光学显微镜技术在活细胞单分子检测中的应用

单分子检测是一门以高度的时间以及空间分辨率研究生物单分子的技术。近来,科学技术的探索发展使我们可以观察、检测甚至操纵单个分子并且研究它们的构象变化和动力学行为。这一发展使得以前被传统系综研究体系平均化所隐藏的新信息被揭示出来。单分子检测技术的发展已经揭开了生命科学研究的新篇章。在本文中,我们将介绍有关活细胞中单分子检测技术的发展以及活细胞内单分子检测的现状。     

荧光单分子检测技术在生命科学中的应用

荧光单分子检测技术是用荧光标记来显示和追踪单个分子的构象变化、动力学,单分子之间的相互作用以及单分子操纵的研究。过去对于生命科学分子机制的研究,都是对分子群体进行研究,然后平均化来进行单分子估测。因此,单个分子的动态性和独立性也被平均化掉而无法表现出来。荧光单分子检测技术真正实现了对单个分子的实时观测,将过去被平均化并隐藏在群体测量中不能获得的信息显示出来。近几年来,荧光单分子检测技术的飞速发展,为生命科学的发展,开辟了全新的研究领域。现就荧光单分子检测技术在研究动力蛋白、DNA转录、酶反应、蛋白质动态性和细胞信号转导方面的应用进展作一综述。     

表观遗传学研究方法进展

表观遗传调控是基因表达调控的重要组成部分,已成为当前研究的热点.目前其研究主要集中在DNA甲基化和组蛋白修饰.针对这两种表观修饰,其研究方法也取得了较太进展,一方面方法的是敏度和特异性都在不断提高;另一方面表现修饰的检测正在逐步从定性检测向定量分析方向发展,从个别位点向高通量检测发展.此外,新一代测序技术的应用特大大推动表观遗传研究的发展,包括单分子实时测序法、单分子纳米孔科序法等.综述目前常用的DNA甲基化、组蛋白修饰研究方法以及最新的单分子测序技术,并对它们在表观遗传修饰检测中的应用作了简要对比分析.     

生物单分子研究的进展

大多数分子生物学实验都代表一种集体平均的测量,所记录的都是复杂系统中大量分子的行为,随着技术的发展,目前已能够对单分子观察、探测、操纵并研究其动态与构象变化。从单分子研究可得到新的信息,这些信息隐藏在集体测量之中,或已被平均掉。这一领域代表着本世纪结构生物学的一个新方向。     

单分子荧光共振能量转移技术

单分子荧光共振能量转移技术（single molecule fluorescence resonance energy transfer,smFRET）通过检测单个分子内的荧光供体及受体间荧光能量转移的效率,来研究分子构象的变化。在单分子探测技术发展之前,大多数的分子实验是探测分子的综合平均效应（ensemble averages）,这一平均效应掩盖了许多特殊的信息。单分子探测可以对体系中的单个分子进行研究,得到某一分子特性的分布状况,也可研究生物分子的动力学反应。介绍了近来单分子荧光共振能量转移技术的进展。     

单域抗体研究进展

近年来用基因工程方法从软骨鱼和骆驼科动物中克隆到的单域抗体（single—domain antibody,sdAb）具有无轻链、单一重链可变区保留了完整的抗原结合活性的特征。这类单域抗体具有分子小、稳定性高、体内组织渗透性好、可溶性好、易表达、抗原识别表位独特的特性,已引起生物技术研究与诊断治疗应用领域的广泛关注,取得了快速发展。综述了这类单域抗体发展历史、分子类别、结构特征、理化特征、分子演化及应用前景。     

生物物理新技术新方法在LB 膜结构与功能研究中的应用

现代生物物理技术与方法的发展为LB膜科学的基础研究提供了丰富的手段,使单分子膜科学取得了令人瞩目的进步。文章就单分子膜研究领域中物理新技术与新方法如LB膜仪、表面粘度计、X射线与中子散射技术、布鲁斯特角光学显微镜、电子显徽镜、原子力显微镜等的基本实验原理及其在单分子膜物理特性与功能研究中的应用作简单介绍;并鉴于各种仪器的优缺点,作者提出了多仪器联合对单分子膜进行研究的方案和前景进行了探讨。     

原子力显微镜单分子力谱研究生物分子间相互作用

原子力显微镜单分子力谱是近年来发展起来的能在单分子水平研究生物分子相互作用的新工具。本文综述了单分子力谱的测定原理、方法及其在研究蛋白．蛋白、蛋白-DNA相互作用,蛋白质去折叠和活细胞上配体／受体结合中的应用进展。     

荔枝次生木质部导管分子及穿孔板观察研究

运用细胞图像分析系统及显微照相的方法,对荔枝次生木质部导管分子进行了观察研究。在荔枝的次生木质部中导管分子存在着多种的样式,并且导管分子穿孔板存在着3种类型:两端均为一个单穿孔板;一端一个单穿孔板另一端两个单穿孔板;一端两个单穿孔板另一端三个单穿孔板。分别对其进行了描述,并从导管分子个体发育与系统发育的角度进行了讨论。     

