21.6 nmol. We demonstrate the practicality of NiP for the rapid determination of Ni2+ levels in mitochondria of living cells. This approach offers advantages by being fast, simple and low cost. [ABSTRACT FROM AUTHOR]

"Bottom-up" method is a popular approach for the preparation of molybdenum disulfide quantum dots (MoS 2 QDs) benefitting from less time consumption and no high-powered sonication required. But the relatively low fluorescent quantum yield of the obtained MoS 2 QDs and the rare study about their in vivo behavior stimulate us to do more research in this area. In this paper, we proposed a "bottom-up" hydrothermal method to prepare MoS 2 QDs with a quantum yield (QY) of 34.55% by optimizing a series of reaction conditions. The successful fluorescence imaging of tumor cells in vitro and in vivo as well as the systematic in vivo behavior study such as biocompatibility, biodistribution and metabolism route provided the good basis for their wider biomedical applications. • MoS 2 QDs prepared with a "bottom-up" approach were proposed. • MoS 2 QDs revealed low cytotoxicity in the long term. • The in vivo behaviors of MoS 2 QDs were studied systemically. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new dual-response sensor was exploited for detecting viscosity and ONOO−. • It had ideal mitochondrion-targeting capability with good pH- and photo-stabilities. • It showed distinct responses to viscosity and ONOO− with high selectivity. • It can realize dual imaging of viscosity and ONOO− in mitochondria. Viscosity and peroxynitrite (ONOO−) are two significant indicators to affect and evaluate the mitochondrial functional status, which are nearly relational with pathophysiological process in many diseases. Developing suitable analytical methods for monitoring mitochondrial viscosity changes and ONOO− is thus of great importance. In this research, a new mitochondria-targeted sensor DCVP-NO 2 for the dual determination of viscosity and ONOO− was exploited based on the coumarin skeleton. DCVP-NO 2 displayed a red fluorescence "turn-on" response toward viscosity along with about 30-fold intensity increase. Meanwhile, it could be used as ratiometric probe for detection of ONOO− with excellent sensitivity and extraordinary selectivity for ONOO− over other chemical and biological species. Moreover, thanks to its good photostability, low cytotoxicity and ideal mitochondrion-targeting capability, DCVP-NO 2 was successfully utilized for fluorescence imaging of viscosity variations and ONOO− in mitochondria of living cells through different channels. In addition, the results of cell imaging revealed that ONOO− would lead to the increase of viscosity. Taken together, this work provides a potential molecular tool for researching biological functions and interactions of viscosity and ONOO− in mitochondria. [ABSTRACT FROM AUTHOR]

