“电子显微学与结构生物学”系列讲座（一）

生物电子显微学是结构生物学的重要分支,主要包括电子晶体学（electron crystallography）、单颗粒技术（single particle technique）,电子断层成像技术（electron tomography）,以及冷冻电镜技术（cryo-EM）,适于从分子到细胞各个结构层次的三维结构研究,与X-射线晶体学和核磁共振谱学一道,是结构生物学的重要研究手段。本次研讨会旨在分析该领域国际发展趋势,促进国内合作研究,整合国内资源,加强人才培养。     

原子力显微技术成像在生物医学中的应用

原子力显微技术利用探针尖端与标本之间相互作用的力场对标本进行三维成像。这种成像可在生理条件下进行 ,可进行动态观察和标本容易制备是有别于其它成像技术如电子显微镜成像等的特点。对于细胞和生物大分子 ,能够在生理条件下成像具有重要意义。它意味着人们在认识生命本质的方法学方面 ,又向前迈出了新的一步。本文简要综述对细胞和生物大分子的成像在生物医学方面的应用。     

结构生物学专题序言

<正>结构生物学是一门研究生物大分子的三维空间结构、动态过程和生物学功能的交叉性学科,结构生物学研究可以提供生物大分子在原子分辨率水平的原子坐标、相互作用的细节信息以及生物大分子在行使其功能时的动态变化,这些结构信息与功能研究相结合,不仅能促进人们对生物大分子的生物功能和分子机理的认识,阐述重要的生物学问题,同时也能为探索与生物大分子功能失调相关疾病的发病机     

膨胀显微成像技术的原理及应用

膨胀显微成像技术（expansion microscopy,ExM）是一种新型超分辨成像技术。该技术借助可膨胀水凝胶均匀地物理放大生物样本,在常规光学成像条件下实现超分辨成像。ExM适用于细胞、组织切片等多种类型生物样本。蛋白质、核酸、脂质等生物大分子均可借助ExM进行超分辨成像。ExM可与共聚焦显微镜、光片显微镜、超高分辨显微镜联合使用,进一步提高成像分辨率。近年来,多种从基础ExM拓展而来的衍生技术进一步促进了该技术的实际应用。本文综述了ExM及其衍生技术的基本原理、ExM与不同成像技术联用的研究进展及ExM在不同类型生物样本中的应用进展,并对ExM技术的发展前景做出展望。     

荧光蛋白研究进展

荧光蛋白在生物学众多研究领域中有着广泛的应用,基于荧光蛋白的分子探针和标记方法已成为活细胞或活体内动态成像研究生物大分子或细胞功能的重要工具。本文对现有荧光蛋白的种类和理化特性,及其在生物学研究中的应用进行了综述介绍。重点介绍了近年来荧光蛋白在亮度、Stokes位移、光谱改变等方面的研究进展,介绍了光转换与光活化荧光蛋白及其在超分辨荧光成像技术中的应用。最后对荧光蛋白未来的发展方向进行了展望。     

低温电子断层扫描术及其应用

低温电子断层扫描术（cryo—electron tomography,cryo—ET）是一种最近才开始应用于探索生物体内部,尤其是细胞内结构的仪器,它将三维成像技术和保持完整细胞结构的样品制备方法有机结合,从而实现了在接近真实状态下高分辨率地观察大分子结构的目的。cryo—ET的出现使许多细胞内部精细结构如细胞器（细胞骨架、核孔复合体、核糖体等）得到了更为详细的阐明,从而为生命研究打开了一扇新的大门,同时cryo—ET也正在逐渐成为细胞生物学研究的必要工具之一。     

超速离心技术

超速离心是生物学研究中的一项重要技术,用于细胞、亚细胞组分和生物大分子(如蛋白质、核酸等)的分离与分析。习惯上把每分钟4,000转的离心机称为低速离心机;每分钟20,000转的离心机称为高速离心     

原子力显微术在生物研究中的应用

原子力显微术不仅能够提供样品表面纳米级别分辨率的三维图像数据,而且能够对pN级微小力进行测量,同时将两者结合发展出的TREC（topography and recognition）显微术还能够在进行高分辨成像的同时实现对特定分子的定位。原子力显微术的这些特点使之成为生物化学、细胞生物学等生物研究的有利工具。本文主要介绍了原子力显微镜高分辨成像和检测生物分子识别的原理,以及TREC显微术在生物学上的应用。     

扫描探针显微镜及其在生物医学研究中的应用

本文介绍了一类可以从原子水平到微米尺寸观察物质结构的三维成像工具——扫描探针显微镜(SPM),重点介绍了扫描隧道显微镜(STM)和原子力显微镜(AFM)的基本原理,以及SPM在细胞生物学、核酸和小分子成像等生物医学研究领域的一些应用。SPM不久将可能成为大多数生命科学实验室的一项重要技术。     

《自然-通讯》：活细胞荧光成像新技术问世

近日来自法国让o皮埃尔o埃贝尔结构生物学研究所的研究人员领导的一个国际研究小组报告称他们设计出了一种新型的荧光蛋白分子。相比于目前采用的荧光蛋白。新荧光分子可在活细胞中发射亮度高3倍的蓝绿色光,大大提高了细胞成像技术的敏感性,从而可以帮助实现更高分辨率的活体内生物过程成像。这一研究成果在线发布在3月20日的《自然一通讯》（Nature Communications）杂志上。     

Application of electron tomography to fungal ultrastructure studies

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.     

