Two-color far-field fluorescence nanoscopy based on photoswitchable emitters

We demonstrate two-color far-field fluorescence microscopy with nanoscale spatial resolution based on the photoswitching of individual fluorescent markers. By enabling, recording, and disabling the emission of the reversibly switchable fluorescent protein rsFastLime and of the organic fluorophore cyanine5, we recorded two-color nanoscale images inside whole cells. The position of individual emitters was determined with a typical accuracy of 20 nm, which largely constitutes the lateral resolution of the system. Photoswitching in two-color colocalization experiments represents a major step towards the application of far-field fluorescence nanoscopy to the study of (biological) samples on the macromolecular level. PACS 33.50.Dq; 42.30.-d; 87.64.-t     

One-pot synthesis of carbon dots co-doped with N and S: high quantum yield governed by molecular state and fluorescence detection of Ag+

Fluorescent carbon-based nanoparticles, called chronically as carbon dots (CDs), were synthesised from citric acid (CA) and 2-Aminothiophenol (2AT) via an N and S co-doped hydrothermal method. After a series of micro-structural characterisation, N and S elements could be sufficiently doped by means of the heteroatom in the CDs solution. The as-prepared CDs solution showed blue colour fluorescence with the highest QY of 78.6%, and study on the UV–visible and PL spectra further revealed that the outstanding fluorescence of as-prepared CDs mainly originates from the generated molecular fluorophores instead of the surface state. Owing to the strong fluorescence, the as-prepared CDs can be used as a sensing probe for the detection of Ag⁺ with high sensitivity and selectivity. However, the changes of fluorescence intensity exhibited the complex nature of the quenching mechanism due to the –SH and –NH₂ groups on the fringes of carbonaceous cores or molecular fluorophores to aggregate into another fluorescent cores with the assistance of Ag⁺ ions, which promises a new approach for efficient detection of Ag⁺ for the application in industrial pollutants.

This figure shows citric acid (CA) and 2-Aminothiophenol (2AT) via an N and S co-doped hydrothermal method to prepare CDs with blue colour fluorescence and the highest QY of 78.6%. Owing to the excellent fluorescence, the as-prepared CDs can be used as a sensing probe for the detection of Ag⁺ with high sensitivity and selectivity, and the changes of fluorescence intensity exhibited the complex nature of the quenching mechanism due to the –SH and –NH₂ groups on the fringes of carbonaceous cores or molecular fluorophores to aggregate into another fluorescent cores with the assistance of Ag⁺ ions, which promises a new approach for efficient detection of Ag⁺ for the application in industrial pollutants.     

Hydrophilic Labeling Reagents of Dipyrrylmethene-BF2 Dyes for Two-Photon Excited Fluorometry: Syntheses and Photophysical Characterization

Recently introduced bioaffinity assay technology, ArcDia TPX, is based on two-photon excited fluorescence (TPE) and it enables separation-free ultra-sensitive immunoassays from microvolumes. Here we present syntheses of novel two-photon excitable fluorescent labeling reagents which have been specially designed to be used as label molecules in the ArcDia TPX assay technique. The labeling reagents are based on dipyrrylmetheneboron difluoride (dipyrrylmethene-BF2) chromophore, which have been substituted with aryl, heteroaryl or arylalkenyl chemical groups to extend the pi-electron conjugation. These substitutions results in a series of dipyrrylmethene-BF2 fluorophores with different photophysical properties. Dipyrrylmethene-BF2 fluorophores have been further substituted with a dipeptide linker unit and finally activated as succinimidyl esters to enable specific coupling with primary amino groups. The dipeptide linker serves as a spacer arm between the label and a target, and enhances the solubility of the label in aqueous solutions. Study of the chemical and photophysical performance of the new labeling reagents is described. The new labeling reagents exhibit high fluorescence quantum yields, and molar absorption coefficients. The results show that the new labels with the hydrophilic dipeptide linker unit provide large two-photon excitation cross-sections, high fluorescence quantum efficiency and good solubility in aqueous solutions. The results suggest that the novel dipyrrylmethene-BF2 labels are highly applicable to bioaffinity assays based on two-photon excitation of fluorescence.     