Luminescent calcium fluoride (CaF 2 ) nanoparticles, because of their excellent biocompatibility, excellent photostability and strong fluorescence, have received increasing attention as drug carriers and bioprobes in cell imaging. Inspired by the role of citrate in the growth of apatite crystals during natural bio-mineralization, uniform and nearly monodisperse Eu 3+ -doped CaF 2 nanoparticles with excellent colloidal stability and high fluorescence in aqueous media have been successfully synthesized in the presence of sodium citrate using a hydrothermal method. X-ray diffraction and transmission electron microscopy show that CaF 2 nanoparticles grown in the presence of sodium citrate are cubes of relatively uniform size (15 nm), and that the Eu 3+ doping level has little effect on size and morphology. Zeta potentials and dynamic light scattering demonstrate that in the synthesis with sodium citrate, the colloidal stability of CaF 2 nanoparticles is greatly improved upon the increase of Eu 3+ doping level. Moreover, aqueous dispersions of these nanoparticles are colloidally stable and can be maintained over a wide range of pH from 5.0 to 11.0 for more than a month. Fluorescence spectra demonstrate that the doped CaF 2 nanoparticles display strong red fluorescence. Fourier transform infrared spectra and thermogravimetric analyses demonstrate the adsorption of substantial quantities of sodium citrate on the surfaces of the CaF 2 nanoparticles. Taken together, such colloidal behavior should be related to strong crystal inhibition of citrate ions and Eu 3+ doping induced promotion thermal-decomplexing between citrate ions and calcium ions. The luminescent CaF 2 nanoparticles obtained using this protocol should be promising candidates for use in many bio-related applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Synthesis of 3-acetylcoumarin derivative, Ruthenium(III) complex and Nano ZnO-Ru(III) complex. • Morphology and particle size of Nano ZnO-Ru(III) complex is observed through SEM and TEM analysis which supported with XRD results. • The DNA cleavage evaluation of compounds showed greater activity. • Ru(III) complex exhibited potent anticancer activity against MCF-7 and K-562 cell lines than the ligand. • The cell imaging of Nano ZnO-Ru(III) complex against L6 rat skeletal muscle cell line showed superior fluorescence at 100 μg/mL. The uniqueness of nanofunctionalized metal complexes would play a prominent role in medicinal chemistry, especially in cancer chemotherapy. Among the metal based chemotherapeutic drugs, ruthenium complexes exhibit different oxidation states, lower toxicity and remarkable antitumor activities than other anticancer drugs. In this investigation bioactive ruthenium(III) complex has been synthesized from novel coumarin derivative and it is functionalized with nanostructured zinc oxide (ZnO) under well defined condition. An octahedral geometry is proposed for the ruthenium complex based on analytical and spectrochemical characterization techniques (UV, FT-IR and NMR). The g|| and g⊥ values obtained from the ESR spectrum indicated high symmetry and octahedral field around ruthenium ion. The XRD patterns of all synthesized compounds explicit average particle sizes are nanometer range. The morphology and particle size of Nano ZnO-Ru(III) complex is also confirmed by using SEM and TEM analysis. The nanofunctionalized material exhibited enhanced fluorescence emissions at 674 nm and 681 nm. The evaluation of DNA cleavage study indicated compounds were effectively cleaved supercoiled pUC18 DNA by gel electrophoresis method. The examinations of in-vitro anticancer activities of compounds were studied against human breast (MCF-7) and leukemia (K-562) cancer cell lines. The ruthenium complex showed greater effect against MCF-7 with < 10 IC50 value. Nowadays, the cell imaging property of nanofunctionalized materials receive more attention. The fluorescence microscopic images of Nano ZnO-Ru(III) complex displayed higher luminescence at 100 μg/ mL against L6 rat skeletal muscle cell line. [ABSTRACT FROM AUTHOR]

Luminescent nanomaterials are encouraging scaffolds for diverse applications such as chemical sensors and biosensors, imaging, drug delivery, diagnostics, catalysis, energy, photonics, medicine, and so on. Carbon dots (CDs) are a new class of luminescent carbonaceous nanomaterial that have appeared recently and reaped tremendous scientific interest. Herein, we have exploited a simple approach to prepare tuneable and highly fluorescent CDs via surface functionalization. The successful synthesis of CDs is manifested from several investigations like high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The CDs exhibit excellent water solubility and with increasing nitrogen content fluorescence quantum yield increases whereas cell toxicity decreases. The CD synthesized at high temperature (180 °C) shows very high quantum yield (more than 56%). The tuneable optical properties of CDs are systematically studied using UV-vis and fluorescence spectroscopy. The cell viability evaluation and in vitro imaging study reveals that the synthesized CDs can be employed as a potential fluorescent probe for bio-imaging without further modification. [ABSTRACT FROM AUTHOR]

Naphthyridine-based fluorescent probe H1 was synthesized and characterized for the quantification and selective detection of Uric Acid ( UA ) in live cell. In presence of UA , H1 forms the specific host-guest complex mainly through intermolecular hydrogen bonding and aromatic stacking which produces “turn-off” fluorescence. The probe and UA is found to be 1:1 stoichiometry on the basis of absorption and fluorescence titrations. The probe H1 has been shown to detect UA up to 0.6 µM at pH 7.4. DFT-TDDFT calculations were performed in order to demonstrate the sensing mechanism and the electronic properties of the receptor-donor complex. The selectivity was evaluated in Vero cells in the presence of UA with other purine derivatives, structurally similar to UA . It was found to exhibit no cytotoxicity effect in tested concentration of H1 and good membrane permeability for the detection of UA in living cell system. The unknown concentration of UA in serum and urine can be measured easily using the fluorescence property of probe H1 . [ABSTRACT FROM AUTHOR]

