Simultaneous wide-field imaging and spectroscopy of localized fluorophores

A method that combines fluorescence imaging and spectroscopy of single molecules at room temperature is presented. This approach allows us to identify a number of imaged molecules unequivocally by simultaneously recording their fluorescence emission spectra. Furthermore, their spectral characteristics not only allow us to separate different fluorescent labels quantitatively and qualitatively but also provide information on the microenvironment of the molecules. This new method was successfully tested on a system of yellow-green and red fluorescent 20-nm latex beads, and its usefulness in studies of biological systems was illustrated for a preparation of combined binary ratio labeling-fluorescence in situ hybridization-stained mouse chromosomes.     

双光子荧光与相干反斯托克斯拉曼散射显微成像技术的实验研究

双光子荧光与相干反斯托克斯拉曼散射同属于三阶非线性效应,二者之间的差异与联系是一个值得研究的问题.本文基于自行搭建的超连续谱近红外宽带相干反斯托克斯拉曼散射显微成像系统进行光谱成像,同时通过理论与实验对比分析了双光子荧光与相干反斯托克斯拉曼散射图像存在差异的原因.结果表明,具有亚微米以上横向分辨率的相干反斯托克斯拉曼散射成像系统,可以使用较大尺寸的荧光珠进行双光子荧光成像,通过解卷积得到双光子荧光成像的系统分辨率,并将它近似等效于相干反斯托克斯拉曼散射成像系统的当下分辨率.如果需要得到相干反斯托克斯拉曼散射成像系准确的分辨率结果,就必须使用尺寸比相干反斯托克斯拉曼散射成像系统实际分辨率小的球形样品进行实验测量.     

Dual-Color Quantum Dot–Encoded Nanoprobe for DNA Assays and Cell Imaging

In this article, a novel dual-color quantum dot–encoded fluorescent nanoprobe was prepared by the reverse microemulsion method and layer-by-layer assembly method. First, red fluorescence–emitting CdTe quantum dots were encapsulated in silica nanoparticles by the reverse microemulsion method. Yellow fluorescence–emitting quantum dots were deposited on the surface of silica nanoparticles to form a dual-color quantum dot@silica beads/quantum dot nanoprobe. Then capture DNA was linked to a QSQ nanoprobe via covalent bonding. We utilized the quantum dot@silica beads/quantum dot nanoprobe to capture and detect the mutant BRAF DNA sequence through the competitive immunoassay method. The resulting quantum dot@silica beads/quantum dot nanoprobe-capture DNA conjugates showed sequence-specific hybridization with target DNA. Furthermore, a multispectral imaging system was utilized to distinguish the quantum dot optical code in the quantum dot@silica beads/quantum dot nanoprobe. The quantum dot@silica beads/quantum dot nanoprobe was used in human osteoblast-like HepG2 cell imaging. The proposed quantum dot@silica beads/quantum dot nanoprobe and decoding analysis method could be used for targeting imaging, biological assays, and early detection of cancer.     

Refocusing and locating effect of fluorescence scattering field

Optical imaging deep inside scattering medium has always been one of the challenges in the field of bioimaging, which significantly drawbacks the employment of con-focal microscopy system. Although a variety of feedback techniques, such as acoustic or nonlinear fluorescence-based schemes have realized the refocusing of the coherent light, the problems of non-invasively refocusing and locating of linearly-excited fluorescent beads inside the scattering medium have not been thoroughly explored. In this paper, we linearly excited the fluorescent beads inside a scattering medium by using our homemade optical con-focal system, collected the fluorescence scattering light as the optimized target, and established a theoretical model of target contrast enhancement, which is consistent with the experimental data. By improving both the cost function and variation rate within the genetic algorithm, we could refocus the fluorescence scattering field while improving the contrast enhancement factor to 12.8 dB. Then, the positions of the fluorescent beads are reconstructed by sub-pixel accuracy centroid localization algorithm, and the corresponding error is no more than 4.2 μ with several fluorescent beads within the field of view. Finally, the main factors such as the number of fluorescent beads, the thickness of the scattering medium, the modulating parameter, the experimental noise and the system long-term stability are analyzed and discussed in detail. This study proves the feasibility of reconstructing fluorescent labeled cells inside biological tissues, which provides certain reference value for deep imaging of biological tissues.     

