单分子定位超分辨显微成像有机荧光探针的研究进展

在生物医学领域,对纳米尺寸级别的微小生物目标进行精确定位研究具有非常重要的意义,而光学显微成像技术为此提供了强有力的工具。 光学显微成像技术受到光学衍射极限的限制,难以分辨尺寸在衍射极限(<200 nm)以下的生物结构,无法直接获取微小生物结构信息,阻碍了生物医学的进一步发展。 近年来,随着纳米分辨显微成像技术的出现,新型荧光探针的开发、成像系统与设备的不断发展及成像算法不断完善地深入结合,促进了光学衍射极限以下尺寸微观目标的研究。 基于单分子定位的超分辨荧光显微成像(SMLM)包括光激活定位成像(PALM)与随机光学重构超分辨成像(STORM),将有机荧光探针与超分辨光学显微成像技术紧密结合在一起,荧光探针的光物理性质直接决定着超分辨成像结果的好坏。 因此,设计不同性能的荧光探针可以实现超精细结构的不同超分辨成像,为研究其生物学功能提供了有力的工具。 本文着重围绕基于SMLM的原理、有机荧光探针的设计要求、用于SMLM的荧光探针种类及其生物应用等方面进行总结综述,指出了单分子定位成像上存在的不足,并对其发展方向进行了展望,希望为对超分辨成像研究感兴趣或初涉该领域的研究者提供成像理论与探针设计方面的帮助。     

超分辨荧光蛋白开发研究获进展

<正>绿色荧光蛋白(GFP)的发明因其能够提供对于活细胞和活体动物的靶向基因修饰标记而获得2008年诺贝尔化学奖。进一步,由基因改造的光激活荧光蛋白(PA-FP)能够提供单分子特性,而实现了超分辨显微,使得这一技术获得2014年诺贝尔化学奖。随后,超分辨的发展向着活细胞动态超高时空分     

受激辐射耗尽超分辨显微成像的研究进展及应用

荧光显微镜凭借其非接触、无损伤、可实时探测生物样品内部等优点,成为生命科学研究中不可或缺的工具,但是Abbe光学衍射极限的存在限制了其对亚细胞尺度生化过程的进一步研究。作为突破光学衍射极限的远场光学显微镜,受激辐射耗尽(Stimulated Emission Depletion,STED)超分辨荧光显微术,因其超高时空分辨率和三维层析能力,成为备受关注的新型成像和分析表征工具。本文结合我们课题组在STED显微成像研究方面的工作,综述了近年来STED显微成像技术的研究进展及其在细胞成像中的应用。     

三维超分辨荧光和形貌显微镜联用系统的研制

搭建了一种联用超分辨荧光和三维形貌显微成像系统(Correlative super-resolution fluorescence and three-dimensional topography imaging microscopy),以聚合纤维状肌动蛋白(F-actin)为例,评价了此联用系统的性能。实验结果表明,此联用仪器具有纳米级分辨率的三维形貌及具体成分的定位分布成像功能,可实现在形貌图中定位具体成分的功能,表明此联用仪器可应用于研究分析细胞骨架及细胞亚结构(如细胞膜蛋白质聚集体的组成和分布特性等)方面,此联用系统在生命科学和材料科学等领域具有广泛的应用前景。     

荧光/化学发光探针成像检测超氧阴离子自由基的研究进展

超氧阴离子自由基(O·-2)是细胞内氧气单电子还原后最先产生的一类含氧的高活性物种(活性氧,ROS),与生命过程息息相关.正常稳态浓度的O·-2起重要的信号调控作用,包括细胞的增殖、分化、自噬等.但O·-2浓度的异常,又与癌症、神经退行性疾病、糖尿病等多种疾病的发生发展密切相关.因此,监测O·-2浓度的变化对揭示相关疾病的机理具有至关重要作用.由于荧光成像检测方法具有诸多优势,发展高灵敏、高选择性检测O·-2的荧光探针成为揭示相关疾病发生发展分子机制的关键切入点.近年来,随着荧光显微技术的发展,研究者开发了多种荧光/化学发光探针,实现了对细胞及活体内O·-2水平的可视化监测.本文综述了近五年用于检测O·-2的分子探针、纳米探针、蛋白探针以及化学发光探针的研究进展,并对其发展前景进行了展望.     