Nitrogen and sulfur co-doped carbon dots (N, S/C-dots) were prepared via one-pot hydrothermal treatment of citric acid and cystamine dihydrochloride. The as-prepared N, S/C-dots exhibited excellent excitation-wavelength-independent photoluminescence property and higher fluorescence quantum yield (QY) of 39.7% compared to C,N - dots (QY = 2.6%) prepared using citric acid and 1,6-diaminohexane dihydrochloride as the precursor. The N, S/C-dots were well-dispersed in aqueous solution and showed good photoluminescence stabilities in different pH and temperature without any surface modification. Additionally, the fluorescence of N, S/C-dots can be quenched based on inner filter effect (IFE) upon the addition of Cr(VI) and showed good selectivity and sensitivity to Cr(VI). The detection for Cr(VI) exhibited a good linear correlation ranging from 1 to 80 μM with a detection limit of 0.86 μM. What’s more, in comparison with other quantum dots and organic dyes, these N, S/C-dots were much more eco-friendly and can be used for multicolor bioimaging. [ABSTRACT FROM AUTHOR]

In general, 4 amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM-DA) dye is used to detect nitric oxide in biological systems through cell imaging. In this study, we have used 96 well plate format to quantify nitric oxide using DAF-FM-DA through a multimode reader (or independently using fluorospectrometer) and could be visualized in a fluorescence microscope. Similar study otherwise will require a high-end instrument. The method has been validated to screen NOS inhibitors in the HEK 293T cell lines over-expressing the NOS isoforms. We observed that the method is very simple to use, adaptive, sensitive and most importantly it saves time. Ethanol (70% [v/v] in distilled water), Nω-Nitro- l -arginine (l -NAME), 7-Nitro-Indazole (7-NI) (Sigma, St. Louis, MO), HEK 293T cell lines (National Centre for Cell Science (NCCS), Pune, India), DMEM (Himedia laboratories Pvt), Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 atmosphere. Hank's Balanced Salt Solution (HBSS) without Phenol Red of pH 7.4 was prepared with the following composition: NaCl, 8.0g, KCl, 0.4g, CaCl 2 , 0.14g, MgSO 4 ⋅7H 2 O, 0.1g, MgCl 2 ·6H 2 O, 0.1g, Na 2 HPO 4 ·2H 2 O, 0.06g, KH 2 PO 4 , 0.06g, glucose, 1.0g, NaHCO 3 , 0.35g, H 2 O, to 1000 ml, Sterilized and refrigerated, Calcium Ionophore A23187 (Sigma Aldrich 52665-69-7) DAF-FM Di Acetate (Molecular Probes Life Technologies), and DAF-FM Di Aceatate was prepared as a stock solution (5 mM) in DMSO, divided into aliquots and stored at −20 °C, followed by dilution to the required concentration in HBSS buffer before use. Neubauer chamber, Microtube centrifuges (1.5 mL), Micropipettors,10,100, and 1000 mL with corresponding tips, multimode reader (Tecan, Synergy-HT), inverted fluorescence microscope (Nikon, eclipse Ti-S), black flat bottom Microplates (96-well) (Corning 3603). Image 1 • A simple, sensitive and adaptive cell-based protocol that requires a microplate reader screen the potential NOS inhibitors. • Enhanced quantum fluorescence of DAF-FM-DA with intracellular NO improves its sensitivity and enzyme activity. • This cell-based assay provides better insight into the bioavailability and inhibitory activity of the potential compounds. [ABSTRACT FROM AUTHOR]