Advances in fluorescence diagnosis to track footprints of cancer progression in vivo

Fluorescence spectroscopy and imaging have been widely used for in vivo cancer diagnosis and therapy monitoring in preclinical models, as well as clinical translation. Great attempts have been made to develop novel fluorescence techniques and improve on existing ones, which can now be used in conjunction with newly developed fluorescent probes for specific cancer imaging. In this review, a broad overview of fluorescence techniques is provided, including photodynamic diagnosis, laser confocal endomicroscopy and fluorescence lifetime imaging, coupled with endogenous and exogenous fluorophores. In particular, endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), are highlighted as they are linked to cellular metabolism in precancer growth. The use of near‐infrared dyes, such as indocynanine green (ICG), for imaging deep‐tissue regions is also reviewed. In addition, diagnostic algorithms used for tissue classification and cancer detection will be discussed. Lastly, emerging technologies in fluorescence diagnosis will also be included.     

多色双光子激发荧光显微技术实验研究

双光子激发荧光(two-photon excited fluorescence, TPEF)显微是一种非线性光学显微技术, 具有高的时间分辨率和空间分辨率、高的信噪比和固有的三维层析分辨能力等优点. 传统的TPEF显微一般采用波长可调谐的超短脉冲激光器作为光源. 在实际应用中, 利用TPEF显微技术研究含有多种荧光团或未知成分的待测样品, 往往需要多次改变激发光的波长以获得对各种荧光团的最佳激发. 为了同时获取不同荧光团的荧光信号, 利用超连续谱激光光源实现了多色TPEF显微成像, 实验中无需调节波长, 能够同时获得具有两种不同发射波长的荧光标记的铃兰根茎切片样品的TPEF图像. 实验结果表明, 与传统的TPEF显微相比, 该方法能够同时获取含有多种荧光团的待测样品的高对比度TPEF图像, 具有系统结构简单、操作简便、信息量大等优点, 在生物医学和材料科学等领域具有广阔的应用前景.     

Obviously Enhanced Fluorescent Signal of Core-Shell Nanostructures through Simultaneous Regulation of Spectral Overlap and Shell Thickness for Imaging and Photothermal Therapy of Ovarian Cancer Cells

In this research, by simultaneously regulating the two major factors affecting the plasmonic enhanced fluorescence (PEF), spectral overlap and the distance between the fluororophores and the noble metal nanoparticles, a significantly enhanced fluorescent signal is achieved. Core-shell nanostructures composed of aspect ratio (AR) adjustable gold nanorods (GNRs) and various thickness of SiO₂ are prepared and the decorated fluorophores are realized optimized PEF. A typical stimuli-responsive conjugated polymer, polydiacetylene (PDA), and a near-infrared (NIR) dye Cy5.5 are selected as fluorophores and their fluorescent signal are enhanced 7.26 and 4.41 times, respectively. Based on the optimized optical properties, a multifunctional antibody modified Mab-Cy5.5-GNRs@SiO₂ is successfully demonstrated the targeting, imaging, and photothermal therapy (PTT) effects on SKOV-3 ovarian cancer cells.     

生物芯片检测系统中荧光信号强度及系统灵敏度分析

从生物芯片荧光信号检测系统结构出发,分析了荧光信号强度与激发光光学系统特性,荧光收集光学系统特性及构成系统的各元件的参量间的关系.建立了荧光信号强度与检测系统中各参量间的表达式,以及对系统灵敏度进行了分析.建立了系统最少可检测的荧光分子数与各参量的函数关系,并对所设计的悬浮生物芯片检测装置的系统灵敏度进行了估算,其灵敏度在曝光时间为1 s时可达7.9个荧光分子/μm2.     

Syntheses of Novel Dipyrrylmethene-BF2 Dyes and Their Performance as Labels in Two-Photon Excited Fluoroimmunoassay

Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications.     

Localized donor-acceptor pairs of fluorophores: Determination of the energy transfer rate by nonlinear fluorimetry

A laser fluorescence method has been developed for determining the energy transfer rate. It is based on the model of collective states of localized donor-acceptor pairs of fluorophores proposed in our previous work and involves nonlinear fluorimetry (other photophysical parameters of the donor and acceptor are simultaneously determined). The proposed approach has been tested on mRFP1 fluorescent (red) protein.     

Development of fluorescent probes for bioimaging applications

Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.     