基于散射信号的超分辨光学相减显微镜

现有的光学超分辨显微成像技术主要依赖于特殊的荧光标记物,其对于大多数非荧光样品的超分辨成像就变得无能为力。因此我们提出将光学相减显微技术应用到非荧光样品的成像当中,利用普通共聚焦光斑和面包圈型光斑分别激发样品的散射光成像,从而得到样品同一区域的两幅图像,再通过图像相减的方法提高了图像空间分辨率。不同于一般的超分辨成像方法,这种光学相减显微镜不需要特殊的样品预处理过程,同时两次成像的激发光强度可以保持在一个较低水平,避免了样品损伤的影响。随后金纳米小球和有机聚合物微丝的散射成像实验证明了光学相减显微镜可以将空间分辨率提高到215 nm (0.33λ, 1λ = 650 nm),并且通过探测散射信号得到更多的样品细节信息。     

超小金纳米簇用于荧光及CT双模态成像的研究

基于金纳米簇强烈的量子限制效应（strong quantum confinement effect,SQCE）,采用一步合成法,制备了同时具有高效近红外荧光与CT双模态成像能力的超小金纳米簇.实验表明,通过优化合成比例以及合成条件,所合成的超小金纳米簇具有很大的斯托克斯（Stokes）位移、较高的荧光强度和X射线吸收效率.除此之外,该超小金纳米簇具有良好的单分散性、稳定性和生物相容性.4T1肿瘤细胞荧光成像实验结果表明,该纳米粒子可被肿瘤细胞快速、高效地摄取.     

时间分辨荧光技术与荧光寿命测量

介绍了时间分辨荧光技术与荧光寿命的测量方法、意义及应用。     

纳米荧光探针用于核酸分子的检测及成像研究

核酸,包括脱氧核糖核酸和核糖核酸,在生物的生长、发育、突变、炎症、癌症等正常或异常的生命活动中发挥着重要的作用,它们的异常表达与多种疾病的发生、发展也密切相关.因此,发展准确、有效的方法实现核酸分子的检测,对深入探究核酸的功能调控以及相关疾病的早期检测与治疗都具有重要的意义.荧光检测法与荧光成像技术具有灵敏度高、时空分辨率高等优点,为实时、准确的检测核酸分子提供了有力的工具.本文着重综述了近年来发展的纳米荧光探针用于疾病相关核酸分子的检测与细胞和活体成像工作的研究进展,最后提出了进一步构建新型纳米荧光探针用于核酸检测面临的挑战、未来发展方向与展望.     

空间分辨荧光分析技术

空间分辨荧光分析技术突破了传统荧光分析的局限,为获得空间定位信息提供了技术保障。系统地综述了构成该技术的共焦荧光法、全内反射荧光法、多光子荧光法以及近场荧光法等4种方法的原理、特点、发展及其应用,并且强调了其在单分子测定中的作用。引用文献64篇。     

Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.     

Submicron hard X-ray fluorescence imaging of synthetic elements

Synchrotron-based X-ray fluorescence microscopy (XFM) using hard X-rays focused into sub-micron spots is a powerful technique for elemental quantification and mapping, as well as microspectroscopic measurements such as μ-XANES (X-ray absorption near edge structure). We have used XFM to image and simultaneously quantify the transuranic element plutonium at the L₃ or L₂-edge as well as Th and lighter biologically essential elements in individual rat pheochromocytoma (PC12) cells after exposure to the long-lived plutonium isotope ²⁴²Pu. Elemental maps demonstrate that plutonium localizes principally in the cytoplasm of the cells and avoids the cell nucleus, which is marked by the highest concentrations of phosphorus and zinc, under the conditions of our experiments. The minimum detection limit under typical acquisition conditions with an incident X-ray energy of 18 keV for an average 202 μm² cell is 1.4 fg Pu or 2.9 × 10⁻²⁰ moles Pu μm⁻², which is similar to the detection limit of K-edge XFM of transition metals at 10 keV. Copper electron microscopy grids were used to avoid interference from gold X-ray emissions, but traces of strontium present in naturally occurring calcium can still interfere with plutonium detection using its L_α X-ray emission.     

可视化分析方法在薄膜基材料荧光传感中的应用

随着光学成像技术的不断突破,荧光可视化已经从简单的肉眼观察逐步向宽场显微、共聚焦显微、超分辨成像等方向发展.然而,荧光可视化在薄膜基材料中的传感应用依然以肉眼观察以及少量的宽场显微为主要分析手段.同时,薄膜基材料结构和性质的可视化分析研究也滞后于荧光可视化技术的发展.基于此,结合本课题组近几年的研究成果,本文系统评述了荧光共聚焦显微技术在薄膜基材料体相分散状态和表面性质的可视化分析中的应用进展,并对当前薄膜基荧光传感材料面临的问题和可能的解决方案进行了简要探讨.     

Multifunctional nanoparticles possessing magnetic, long-lived fluorescence and bio-affinity properties for time-resolved fluorescence cell imaging

Multifunctional nanoparticles possessing magnetic, long-lived fluorescence and bio-affinity properties have been prepared by copolymerization of a conjugate of (3-aminopropyl)triethoxysilane bound to a fluorescent Eu³⁺ complex, 4,4′-bis(1″,1″,1″-trifluoro- 2″,4″-butanedion-4″-yl)chlorosulfo-o-terphenyl-Eu³⁺ (APS-BTBCT-Eu³⁺), free (3-aminopropyl)triethoxysilane (APS) and tetraethyl orthosilicate (TEOS) in the presence of poly(vinylpyrrolidone) (PVP) stabilized magnetic Fe₃O₄ nanoparticles (10 nm) with aqueous ammonia in ethanol. The nanoparticles were characterized by transmission electron microscopy (TEM), spectrofluorometry and vibrating sample magnetometry methods. The direct-introduced amino groups on the nanoparticle's surface by using free APS in nanoparticle preparation facilitated the surface modification and bioconjugation of the nanoparticles. The nanoparticle-labeled transferrin was prepared and used for staining the cultured Hela cells. A time-resolved fluorescence imaging technique that can fully eliminate the fast-decaying background noises was developed and used for the fluorescence imaging detection of the cells. A distinct image with the high ratio of signal to noise (S/N) was obtained.     