A fluorescence "turn-on" probe for Cu (Ⅱ) ions was prepared based on the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene. A strong fluorescent flavonoid intermediate was formed and verified by the NMR and ESI-MS experiments. The water-soluble and pH dependence experiments were performed to confirm the optimal solvent condition (CH 3 CN: HEPES = 1:1, v/v, pH = 7.2–7.4). The dynamic experiments indicated that the formation process of the intermediate catalyzed by Cu(Ⅱ) ions was probably pseudo-first-order reaction process. The probe showed good selectivity toward copper ions and almost no interference except Ag+ ions by the selectivity and competitive experiments. The HeLa cells were used in the cell fluorescence imaging tests and it was demonstrated that the probe could be used in the phycological condition and showed weak cytotoxicity by the MTT experiments. [Display omitted] • A Cu2+ ions biosensor was constructed by the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene. • The probe exhibited high selectivity for Cu2+ and could not be disturbed even if high concentration of GSH existed. • The mechanism probably involved the procedure of copper-mediated cyclization to generate the flavonoid intermediate. • The probe can be used to detect Cu2+ ions in physiological environment proven by the cell image experiments. [ABSTRACT FROM AUTHOR]

Carbon dots (CDs) are a new representative in carbonaceous family and have initiated remarkable research interests over the past one decade in a large variety of fields. Herein, we have utilized a facile, one-step carbonization method to prepare fluorescent carbon dots using poly(vinyl alcohol) (PVA) both as a carbon source and as a surface passivating agent. The as prepared CDs emit bright blue fluorescence under ultraviolet illumination. The structure and optical properties of the CDs are thoroughly investigated by several methods such as high-resolution transmission electron microscopy; dynamic light scattering; UV–vis, fluorescence and Fourier transform infrared spectroscopy. The CDs exhibit excellent water solubility and demonstrate average hydrodynamic diameter of 11.3 nm, holding great promise for biological applications. The biocompatibility evaluation and in vitro imaging study reveals that the synthesized CDs can be used as effective fluorescent probes in bio-imaging without noticeable cytotoxicity. In addition, a unique sensor for the detection of vitamin B 2 in aqueous solution is proposed on the basis of spontaneous fluorescence resonance energy transfer from CD to vitamin B 2 . These findings therefore suggest that the CDs can find potential applications in cellular imaging along with sensing of vitamin B 2 . [ABSTRACT FROM AUTHOR]

Viruses represent an important class of pathogens that have had an enormous impact on the health of the human race. They are extraordinarily diverse; viral particles can range in size from ∼80 nm to ∼10 μm in length, and contain genomes with RNA or DNA strands. Regardless of their genome type, RNA species are frequently generated as a part of their replication process, and for viruses with RNA genomes, their loading into the virion represents a critical step in the creation of infectious particles. RNA imaging tools represent a powerful approach to gain insight into fundamental viral processes, including virus entry, replication, and virion assembly. Imaging viral processes in live cells is critical due to both the heterogeneity of these processes on a per cell basis, and the inherent dynamics of these processes. There are a number of methods for labeling RNA in live cells; we’ll introduce the myriad of methods and then focus on one approach for labeling viral RNA, using multiply-labeled tetravalent RNA imaging probes (MTRIPs), which do not require engineering of the target RNAs. We feel this approach is advantageous given many viral genomes may not tolerate large nucleotide insertions into their sequences. [ABSTRACT FROM AUTHOR]

Free cholesterol in mammalian cells resides mostly in the plasma membrane, where it plays an important role in cellular homeostasis. We synthesized a new fluorescent cholesterol analogue that retained an intact alkyl chain and the sterane backbone of cholesterol. The hydroxyl group of cholesterol was converted into an amino group that was covalently linked to the fluorophore tetramethylrhodamine to retain the ability to form hydrogen bonds with adjacent molecules. Incubating live MDCK (Madin–Darby canine kidney) cells with our fluorescent cholesterol analogue resulted in the generation of intense signals that were detected by microscopy at the plasma membrane. Incubation with the analogue exerted minimal, if any, influence on cell growth, indicating that it could serve as a useful tool for analyzing free cholesterol at the plasma membrane. [ABSTRACT FROM AUTHOR]