Full-field optical sectioning and three-dimensional localization of fluorescent particles using focal plane modulation

We present a technique for imaging fluorescent particles based on the axial modulation of the objective's focal plane position. This technique provides full-field optical sectioning and can be used to localize the fluorophores in three dimensions. We describe the technique and apply it to image 200 nm diameter fluorescent beads immobilized in a gel. We show that full-field optical sectioning is obtained and that the beads are localized with a precision of 10 nm in the transverse plane and 14 nm in the axial direction.     

点击化学合成具有聚集诱导荧光特性的双响应性聚合物凝胶

本文合成了巯基功能化的四苯基乙烯并利用thiol-ene点击化学合成得到聚合物凝胶. 基于四苯基乙烯的聚集诱导发光特性，聚合物凝胶的合成过程可以通过四苯基乙烯荧光发射性能的变化进行监测. 此外，由于凝胶的还原和酸性环境响应性，在二硫苏糖醇和三氟乙酸存在下，凝胶的荧光发射会被猝灭，这种双重刺激响应性使其在荧光传感成像、癌症诊断和自愈合材料方面具有潜在的应用价值.     

Fiber-optic scanning two-photon fluorescence endoscope

We report on the development of a miniature, flexible, fiber-optic scanning endoscope for two-photon fluorescence imaging. The endoscope uses a tubular piezoelectric actuator for achieving two-dimensional beam scanning and a double-clad fiber for delivery of the excitation light and collection of two-photon fluorescence. Real-time imaging of fluorescent beads and cancer cells has been performed.     

Improved Fluorescent Proteins for Single-Molecule Research in Molecular Tracking and Co-Localization

Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and > 50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores. Authors contributed equally to this article.     

Sulfur doped carbon nitride quantum dots with efficient fluorescent property and their application for bioimaging

Heteroatom doping can drastically alter electronic characteristics of carbon nitride quantum dots, thus resulting in unusual properties and related applications. Herein, we used sulfur as the doping element and investigated the influence of doping on the electronic distribution of carbon nitride and the corresponding fluorescent property. A simple synthetic strategy was applied to prepare sulfur-doped carbon nitride (S-g-C₃N₄) quantum dots through ultrasonic treatment of bulk S-g-C₃N₄. Characterization results demonstrated that the prepared S-g-C₃N₄ quantum dots with an average size of 2.0 nm were successfully prepared. Fluorescent properties indicated that S-g-C₃N₄ quantum dots have an emission peak at 460 nm and cover the emission spectra region up to 550 nm. Furthermore, the fluorescent intensity is greatly increased due to the sonication of bulk S-g-C₃N₄ into quantum dots. As a result, S-g-C₃N₄ quantum dots not only show a blue cell imaging, but have a bright green color. Therefore, S-g-C₃N₄ quantum dot is a promising candidate for bioimaging benefiting from the efficient fluorescent property, good biocompatibility, and low toxicity.     

Characterization of a magnetic carrier encapsulating europium and ferrite nanoparticles for biomolecular recognition and imaging

In this study, FG beads (ferrite nanoparticles in the core covered with poly-(styrene-co-glycidyl methacrylate)) were made into fluorescent magnetic carriers (FMCs) containing the fluorescent substance, europium ion (Eu³⁺) complex. The developed FMCs showed several notable features such as high fluorescence intensity and high dispersibility in water. More importantly, FMCs did not leak Eu³⁺ complex. It is expected that the FMCs will be a useful tool for biomolecular recognition and imaging and contribute to advancement of a wide range of research fields, including cell biology and molecular imaging.     

Dual-color prism-type TIRFM system for direct detection of single-biomolecules on nanoarray biochips

This study examined the applicability of a prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) system for the simultaneous detection of nano biomolecules on nanoarray biochips at the single-molecule level. The dual-color TIRFM system with two individual laser beams and a high-sensitivity camera was used for the simultaneous dual-color detection of two different nano beads (i.e. 20 nm yellow–green and crimson fluorescent FluoSpheres beads), and single-protein molecules labeled with different fluorescent dyes (i.e. actin from rabbit muscle conjugated with Alexa Fluor^® 488 and Alexa Fluor^® 633 goat anti-rabbit IgG) without a time-delay and the need to move the sample. When this system was applied to two different single-protein molecules labeled with different fluorescent dyes on the GPTS/CHI/GA-modified glass nanoarray chip, the full images of the biomolecules at the single-molecule level were obtained simultaneously in two different colors using a Dual-View™. The dual-color TIRFM system is quite suitable for the biological imaging at the single-molecule level on nanoarray biochips. This study provides a benchmark for directly monitoring the interactions and detecting the colocalization of two different nano biomolecules, and can be applied to the development of a nanoarray biochip at the single-molecule level.     