Multiple-axis tomography: applications to basal bodies from Paramecium tetraurelia

BACKGROUND INFORMATION: Transmission electron tomography is becoming a powerful tool for studying subcellular components of cells. Classical approaches for electron tomography consist of recording images along a single-tilt axis. This approach is being improved by dual-axis reconstructions and/or high-tilt devices (tilt angle>+/-60 degrees) on microscopes to compensate part of the information loss due to the 'missing wedge' phenomena. RESULTS: In the present work we have evaluated the extension of the dual-axis technique to a multiple-axis approach, and we demonstrate a freely available plug-in for the Java-based freeware image-analysis software ImageJ. Our results from phantom and experimental data sets from Paramecium tetraurelia epon-embedded sections have shown that multiple-axis tomography achieves results equivalent to those obtained by dual-axis approach without the requirement for high-tilt devices. CONCLUSIONS: This new approach allows performance of high-resolution tomography, avoiding the need for high-tilt devices, and therefore will increase the access of electron tomography to a larger community.     

生物三维电子显微学进入全新高速发展时期

生物三维电子显微学主要由三个部分组成——电子晶体学、单颗粒技术和电子断层成像术,其结构解析对象的尺度范围介于x射线晶体学与光学显微镜之间,适合从蛋白质分子结构到细胞和组织结构的解析。以冷冻电镜技术与三维重构技术为基础的低温电子显微学代表了生物电子显微学的前沿。低温单颗粒技术对于高度对称的病毒颗粒的解析最近已达到3．8A分辨率,正在成为解析分子量很大的蛋白质复合体高分辨结构的有效技术手段。低温电子断层成像技术目前对于真核细胞样品的结构解析已达到约40A的分辨率,在今后5年有望达到20A。这样,把x射线晶体学、NMR以及电镜三维重构获得的蛋白质分子及复合体的高分辨率的结构,锚定到较低分辨率的电子断层成像图像中,从而在细胞水平上获得高精确的蛋白质空间定位和原子分辨率的蛋白质相互作用的结构信息。这将成为把分子水平的结构研究与细胞水平的生命活动衔接起来的可行途径。     

Reprint of "On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography" [J. Struct. Biol. 159 (2007) 507-522

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15–20 mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.     

On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15–20 mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.     

Serial block face scanning electron microscopy—the future of cell ultrastructure imaging

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.     

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.     

冷冻电子断层成像技术及其在生物研究领域的应用

冷冻电子断层成像可以在纳米级尺度上研究那些结构不具有均一性的分子、病毒、细胞器以及它们之间组成的复合体的三维结构。在过去的十年中,电子显微镜硬件、冷冻制样设备和技术,以及自动化断层数据收集方法的进步使得本研究领域得到快速发展。本文对冷冻电子断层成像的方法,包括基本原理、样品制备、断层数据采集和图像处理、三维重构以及重建信息的理解和展示、近年来在生物样品领域的一些典型应用以及前景作一简单介绍。     

Heterocellular communication in the heart: electron tomography of telocyte-myocyte junctions

Myocardium is composed of two main cell populations: cardiomyocytes (CMs) and interstitial cells (e.g. fibroblasts, immunoreactive cells, capillaries). However, very recently we have showed that a novel type of interstitial cell called telocytes (TCs) does exist in epi-, myo- and endocardium. They have very long and thin telopodes (Tp) formed by alternating podomeres and podoms. Heterocellular communication between TCs and CMs it is supposed to occur by shed vesicles and close apposition. If TCs have to play a role in cardiac physiology it is expected to develop direct and unambiguous contacts with CMs. Because a clear membrane-to-membrane junction has not been reported by electron microscopy we have investigated the heterocellular communication in the mouse heart by electron tomography. This advanced technique showed that small dense structures (10-15 nm nanocontacts) directly connect TCs with CMs. More complex and atypical junctions could be observed between TCs and CMs at the level of intercalated discs. This study proves that TCs and CMs are directly connected and might represent a 'functional unit'.     

Cryo electron tomography of vitrified fibroblasts: microtubule plus ends in situ

Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 nm over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5–3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beck, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759–765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex.