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Sample-scan phase contrast imaging was demonstrated by producing and coherently recombining light from a pair of axially offset focal planes. Placing a homogeneous medium in one of the two focal planes enables quantitative phase imaging using only common-path optics, recovering absolute phase without halo or oblique-illumination artifacts. Axially offset foci separated by 70 µm with a 10x objective were produced through polarization wavefront shaping using a matched pair of custom-designed microretarder arrays, compatible with retrofitting into conventional commercial microscopes. Quantitative phase imaging was achieved by two complementary approaches: i) rotation of a half wave plate, and ii) 50 kHz polarization modulation with lock-in amplification for detection.
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A single fixed optic is combined with the sample translation capabilities inherent to most microscopes to achieve precise polarization-dependent second harmonic generation microscopy measurements of thin tissue sections. Although polarization measurements have enabled detailed structural analysis of collagen, challenges in integrating rotation stages or fast electro-optic/photoelastic modulation have complicated the retrofitting of existing systems for precise polarization analysis. Placing a static microretarder array in the rear conjugate plane resulted in spatially encoded polarization modulation across the field of view. A complete set of polarization rotation measurements was acquired at each pixel by sample translation, recovering local-frame tensors relating to structure within collagenous tissue.
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A Mueller tensor mathematical framework was applied for predicting and interpreting the second harmonic generation (SHG) produced with an unpolarized fundamental beam. In deep tissue imaging through SHG and multiphoton fluorescence, partial or complete depolarization of the incident light complicates polarization analysis. The proposed framework has the distinct advantage of seamlessly merging the purely polarized theory based on the Jones or Cartesian susceptibility tensors with a more general Mueller tensor framework capable of handling partial depolarized fundamental and/or SHG produced. The predictions of the model are in excellent agreement with experimental measurements of z-cut quartz and mouse tail tendon obtained with polarized and depolarized incident light. The polarization-dependent SHG produced with unpolarized fundamental allowed determination of collagen fiber orientation in agreement with orthogonal methods based on image analysis. This method has the distinct advantage of being immune to birefringence or depolarization of the fundamental beam for structural analysis of tissues.
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Intracellular pH plays a vital role in cell biology, including signal transduction, ion transport and homeostasis. Herein, a ratiometric fluorescent silica probe was developed to detect intracellular pH values. The pH sensitive dye fluorescein isothiocyanate isomer I (FITC), emitting green fluorescence, was hybridized with reference dye rhodamine B (RB), emitting red fluorescence, as a dual-emission fluorophore, in which RB was embedded in a silica core of â¼40 nm diameter. Moreover, to prevent fluorescence resonance energy transfer between FITC and RB, FITC was grafted onto the surface of core-shell silica colloidal particles with a shell thickness of 10-12 nm. The nanoprobe exhibited dual emission bands centered at 517 and 570 nm, under single wavelength excitation of 488 nm. RB encapsulated in silica was inert to pH change and only served as reference signals for providing built-in correction to avoid environmental effects. Moreover, FITC (λem = 517 nm) showed high selectivity toward H(+) against metal ions and amino acids, leading to fluorescence variation upon pH change. Consequently, variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor. The specific nanoprobe showed good linearity with pH variation in the range of 6.0-7.8. It can be noted that the fluorescent silica probe demonstrated good water dispersibility, high stability and low cytotoxicity. Accordingly, imaging and biosensing of pH variation was successfully achieved in HeLa cells.
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Técnicas Biosensibles , Fluoresceína-5-Isotiocianato , Nanopartículas , Dióxido de Silicio , Colorantes Fluorescentes , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Espectrometría de FluorescenciaRESUMEN
It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu(2+), to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu(2+), and pH/Cu(2+) with different ratiometric fluorescent signals. First, 2,2',2â³-(2,2',2â³-nitrilotris(ethane-2,1-diyl)tris((pyridin-2-yl-methyl)azanediyl))triethanethiol (TPAASH) was designed for specific recognition of Cu(2+), as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu(2+), and pH/Cu(2+) excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu(2+) against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic-organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu(2+) and in understanding the interplaying roles of Cu(2+) and pH in live cells by ratiometric multicolor fluorescent imaging.
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Técnicas Biosensibles/métodos , Cobre/análisis , Cobre/química , Colorantes Fluorescentes/química , Imagen Molecular/métodos , Supervivencia Celular , Color , Cumarinas/química , Diseño de Fármacos , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/síntesis química , Oro/química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/química , Espectrometría de FluorescenciaRESUMEN
Nanoparticles are promising scaffolds for applications such as imaging, chemical sensors and biosensors, diagnostics, drug delivery, catalysis, energy, photonics, medicine, and more. Surface functionalization of nanoparticles introduces an additional dimension in controlling nanoparticle interfacial properties and provides an effective bridge to connect nanoparticles to biological systems. With fascinating photoluminescence properties, carbon dots (C-dots), carbon-containing nanoparticles that are attracting considerable attention as a new type of quantum dot, are becoming both an important class of imaging probes and a versatile platform for engineering multifunctional nanosensors. In order to transfer C-dots from proof-of-concept studies toward real world applications such as in vivo bioimaging and biosensing, careful design and engineering of C-dot probes is becoming increasingly important. A comprehensive knowledge of how C-dot surfaces with various properties behave is essential for engineering C-dots with useful imaging properties such as high quantum yield, stability, and low toxicity, and with desirable biosensing properties such as high selectivity, sensitivity, and accuracy. Several reviews in recent years have reported preparation methods and properties of C-dots and described their application in biosensors, catalysis, photovoltatic cells, and more. However, no one has yet systematically summarized the surface engineering of C-dots, nor the use of C-dots as fluorescent nanosensors or probes for in vivo imaging in cells, tissues, and living organisms. In this Account, we discuss the major design principles and criteria for engineering the surface functionality of C-dots for biological applications. These criteria include brightness, long-term stability, and good biocompatibility. We review recent developments in designing C-dot surfaces with various functionalities for use as nanosensors or as fluorescent probes with fascinating analytical performance, and we emphasize applications in bioimaging and biosensing in live cells, tissues, and animals. In addition, we highlight our work on the design and synthesis of a C-dot ratiometric biosensor for intracellular Cu(2+) detection, and a twophoton fluorescent probe for pH measurement in live cells and tissues. We conclude this Account by outlining future directions in engineering the functional surface of C-dots for a variety of in vivo imaging applications, including dots with combined targeting, imaging and therapeutic-delivery capabilities, or high-resolution multiplexed vascular imaging. With each application C-dots should open new horizons of multiplexed quantitative detection, high-resolution fluorescence imaging, and long-term, real-time monitoring of their target.
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Técnicas Biosensibles/métodos , Carbono/química , Ingeniería/métodos , Nanopartículas/química , Imagen Óptica/métodos , Animales , Humanos , Propiedades de SuperficieRESUMEN
In this work, we developed a new two-photon fluorescent probe, ATD (ATD = amino triphenylamine dendron), by combining a two-photon fluorophore 4-(bis(4-(4-(diphenylamino)styryl)-phenyl)amino) benzaldehyde (TD) with a specific recognition molecule for Hg(2+)- phenyl thiourea (PT) - for the determination of Hg(2+). The designed fluorescent probe emitted at â¼455 nm upon one-photon and two-photon excitation at 400 nm and 800 nm, respectively. The blue fluorescence obviously dropped with the continuous addition of Hg(2+), and demonstrated a good linearity with the concentration of Hg(2+) in a wide dynamic range of 5 nM-1.0 µM. The detection limit achieved was 0.49 nM (â¼0.2 ppb), which is much lower than the standard levels required by the U.S. Environmental Protection Agency (EPA) and World Health Organization (WHO). Furthermore, this probe featured high selectivity for Hg(2+) detection over other metal ions such as Cd(2+), Ag(+), Pd(2+), and so on, due to the specific Hg(2+) recognition by the PT molecule. Meanwhile, the probe demonstrated long-term stability with respect to pH and illumination. As a result, the developed two-photon fluorescent probe, with high sensitivity and selectivity, was successfully applied for the on-site determination of Hg(2+) in environmental water samples.