人体血清中胆红素荧光光谱初步分析

利用分子荧光和紫外-可见吸收光谱仪分别测定了在模拟生理条件下的胆红素、胆红素和牛血清白蛋白混合溶液的荧光光谱和吸收光谱,同时也测定了一些体检者血清样品的荧光发射光谱。实验结果表明:不同情况下的胆红素溶液具有不同的荧光光谱和吸收光谱;血清白蛋白对胆红素荧光具有增敏效应。此外,初步认为所测得的人体血清样品位于约524 nm处的荧光不是主要来自于人体血清中白蛋白结合形式的胆红素组分贡献。     

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Fluorescein,Quinine Sulfate and Green Fluorescent Protein

It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10^??3 ??10^??5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-photon excitation and lifetime data was obtained in the time-domain using time-correlated single-photon counting.     

Measurement of surface concentration of fluorophores by fluorescence fluctuation spectroscopy

Fluorescence fluctuation spectroscopy is applied to study molecules passing through a small observation volume, usually subjected to diffusive or convective motion in a liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or embedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres) and DNA biochips.     

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.     

Application and New Developments in Fluorescence Spectroscopic Techniques in Studying Individual Molecules

Abstract

Techniques based on fluorescence have played a variety of roles in chemistry, physics, spectroscopy, medicine, nanotechnology, and biotechnology due to their high selectivity, sensitivity, simplicity, and fastness in spectroscopic and imaging measurements. While detecting fluorescence from individual molecules by fluorescence‐based techniques, poor signal, limited lifespan of fluorophores, trade‐off between time resolution, and the level of detail of information were few major concerns. Ultrasensitive detectors permit the combination of the high time resolution of single photon counting devices with the large field of view and spectral resolution allowed by two‐dimensional detectors. Photobleaching and on‐off blinking of fluorophores can be improved dramatically by chemical modifications or changing the reagents. New ways of controlling local fields such as optic, electric, magnetic, chemical, or biochemical environments take advantage of the noninvasiveness and high temporal and spatial resolution of single‐molecule fluorescence (SMF) to get a direct feedback of events at the nanometer scale in various domains of research. Some of the applications and new developments in fluorescence spectroscopic techniques in detecting, investigating, and/or manipulating individual molecules have been discussed.     

Development of a Fluorescent Enzyme-Linked DNA Aptamer-Magnetic Bead Sandwich Assay and Portable Fluorometer for Sensitive and Rapid Leishmania Detection in Sandflies

A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (～100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.     

Confocal fluorescence polarization microscopy for linear unmixing of spectrally similar labels

Studies of biological samples often call for simultaneous identification of multiple molecular or structural components. Multiple labelling fluorescence techniques are a powerful way of achieving this. However, the ability to distinguish a number of fluorescent probes unambiguously can be restricted by the fact that fluorescence spectra are generally broad and overlapping. Recently a technique known as linear unmixing has been combined with spectral imaging to discriminate between multiple fluorophores. In this study a scheme is proposed whereby fluorescence polarization information is used to expand the capability of the linear unmixing technique to accommodate additional fluorescent probes. As a proof-of-concept, it is shown that this polarization-based technique can be used to divide the signals generated by two spectrally similar fluorescent probes into their separate components.     

Double Labeling and Simultaneous Detection of B- and T Cells Using Fluorescent Nano-Crystal (q-dots) in Paraffin-Embedded Tissues

A double immunohistochemical technique for the simultaneous detection of T- and B cells in paraffin-embedded mice tissues have been developed. This procedure is based on using fluorescent nano-crystals (q-dots). The benefit of using q-dots evolves from their unique fluorescence characteristics advantages: such as broad excitation spectrum, narrow emission band and high photo-bleaching threshold compare to organic fluorophores. T cells antigens (CD3) were stained using antibody-coated q-dots with max emission at 655 nm (GαRb-QD655). B cells antigens (CD45R/B220) were stained using streptavidin-coated q-dots with max emission at 585 nm (SA-QD585). The simultaneous detection of T- and B cells was demonstrated in paraffin-embedded lymph node using standard fluorescence microscope.     

Single-molecule detection using continuous wave excitation of two-photon fluorescence

Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.     

丹酰氯SiO2纳米发光标记物的制备

通过合成丹酰氯的荧光单体硅酯前驱物,采用油包水的反相微乳液法,以丹酰氯的荧光单体硅酯前驱物为核材料,成功地制备了丹酰氯的荧光纳米颗粒,克服了传统方法制备核壳荧光纳米颗粒中存在的荧光染料泄漏问题.通过透射电子显微镜表征该纳米粒子呈球形,大小均匀,直径为40nm左右.所制得纳米颗粒荧光性质稳定,受外界环境的影响小,且潜在生物亲和性,是一种新型的荧光标记物.