We can learn much about cell function by imaging and quantifying sub-cellular structures, especially if this is done non-destructively without altering said structures. Soft X-ray tomography (SXT) is a high-resolution imaging technique for visualizing cells and their interior structure in 3D. A tomogram of the cell, reconstructed from a series of 2D projection images, can be easily segmented and analyzed. SXT has a very high specimen throughput compared to other high-resolution structure imaging modalities; for example, tomographic data for reconstructing an entire eukaryotic cell is acquired in a matter of minutes. SXT visualizes cells without the need for chemical fixation, dehydration, or staining of the specimen. As a result, the SXT reconstructions are close representations of cells in their native state. SXT is applicable to most cell types. The deep penetration of soft X-rays allows cells, even mammalian cells, to be imaged without being sectioned. Image contrast in SXT is generated by the differential attenuation soft X-ray illumination as it passes through the specimen. Accordingly, each voxel in the tomographic reconstruction has a measured linear absorption coefficient (LAC) value. LAC values are quantitative and give rise to each sub-cellular component having a characteristic LAC profile, allowing organelles to be identified and segmented from the milieu of other cell contents. In this chapter, we describe the fundamentals of SXT imaging and how this technique can answer real world questions in the study of the nucleus. We also describe the development of correlative methods for the localization of specific molecules in a SXT reconstruction. The combination of fluorescence and SXT data acquired from the same specimen produces composite 3D images, rich with detailed information on the inner workings of cells. [ABSTRACT FROM AUTHOR]

In Brome mosaic virus , it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. [ABSTRACT FROM AUTHOR]

There are many challenges to live Caenorhabditis elegans imaging including the high motility of the animals and sustaining their viability for extended periods of time. Commonly used anesthetics to immobilize the C. elegans for imaging purpose prevents feeding of the animals and can cause cellular physiologic changes. Here we present three adapted or novel methodologies to image live C. elegans over different imaging microscopy equipment to allow for visualization of animals by DIC and fluorescence without the use of microfluidic technologies. The methods present here use common microscopy consumables and equipment found in many imaging core facilities and can be easily adapted to fit on multiple microscopy systems. [ABSTRACT FROM AUTHOR]

Abstract: Of all the ways in which complex materials (including many biological systems) can be explored, imaging is perhaps the most powerful because delivering high information content directly. This is particular relevant in aspects of cellular localization where the physical proximity of molecules is crucial in biochemical processes. A great deal of effort in imaging has been spent on enabling chemically selective imaging so that only specific features are revealed. This is almost always achieved by adding fluorescent chemical labels to specific molecules. Under appropriate illumination conditions only the molecules (via their labels) will be visible. The technique is simple and elegant but does suffer from fundamental limitations: (1) the fluorescent labels may fade when illuminated (a phenomenon called photobleaching) thereby constantly decreasing signal contrast over the course of image acquisition. To combat photobleaching one must reduce observation times or apply unfavourably low excitation levels all of which reduce the information content of images; (2) the fluorescent species may be deactivated by various environmental factors (the general term is fluorescence quenching); (3) the presence of fluorescent labels may introduce unexpected complications or may interfere with processes of interest (4) Some molecules of interest cannot be labelled. In these circumstances we require a fundamentally different strategy. One of the most promising alternative is based on a technique called Coherent Anti-Stokes Raman scattering (CARS). CARS is a fundamentally more complex process than is fluorescence and the experimental procedures and optical systems required to deliver high quality CARS images are intricate. However, the rewards are correspondingly very high: CARS probes the chemically distinct vibrations of the constituent molecules in a complex system and is therefore also chemically selective as are fluorescence-based methods. Moreover,the potentially severe problems of fluorescence bleaching and quenching are circumvented and high-resolution three dimensional images can be obtained on completely unlabelled specimens. We review here aspects of CARS and Multiphoton fluorescence techniques to cellular localization and measurement. [Copyright &y& Elsevier]

Highlights: [•] A simple one step method was developed to prepare CNP from biomaterials. [•] Cell uptake of CNP is a concentration-dependent manner. [•] CNP showed poor colocalization with endosomes and mitochondria. [•] CNP could adsorb serum protein. [•] CNP showed well hemocompatibility and low cytotoxicity. [Copyright &y& Elsevier]