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Agua Potable/análisis , Colorantes Fluorescentes/química , Lagos/análisis , Mercurio/análisis , Espectrometría de Fluorescencia/métodos , Benzaldehídos/química , Cationes Bivalentes/análisis , Límite de Detección , Tiourea/análogos & derivadosRESUMEN
Determination of hydroxyl radical ((â¢)OH) with high sensitivity and accuracy in live cells is a challenge for evaluating the role that (â¢)OH plays in the physiological and pathological processes. In this work, a ratiometric fluorescence biosensor for (â¢)OH was developed, in which gold nanocluster (AuNC) protected by bovine serum albumin was employed as a reference fluorophore and the organic molecule 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) acted as both the response signal and specific recognition element for (â¢)OH. In the absence of (â¢)OH, only one emission peak at 637 nm ascribed to AuNCs was observed, because HPF was almost nonfluorescent. However, fluorescence emission at 515 nm attributed to the HPF product after reaction with (â¢)OH--dianionic fluorescein--gradually increased with the continuous addition of (â¢)OH, while the emission at 637 nm stays constant, resulting in a ratiometric determination of (â¢)OH. The developed fluorescent sensor exhibited high selectivity for (â¢)OH over other reactive oxygen species (ROS), reactive nitrogen species (RNS), metal ions, and other biological species, as well as high accuracy and sensitivity with low detection limit to â¼0.68 µM, which fulfills the requirements for detection of (â¢)OH in a biological system. In addition, the AuNC-based inorganic-organic probe showed long-term stability against light illumination and pH, good cell permeability, and low cytotoxicity. As a result, the present ratiometric sensor was successfully used for bioimaging and monitoring of (â¢)OH changes in live cells upon oxidative stress.
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Técnicas Biosensibles/métodos , Fluoresceínas/química , Oro/química , Radical Hidroxilo/análisis , Radical Hidroxilo/química , Nanoestructuras/química , Animales , Bovinos , Supervivencia Celular , Células HeLa , Humanos , Espectrometría de FluorescenciaRESUMEN
In this article, a ratiometric fluorescent biosensor for O2(â¢-) was developed, by employing carbon dots (C-Dots) as the reference fluorophore and hydroethidine (HE), a specific organic molecule toward O2(â¢-), playing the role as both specific recognition element and response signal. The hybrid fluorescent probe CD-HE only emitted at 525 nm is ascribed to C-Dots, while HE was almost nonfluorescent, upon excitation at 488 nm. However, after reaction with O2(â¢-), a new emission peak ascribed to the reaction products of HE and O2(â¢-) was clearly observed at 610 nm. Meanwhile, this peak gradually increased with the increasing concentration of O2(â¢-) but the emission peak at 525 nm stayed constant, leading to a ratiometric detection of O2(â¢-). The inorganic-organic fluorescent sensor exhibited high sensitivity, a broad dynamic linear range of ~5 × 10(-7)-1.4 × 10(-4) M, and low detection limit down to 100 nM. The present probe also showed high accuracy and excellent selectivity for O2(â¢-) over other reactive oxygen species (ROS), metal ions, and so on. Moreover, the C-Dot-based inorganic-organic probe demonstrated long-term stability against pH changes and continuous light illumination, good cell-permeability, and low cytotoxicity. Accordingly, the developed fluorescent biosensor was eventually applied for intracellular bioimaging and biosensing of O2(â¢-) changes upon oxidative stress.