Three-dimensional holographic fluorescence microscopy

Most commonly used methods for three-dimensional (3D) fluorescence microscopy make use of sectioning techniques that require that the object be physically scanned in a series of two-dimensional (2D) sections along the z axis. The main drawback in these approaches is the need for these sequential 2D scans. An alternative approach to fluorescence imaging in three dimensions has been developed that is based on optical scanning holography. This novel approach requires only a 2D scan to record 3D information. Holograms of 15-microm fluorescent latex beads with longitinal separation of ~2 mm have been recorded and reconstructed. To our knowledge, this is the first time holograms of fluorescent specimens have been recorded by an optical holographic technique.     

Imaging contrast effects in alginate microbeads containing trapped emulsion droplets

This study focuses on spherical microparticles made of cross-linked alginate gel and microcapsules composed of an oil-in-water emulsion where the continuous aqueous phase is cross-linked into an alginate gel matrix. We have investigated the use of these easily manufactured microbeads as contrast agents for the study of the flow properties of fluids using nuclear magnetic resonance imaging. Results demonstrate that combined spin-spin (T(2)) relaxation and diffusion contrast in proton NMR imaging can be used to distinguish among rigid polymer particles, plain alginate beads, and alginate emulsion beads. Multi-echo CPMG spin-echo imaging indicates that the average spin-lattice (T(1)) and spin-spin (T(2)) relaxation times of the plain alginate and alginate emulsion beads are comparable. Meanwhile, diffusion-weighted imaging produces sharp contrast between the two types of alginate beads, due to restricted diffusion inside the embedded oil droplets of the alginate emulsion beads. While the signal obtained from most materials is severely attenuated under applied diffusion gradients, the alginate emulsion beads maintain signal strength. The alginate emulsion beads were added to a suspension and imaged in an abrupt, annular expansion flow. The emulsion beads could be clearly distinguished from the surrounding suspending fluid and rigid polystyrene particles, through either T(2) relaxation or diffusion contrast. Such a capability allows future use of the alginate emulsion beads as tracer particles and as one particle type among many in a multimodal suspension where detailed concentration profiles or particle size separation must be quantified during flow.     

金属-介质-金属渔网超表面结构调控的荧光辐射：空间选择性激发和双增强

增强荧光辐射在生物成像、高灵敏探测、集成光源等方面都具有重要的应用价值.金属纳米颗粒的周围或者金属纳米结构的间隙都可以产生强的电磁场,相应的,这些结构附近的局域态密度也被极大地增强.虽然增强荧光辐射已经在多种金属纳米颗粒和颗粒对中被证明,但是利用金属纳米结构对荧光分子的吸收和辐射过程同时进行调制仍然是一个有挑战的问题.本文研究了金属-介质-金属超表面对荧光辐射的调控,其中局域表面等离激元(LSP)和磁等离激元(MPP)分別与于分子的吸收和辐射过程发中耦合相互作用.对于吸收过程,LSP的耦合作用使得可以通过旋转泵浦激光的偏振态来实现荧光分子的空间选择激发.此外,MPP模式的偏振依赖特性使得矩形渔网结构中的荧光分子的辐射波长和偏振态也受到调控.实验观测结果经过了时域有限差分模拟的验证.本文报道的纳米结构在光辐射器件和纳米尺度集成光源等方面都具有潜在的应用价值.     

Green and Cost Effective Synthesis of Fluorescent Carbon Quantum Dots for Dopamine Detection

Carbon quantum dots (CQDs) due to its high fluorescent output is evolving as novel sensing material and is considered as future building blocks for nano sensing devices. Hence, in this investigation we report microwave assisted preparation and multi sensing application of CQDs. The microwave derived CQDs are characterized by Dynamic Light Scattering (DLS) experiment and Fourier Infrared spectra (FTIR) to investigate the size distribution and chemical purity respectively. Fluorescent emission spectra recorded at varying pH shows varying fluorescence emission intensities. Further, emission spectra recorded at different temperatures shows that fluorescence emission of CQDs greatly depends on temperature. Therefore, we demonstrate the pH and temperature sensing characteristics of CQDs by fluorescence quenching behaviour. In addition, the interaction and sensing behaviour of CQDs for dopamine is also presented in this work with a detection limit of 0.2 mM. The steady state and time-resolved methods have been employed in fluorescence quenching methods for sensing dopamine through CQDs at room temperature. The bimolecular quenching rate constants for different concentration have been measured. The interaction between CQDs and dopamine indicates fluorescence quenching method is an elegant process for detecting dopamine through CQDs.     