In vivo fluorescence imaging using two excitation and/or emission wavelengths for image contrast enhancement

Steady-state fluorescence imaging can be used in conjunction with selective exogenous or endogenous fluorescent compounds for the diagnosis of skin lesions, for example cancer. Depending on the excitation and emission properties of the fluorescent compound used, various excitation and/or emission wavelengths can be chosen in order to allow fluorescence imaging. Unwanted background signals such as autofluorescence and scattering can decrease the image quality and, hence, the diagnosis potential of this imaging method. We have used an inexpensive dual excitation and/or emission wavelength approach in order to suppress the unwanted background signal and allow contrast enhanced fluorescence imaging. One excitation and/or emission wavelength is at the corresponding maximum of the fluorescent compound, while the second is at a nearby excitation/emission minimum. The first image contains the emission from the fluorescent compound used combined with the signal from the unwanted background. The second image provides an image of just the unwanted background signal. The difference of both images taken, thus gives a contrast enhanced image of the skin lesion. The method relies on the assumption that the background signal does not change significantly due to the small changes in wavelength for excitation or emission. Image ratio methods have already been applied towards diagnosis of basal cell carcinomas after administration of aminolevulinic acid-induced protoporphyin IX. In this study, we describe in vivo measurements in mice where the second image, usually the background signal only, contains new unwanted image data. This simple method can successfully resolve the desired image, thus demonstrating the versatility of the image processing procedure.     

A water‐soluble,AIE‐active polyelectrolyte for conventional and fluorescence lifetime imaging of mouse neuroblastoma neuro‐2A cells

A new conjugated polyelectrolyte containing tetraphenylethene units in the backbone is synthesized and characterized. This polyelectrolyte is water‐soluble and exhibits aggregation‐induced emission (AIE) behavior. It is biocompatible and can be directly used in conventional and fluorescence lifetime imaging of mouse neuroblastoma neuro‐2A cells, providing useful information of cellular morphology and intracellular aggregation or motion. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 672–680     

The art of fluorescence imaging with chemical sensors

Fluorescence imaging techniques involving chemical sensors are essential tools in many fields of science and technology because they enable the visualization of parameters which exhibit no intrinsic color or fluorescence, for example, oxygen, pH value, CO(2), H(2)O(2), Ca(2+), or temperature, to name just a few. This Review aims to highlight the state of the art of fluorescence sensing and imaging, starting from a comprehensive overview of the basic functional principles of fluorescent probes (or indicators) and the design of sensor materials. The focus is directed towards the progress made in the development of multiple sensors and methods for their signal read out. Imaging methods involving optical sensors are applied in quite diverse scientific areas, such as medical research, aerodynamics, and marine research.     

Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.     

SCOTfluors: Small,Conjugatable, Orthogonal,and Tunable Fluorophores for In Vivo Imaging of Cell Metabolism

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small‐sized multi‐colored fluorophores for real‐time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.     

Time-resolved fluorescence spectroscopy and imaging of proteinaceous binders used in paintings

The differentiation of proteins commonly found as binding media in paintings is presented based on spectrally resolved and time-resolved laser-induced fluorescence (LIF) and total emission spectroscopy. Proteins from eggs and animal glue were analysed with pulsed laser excitation at 248 nm (KrF excimer) and 355 nm (third harmonic of Nd:YAG) for spectrally resolved measurements, and at 337 nm (N₂) and 405 nm (N₂ pumped dye laser) for spectrally resolved lifetime measurements and fluorescence lifetime imaging (FLIM). Total emission spectra of binding media are used for the interpretation of LIF spectra. Time-resolved techniques become decisive with excitation at longer wavelengths as fluorescence lifetime permits the discrimination amongst binding media, despite minimal spectral differences; spectrally resolved measurements of fluorescence lifetime have maximum differences between the binding media examined using excitation at 337 nm, with maximum observed fluorescence at 410 nm. FLIM, which measures the average lifetime of the emissions detected, can also differentiate between media, is non-invasive and is potentially advantageous for the analysis of paintings. Figure The fluorescence of solid ox glue extracted from collagen can be visualised in this Total Fluorescence Spectrum; three different peaks from multiple fluorophores are present and allow the discrimination between collagen- and non-collagen proteinaceous binding media found in paintings