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Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Imagen Molecular/métodos , Superóxidos/análisis , Apoptosis/efectos de los fármacos , Técnicas Biosensibles/instrumentación , Carbono , Colorantes Fluorescentes/toxicidad , Células HeLa/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Límite de Detección , Estrés Oxidativo , Fenantridinas/química , Puntos Cuánticos/química , Especies Reactivas de Oxígeno/análisis , Sensibilidad y Especificidad , Espectrometría de FluorescenciaRESUMEN
Determination of hydrogen sulfide (H2S) in live cells and tissues is still a challenge for evaluating the key roles that H2S plays in physiological and pathological processes. In this work, a "turn-on" two-photon fluorescent (TPF) sensor for H2S is developed, in which carbon nanodot (C-Dot) was employed as a two-photon fluorophore due to its large two-photon absorption cross-section (σ), and AE-TPEA-Cu(2+) complex [AE-TPEA = N-(2-aminoethyl)-N,N,N'-tris(pyridin-2-ylmethyl)ethane-1,2-diamine] was first designed as a specific receptor for H2S. The fluorescence of C-Dot conjugated with AE-TPEA (C-Dot-TPEA) was quenched upon the addition of Cu(2+). Then, the fluorescence was restored after the addition of H2S, because Cu(2+) could be released from TPEA binding site when H2S interacted with the Cu(2+) ion. The designed C-Dot-TPEA-Cu(2+) fluorescent sensor exhibited high specificity for H2S over biothiols, sulfur-containing compounds, reactive oxygen species (ROS), and other biological interferences. Meanwhile, a broad linear range from 5 µM to 100 µM was obtained and the detection limit was achieved to 0.7 µM. In addition, the C-Dot-based TPF probe exhibited bright two-photon fluorescence, favourable photostability against light illumination and pH change, and low cytotoxicity. Accordingly, the nanohybridized TPF sensor with high selectivity and sensitivity, as well as the fascinating properties of C-Dot themselves, successfully provided a new way for TPF imaging and biosensing of H2S in live cells and tissues. We believe this is the first report of TPF imaging and biosensing of H2S in live cells and tissues using a specially engineered C-Dot-based nanosystem.
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Técnicas Biosensibles , Carbono/química , Colorantes Fluorescentes/química , Sulfuro de Hidrógeno/análisis , Nanoestructuras , Animales , Apoptosis , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Microscopía Electrónica de Transmisión , Fotones , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E2Zn2SOD (ATD = amino triphenylamine dendron, QD = CdSe/ZnSe quantum dot, E2Zn2SOD = Cu-free derivative of bovine liver copper-zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu(2+) level with clear red-to-yellow color change based on specific biomolecular recognition of E2Zn2SOD for Cu(2+) ion. The inorganic-organic nanohybrided fluorescent probe features two independent emission peaks located at 515 nm for ATD and 650 nm for QDs, respectively, under two-photon excitation at 800 nm. Upon addition of Cu(2+) ions, the red fluorescence of QDs drastically quenches, while the green emission from ATD stays constant and serves as a reference signal, thus resulting in the ratiometric detection of Cu(2+) with high accuracy by two-photon microscopy (TPM). The present probe shows high sensivity, broad linear range (10(-7)-10(-3) M), low detection limit down to â¼10 nM, and excellent selectivity over other metal ions, amino acids, and other biological species. Meanwhile, a QD-based inorganic-organic probe demonstrates long-term photostability, good cell-permeability, and low cytotoxicity. As a result, the present probe can visualize Cu(2+) changes in live cells by TPM. To the best of our knowledge, this is the first report for the development of a QD-based two-photon ratiometric fluorescence probe suitable for detection of Cu(2+) in live cells.
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Técnicas Biosensibles/métodos , Cobre/metabolismo , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Aminas/química , Animales , Bovinos , Supervivencia Celular , Colorantes Fluorescentes/química , Células HeLa , Humanos , Puntos Cuánticos , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismoRESUMEN
The depth of field (DoF) was extended 2.8-fold to achieve rapid crystal screening by retrofitting a custom-designed micro-retarder array (µRA) in the optical beam path of a nonlinear optical microscope. The merits of the proposed strategy for DoF enhancement were assessed in applications of second-harmonic generation imaging of protein crystals. It was found that DoF extension increased the number of crystals detected while simultaneously reducing the number of `z-slices' required for screening. Experimental measurements of the wavelength-dependence of the extended DoF were in excellent agreement with theoretical predictions. These results provide a simple and broadly applicable approach to increase the throughput of existing nonlinear optical imaging methods for protein crystal screening.