Abstract: There is increasing interest in using live cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of fluorescent signals from multiple probes is that emission spectra of many fluorophores overlap, often with maxima that are only a few nanometers apart. Spectral acquisition of mixed fluorescence signals captured within a dedicated scanning range can be used to quantitatively separate signals into component spectra. We report here the development of a novel live cell application of spectral unmixing for the simultaneous monitoring of intracellular events reported by closely-emitting fluorophores responding dynamically to external stimuli. We validate the performance of dynamic spectral unmixing microscopy (DynSUM) using genetically encoded sensors to simultaneously monitor changes in glutathione redox potential (Egsh) and H2O2 production in living cells exposed to oxidizing and reducing agents. We further demonstrate the utility of the DynSUM approach to observe the relationship between the increases in Egsh and H2O2 generation induced in airway epithelial cells exposed to an environmental electrophile. [Copyright &y& Elsevier]

At present, the early diagnosis and treatment of NSCLC has become an international research hotspot. However, how to realize the organic combination of highly sensitive and high-resolution tumor imaging diagnosis and effective treatment, and to provide effective information for the diagnosis and treatment of cancer is still a major problem in the integration of cancer diagnosis and treatment. In this study, based on the Crizotinib has a good targeted inhibitory effect on ALK positive tumor cells, the near-infrared targeted fluorescent dye IR-780 was covalently bound with the drug molecule Crizotinib, thus the near-infrared fluorescent probe IR-780-Crizotinib targeting ALK positive tumor cells was synthesized. The probe structure is confirmed by NMR and MS. The optical properties of the fluorescent probe and the imaging process in ALK positive tumor-bearing mice were analyzed using ultraviolet spectrophotometer, near-infrared fluorescence spectrometer, and near-infrared fluorescence imaging system. The results show that the probe had better photoactivity. In vivo imaging shows that the probe maintained the biological activity of Crizotinib, effectively targeting the tumor site involved with clear imaging, and ultimately excreted from the body. It was confirmed that the probe could be used for the tracking, positioning and targeted therapy of nude mice with ALK positive tumors in vivo, thus exploring a new approach for the clinical application of near-infrared fluorescent probe to detect ALK positive tumors in the future. • We report a near-infrared fluorescent probe for targeted imaging of non-small cell lung cancer in vitro and in vivo. • The probe was synthesized based on the NIR fluorescent probe IR-780 and crizotinib, an ALK-positive NSCLC inhibitor. • The probe can respond to ALK positive NSCLC and shows high fluorescence imaging, sensitivity and targeting. [ABSTRACT FROM AUTHOR]

[Display omitted] • Good selective sensing glutathione over cysteine/homocysteine at physiological pH (6.0–8.0). • Improved brightness and water solubility. • Fast substitution reaction induced between brominated group and -SH group. • Excellent performance for living cell imaging and human tumor diagnosis. The development of novel fluorescent dyes for bio-thiol is of great importance in biological, clinical and pharmaceutical sciences. Given the importance of bio-thiol anticipating in numerous physiological processes, there is a great need to construct fluorescent biosensors with high quality to detect them. Fluorophores, especially those used in bio-system, usually require high-quality properties such as high brightness, good water solubility, bio-compatible and photostability. Herein, we reported a novel fluorescent probe based on piperazine-coumarin scaffold with enhanced brightness and solubility. To further demonstrate the potential clinical applications, we performed living cell fluorescence image and human esophageal carcinoma diagnosis. The result indicated that we were able to distinguish pathological tissue from normal tissue by applying this probe. Thus, we hope this design will be helpful to develop high-quality fluorophores for clinical diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells. [Copyright &y& Elsevier]

Abstract: In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40nm silica cores and 10nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells. [Copyright &y& Elsevier]