单分子测序与个体医学

目录一、前言二、基因组学的产生、发展及研究方法的演进三、基因芯片技术四、新一代基因测序技术(一)新一代测序技术的技术对比(二)单分子测序技术的基本原理(三)单分子测序技术所面临的挑战五、单分子测序技术的应用(一)在基因组学和系统生物学研究领域的应用(二)在基因表达图谱和单核苷酸多态性等研究领域的应用(三)在个体医学领域的应用六、结语     

Stochastic signal inputs for chemotactic response in Dictyostelium cells revealed by single molecule imaging techniques

Chemotactic cells can exhibit extreme sensitivity to chemical gradients. Theoretical estimations of the signal inputs required for chemotaxis suggest that the response can be achieved under the strong influence of stochastic input noise generated by the receptors during the transmembrane signaling. This arises a fundamental question regarding the mechanisms for directional sensing: how do cells obtain reliable information regarding gradient direction by using stochastically operating receptors and the downstream molecules? To address this question, we have developed single molecule imaging techniques to visualize signaling molecules responsible for chemotaxis in living Dictyostelium cells, allowing us to monitor the stochastic signaling processes directly. Single molecule imaging of a chemoattractant bound to a receptor demonstrates that signal inputs fluctuate with time and space. Downstream signaling molecules, such as PTEN and a PH domain-containing protein that are constituent parts of chemotactic signaling system, can also be followed at single molecule level in living cells, illuminating the stochastic nature of chemotactic signaling processes. In this report, we start with a brief introduction of chemotactic response of the eukaryotic cells, followed by an explanation for single molecule imaging techniques, and finally discuss these applications to chemotactic signaling system of Dictyostelium cells.     

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells. In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-l-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.     

Single molecule nanomanipulation of biomolecules

The development of nanomanipulation techniques has given investigators the ability to manipulate single biomolecules and to record mechanical events of biomolecules at the single molecule level. The techniques were developed to elucidate the mechanism of molecular motors. We can directly monitor the unitary process of the mechanical work and the energy conversion processes by combining these techniques with the single molecule imaging techniques. Our results strongly suggest that the sliding movement of the actomyosin motor is driven by Brownian movement. Other groups have reported data that are more consistent with the lever arm model. These methods and imaging techniques enable us to monitor the behavior of biomolecules at work and will be applied to other molecular machines.     

Single molecule microscopy of biomembranes (review)

Recent advances in the development of new microscopy techniques with a sensitivity of a single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are analysed here in terms of their applicability to the in vivo visualization of cellular processes on the molecular scale, in particular of processes in cell membranes. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultrasensitive methodology as a general tool to study local processes and heterogeneities in living cells.     

Single molecule microscopy of biomembranes (Review)

Recent advances in the development of new microscopy techniques with a sensitivity of a single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are analysed here in terms of their applicability to the in vivo visualization of cellular processes on the molecular scale, in particular of processes in cell membranes. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultrasensitive methodology as a general tool to study local processes and heterogeneities in living cells.     

Multicolour single molecule imaging in cells with near infra-red dyes

Background

The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging.

Methodology/Principal Findings

A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells.

Conclusions/Significance

We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.     

Study on the compounds containing 19F and 10B atoms in a single molecule for the application to MRI and BNCT

Magnetic resonance imaging (MRI) and boron-neutron capture therapy (BNCT) are quite attractive techniques for diagnosis and treatment of cancer, respectively. In order to progress the study on both MRI and BNCT, the novel compounds containing 19F and 10B atoms in a single molecule were designed and synthesized. In the present paper, the syntheses and the internalization rates into tumor cells of these compounds are elucidated.     

FRET imaging

F?rster (or Fluorescence) Resonance Energy Transfer (FRET) is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range. We introduce a revised photophysical framework for the phenomenon and provide a systematic catalog of FRET techniques adapted to imaging systems, including new approaches proposed as suitable prospects for implementation. Applications extending from a single molecule to live cells will benefit from multidimensional microscopy techniques, particularly those adapted for optical sectioning and incorporating new algorithms for resolving the component contributions to images of complex molecular systems.     

Single Molecule Probe Scanning Optical Force Imaging Microscopefor Viewing Live Cells

We have developed an imaging system that combines the soft compliance of an optical trap with the sensitivity of single particle tracking to image forces on/in live cells using a single molecule probe. The probe used is a single (or few) molecule of interest that is conjugated with a single 40 nm colloidalgold probe. The colloidal gold/membrane protein complex, freely diffusing on a live cell, is held in a laser trap while the cell is scanned underneath. Computer control allows for synchronization of the cell scan and capture of the probe position. Resistance to the dragging of the probe images a fine structure of barriers in the membrane of live cells.