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Imagen Óptica/métodos , Proteínas/químicaRESUMEN
The dysregulated pH is working as a mark of cancer. It is a challenge for developing a biosensor for targeted imaging in cancer cells and monitoring of intracellular pH. Here, a ratiometric fluorescence biosensor for pH determination was developed with targeted imaging into folate acceptor (FR)-rich cancer cells at the same time. AuNCs protected by bovine serum albumin (BSA) worked as reference fluorophore and fluorescein-isothiocyanate (FITC) acted as the response signal for pH. For targeted imaging of cancer cells, the AuNCs were simultaneously conjugated with folic acid (FA). The developed ratiometric biosensor can monitor pH with a wide linear range from 6.0-7.8 with a pKa at 6.84. Under every different pH condition, the probe showed high selectivity over various metal ions and amino acids with its fluorescence ratio stayed almost constant (<5%). It also showed good cyclic accuracy when pH switched between 6.0 and 8.0, as well as low cytotoxicity. The AuNC-based inorganic-organic nanohybrid biosensor showed good cell-permeability, low cytotoxicity, and long-term photostability. Accordingly, the pH biosensor was employed to gain targeted imaging in FR(+ve) Hela cells with FR(-ve) lung carcinoma cells A549 as comparison, and achieved to monitor the pH changes in Hela cells.
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Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Receptores de Folato Anclados a GPI/análisis , Oro/química , Nanopartículas del Metal/química , Neoplasias/diagnóstico por imagen , Imagen Óptica/métodos , Células A549 , Fluoresceína-5-Isotiocianato/química , Ácido Fólico/química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/ultraestructura , Espectrometría de Fluorescencia/métodosRESUMEN
Extracellular pH plays a vital role no matter in physiological or pathological studies. In this work, a hydrogel, CD@Nile-FITC@Gel (Gel sensor), entrapping the ratiometric fluorescent probe CD@Nile-FITC was developed. The Gel sensor was successfully used for real-time extracellular pH monitoring. In the case of CD@Nile-FITC, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was chosen as the response signal for H(+) and Nile blue chloride (Nile) as the reference signal. The developed fluorescent probe exhibited high selectivity for pH over other metal ions and amino acids. Meanwhile, the carbon-dots-based inorganic-organic probe demonstrated excellent photostability against long-term light illumination. In order to study the extracellular pH change in processes of cell proliferation and metabolism, CD@Nile-FITC probe was entrapped in sodium alginate gel and consequently formed CD@Nile-FITC@Gel. The MTT assay showed low cytotoxicity of the Gel and the pH titration indicated that it could monitor the pH fluctuations linearly and rapidly within the pH range of 6.0-9.0, which is valuable for physiological pH determination. As expected, the real-time bioimaging of the probe was successfully achieved.
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Rastreo Celular/métodos , Colorantes Fluorescentes/química , Espectrometría de Fluorescencia/métodos , Fracciones Subcelulares/fisiología , Fracciones Subcelulares/ultraestructura , Proliferación Celular/fisiología , Fluoresceína-5-Isotiocianato , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Oxazinas/química , Puntos Cuánticos , Fracciones Subcelulares/químicaRESUMEN
Development of sensitive and selective probes for cupric ions (Cu(2+)) at cell and tissue level is a challenging work for progress in understanding the biological effects of Cu(2+). Here, we report a ratiometric two-photon probe for Cu(2+) based on the organic-inorganic hybrids of graphene quantum dots (GQDs) and Nile Blue dye. Meanwhile, Cu-free derivative of copper-zinc superoxide dismutase (SOD) - E2Zn2SOD is designed as the unique receptor for Cu(2+) and conjugated on the surface of GQDs. This probe shows a blue-to-yellow color change in repose to Cu(2+), good selectivity, low cytotoxicity, long-term photostability, and insensitivity to pH over the biologically relevant pH range. The developed probe allows the direct visualization of Cu(2+) levels in live cells as well as in deep-tissues at 90-180 µm depth through the use of two-photon microscopy. Furthermore, the effect of ascorbic acid is also evaluated on intracellular Cu(2+) binding to E2Zn2SOD by this probe.
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Cationes/química , Cobre/química , Colorantes Fluorescentes/química , Imagen Óptica , Fotones , Línea Celular Tumoral , Grafito/química , Humanos , Imagen Óptica/métodos , Puntos Cuánticos/químicaRESUMEN
A carbon dot (C-Dot)-based two-photon fluorescent probe has been developed for the monitoring of pH changes across a broad range with high sensitivity and selectivity. The inorganic-organic probe also shows good biocompatibility and cell permeability, and thus can be successfully applied in bioimaging and biosensing of physiological pH in living cells, as well as living tissues at a depth of 65-185 µm.