Abstract: Multifunctional nanoprobes combining magnetic nanoparticles with organic dyes have attracted tremendous interest due to their promising applications in biomedical field. Here we demonstrate a facile and general strategy for the fabrication of robust fluorescent magnetic nanoprobes with high payloads of dye molecules and their use as multimodal nanoprobes for cell imaging. These nanoprobes not only effectively keep photochemical stability of dyes, but also provide a platform for grafting other functional or targeted moieties into silica surface via primary amines. Moreover, the nanoprobes are uniformly spherical morphology and can be dispersed well in aqueous solution, which are very desirable for biomedical applications. Importantly, this method can be extended to synthesize other bifunctional nanoprobes by using the dyes with isothiocyanate group. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization is a widely used technique in cell biology providing insight into the spatial organization of specific RNA transcripts in the cell nucleus. However, to further investigate the dynamics of the transcription process and the transport rates of RNAs through the nucleus, RNAs need to be visualized and tracked in the living cell. In past years, various methods have been developed with the aim of tagging specific RNAs with a fluorescent moiety without interfering with cell vitality. These methods include the delivery of probes into a living cell, the in vivo hybridization of fluorescent oligonucleotide probes to endogenous RNAs, and the microscopic imaging of the tagged RNAs in living cells. In this article, we review a number of methods for tagging and visualizing endogenous RNAs in living cells. In addition, a protocol is described that allows detection of various RNA types using fluorochrome-labeled 2′-O-methyl oligoribonucleotide (2′-OMe RNA) probes. Compared with conventional oligodeoxynucleotide probes, 2′-OMe RNA probes are not degraded by nucleases, form stable hybrids with structured RNAs, and do not interfere with cell vitality. [Copyright &y& Elsevier]

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100–300 mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons. [Copyright &y& Elsevier]

A newly synthesized compound, 5-methyl-1-phenyl-1H-1,2,3-triazole-4- carboxylic acid (MPC) was analyzed for its quantum chemical parameters and theoretical spectrum by computational chemistry. The calculated spectrum was in accord with the experimental measurements in a great degree. Then MPC was successfully designed and synthesized to a novel rhodamine B derivative RMPC. The RMPC exhibited about a 4000-fold increase in fluorescence intensity in the presence of Hg2+ ions over most other competitive metal ions. The triazole appended colorless chemodosimeter RMPC turns to pink upon the complex formation only with Hg2+ ions as a 1: 2 M ratio and enables naked-eye detection. The coordination mechanism of turning on/off fluorescence for Hg2+ ions were well proposed by explaining Hg2+ inducing the ring-opened rhodamine B moiety. The fluorescence imaging experiments of Hg2+ in HeLa cell demonstrated that the probe was labeled and it could be used in biological systems. Image 1 • The structural and photophysical properties of a new 1, 2, 3-triazole MPC were computed. • zMPC was synthesised a novel rhodamine based fluorescent chemosensors RMPC. • MPC selectively responds to Hg2+ by off-on fluorescence mechanisms with "naked-eye" detection of Hg2+. [ABSTRACT FROM AUTHOR]

Novel 4-amino-1,8-naphthalimides are demonstrated as anticancer and fluorescent imaging agents with K562 and MCF-7 cancer cells. • Compound 1 is a dual-functional aminonaphthalimide derivative with cytotoxic and fluorescent cell imaging properties. • The aminonaphthalimide derivatives showed anti-proliferative effects against human cancer cell lines K562 and MCF-7. • Phase contrast microscopy and fluorescent staining provide evidence for apoptotic cell death. Novel water-soluble 4-aminonaphthalimides were synthesised and their cellular fluorescent imaging, cytotoxicity and ability to induced apoptosis evaluated. The lead compound 1 was designed from the cross-fertilisation of the basic hydrophilic amino pharmacophore of mitoxantrone, and an aminonaphthalimide scaffold of the drug candidate, amonafide. The compounds are also fluorescent pH probes based on photoinduced electron transfer (PET) and internal charge transfer (ICT). The compounds are sensitive to solvent polarity with large Stoke shifts (>90 nm) and provide emissive-coloured solutions (blue to yellow). Excited state p K a s of 9.0–9.3 and fluorescence quantum yields of 0.47–0.58 were determined in water. The cytotoxicity and cellular fluorescent imaging properties of the compounds were tested on human cancer cell lines K562 and MCF-7 by the MTT assay, phase contrast and fluorescence microscopy. Compounds 1 and 3 with flexible aminoalkyl chains exhibited GI 50 comparable to amonafide, while 2 and 4 with a rigid piperazine moiety and butyl chain are less cytotoxic. Fluorescence microscopy with 1 allowed for the visualization of the intracellular microenvironment exemplifying the potential utility of such hybrid molecules as anticancer and fluorescent cellular imaging agents. [ABSTRACT FROM AUTHOR]