Retroemission by a glass bead monolayer for high-sensitivity, long-range imaging of upconverting phosphors

We introduce a retroemitter (REM) device comprising a planar glass bead set placed on a luminescent material substrate, which converges an excitation beam into a set of foci (voxels). The in-voxel emission is collimated by the beads, and propagates upstream over the long range, unlike the out-of-voxel emission spreading in all angles. The REM signal contrast is characterized as a function of incidence and observation angles and propagation distance. REM signal contrasts of approximately 20 and 1600 were found for the organic fluorescent dye and upconverting phosphor substrates, respectively. In the latter case, nonlinear optical signal enhancement plays a role in addition to the retroemission effect. This allows centimeter-scale REM patterns to be read out at the meter-scale distance using eye-safe sub-mW/cm(2) excitation intensities.     

Time-resolved imaging of latent fingerprints with nanosecond resolution

Imaging of latent fingerprints using time-resolved (TR) method offers a broader platform to eliminate the unwanted background emission. In this paper, a novel TR imaging technique is demonstrated and implemented, which facilitates the detection of latent fingerprints with nanosecond resolution. Simulated experiments were carried out with two overlapping fingerprints treated with two fluorescent powders having different lifetimes in nanosecond range. The dependence of the fluorescence emission intensity in nanosecond resolution of TR imaging is also revealed.     

Synthesis,Spectroscopic Properties,and Biological Applications of Eight Novel Chlorinated Fluorescent Proteins-labeling Probes

Eight novel chlorinated fluorescent proteins-labeling probes with a linker and reactive group were prepared in 7 steps by the reaction of chlorinated resorcinols with 3, 6-dichloro-4-carboxyphthalic anhydride in the presence of methanesulfonic acid. Structures of target compounds and intermediates were determined via IR, MS, ¹H NMR and element analysis. The spectral properties of the chlorinated fluoresceins were studied. These fluorescent probes showed absorbance peaks at 508–536 nm and fluorescence peaks at 524–550 nm. It was found that they have absorption and emission maxima at long wavelengths and high fluorescence quantum yields. Emission spectra of chlorinated fluoresceins shifted towards long wavelength with increase in chlorine. The probes were used for fluorescence imaging of cells in order to investigate whether they can conjugate to cells. The fluorescence imaging of living cells showed that they were localized in cell nucleus. However, they were localized in cytosol of chemically fixed cells. These probes will be useful reagents for the preparation of stable fluorescent conjugates.     

Synthesis and Characterization of Pressure and Temperature Dual‐Responsive Polystyrene Microbeads

A facile approach to the synthesis of pressure and temperature dual‐responsive polystyrene (PS) microbeads with controlled sizes via dispersion polymerization is described. Three different luminophors are selected and directly introduced into the reaction system and thus incorporated into the resultant PS microbeads during polymerization. By manipulating the reaction conditions, including concentrations of the initiator and monomer, polarity of the reaction medium, and injection rate for the monomer, uniform PS microbeads with sizes ranging from 1 to 5 μm are obtained. When a light source centered at 365 nm is used to excite all the luminophors in the PS beads, three distinct and resolvable emission peaks corresponding well with the luminophors are observed. By taking advantage of their sensitive responses to both pressure and temperature, the PS beads can be utilized for quantitative measurements of these two stimulations simultaneously. The PS beads loaded with multiple luminophors have the ability to serve as building blocks for the fabrication of novel sensing and imaging devices and therefore provide a promising strategy for the study of aerodynamics.     

Imaging of Bacterial and Fungal Cells Using Fluorescent Carbon Dots Prepared from <Emphasis Type="Italic">Carica papaya</Emphasis> Juice

In this paper, we have described a simple hydrothermal method for preparation of fluorescent carbon dots (C-dots) using Carica papaya juice as a precursor. The synthesized C-dots show emission peak at 461 nm with a quantum yield of 7.0 %. The biocompatible nature of C-dots was confirmed by a cytotoxicity assay on E. coli. The C-dots were used as fluorescent probes for imaging of bacterial (Bacillus subtilis) and fungal (Aspergillus aculeatus) cells and emitted green and red colors under different excitation wavelengths, which indicates that the C-dots can be used as a promising material for cell imaging.