Efficient three-dimensional reconstruction of synapse with high-voltage electron microscopy

Three-dimensional (3-D) information on nervous tissue is essential for the understanding of brain function. Especially, 3-D synaptic analyses on serial ultrathin sections with transmission electron microscopy (TEM) have contributed to the knowledge on neural plasticity associated with various pathophysiological conditions. The 3-D reconstruction procedures, however, not only require a great amount of expertise but also include time-consuming processes. Here, we carried out computer-assisted 3-D reconstruction of parallel fibre-Purkinje cell synapses based on 250 nm serial sections using high-voltage electron microscopy (HVEM). The 3-D synapse models were constructed more efficiently and rapidly compared with conventional serial TEM reconstruction. This result suggests that 3-D reconstruction with thicker sections and HVEM is a useful method to study synaptic connectivity.     

Computer-assisted simulation of high-voltage electron microscopy using serial images recorded by conventional transmission electron microscopy

Here we describe a computer-assisted method which, based on conventional transmission electron microscopy, renders simulated high-voltage electron micrographs. We perform arithmetic minimum filtering on stacks of aligned serial transmission electron microscopic images. In this way, the structural information of the separate images is fused into one compound image that highlights organization patterns otherwise easily overlooked or impossible to comprehend. Our method, like high-voltage electron microscopy, offers the possibility to build stereo-pairs for high-resolution three-dimensional analysis of tissue layers 1-2 microm thick. The use of background elimination and the development of a depth enhancement routine improved the three-dimensional effect and facilitated the analysis of the interior of objects. As an example, we use the method to display the distribution of axonal organelles at nodes of Ranvier and the shape and contents of a highly branched hippocampal dendritic spine.     

Three-dimensional reconstruction by scanning electron microscopy from serial epoxy resin semi-thin sections after ion-etching

Three-dimensional reconstruction was performed using scanning electron micrographs of serial semi-thin sections of Epon embedded specimens. Connective tissue in a rabbit ear chamber was fixed in glutaraldehyde and osmium tetroxide, and then embedded in Epon. One-microm-thick serial sections were cut with a diamond knife, mounted on glass slides and stained with toluidine blue. After observation with a light microscope, the sections were ion-etched with an ion-spatter coater. Following double staining with uranyl acetate and lead citrate, the consecutive sections were ion-coated with platinum. Each serial section was photographed with a scanning electron microscope. Profiles of a blood vessel and fibroblasts were digitized with a computer and computer reconstruction of the blood vessel was performed. Three-dimensional reconstructions showed that the newly formed blood vessel was a cylinder-like, bare endothelial tube with a rather smooth outer surface. Fibroblasts were situated around the endothelial tube. Several openings were found in the endothelial tube, suggesting the morphological feature of high permeability and fragility in newly formed blood vessels. The availability of three-dimensional reconstruction from scanning electron micrographs of serial semi-thin epoxy resin sections was discussed; structures of interest can be reconstructed (1) quickly and easily, (2) without skilful techniques, and (3) almost at the level of ultrastructure.     

Measurement of the local latch-up sensitivity by means of computer-controller scanning electron microscopy

The scanning electron microscopy (SEM) technique for the study of the local sensitivity to latch-up of CMOS integrated circuits is discussed. The technique is independent of a particular electric firing mechanism of latchup and does not require in-depth electrical characterization of the IC before the analysis. The electron beam in the SEM is adopted as a localized current injector, and the injected carriers are used to induce the latch-up state rather than to visualize its paths. A minicomputer-based system drives the beam position and automatically blanks the beam if the scan path has to cross areas which should be protected from charge injection. An example of application is described.<>     

Imaging the interaction between dengue 2 virus and human blood platelets using atomic force and electron microscopy

Thrombocytopenia is frequently associated with dengue virus infection. Host factors such as anti-platelet immunopathogenic processes have been implicated in the origin of dengue-associated thrombocytopenia but the role of dengue virus in directly interacting with platelets and altering their hemostatic property remains incompletely understood. In the present study, we examined the effect of dengue 2 virus on the morphology and physiological activation profile of normal human platelets using atomic force microscopy, electron microscopy and flowcytometry. Platelets obtained from healthy donors were exposed to a cell culture-adapted 10(4) LD(50) dengue 2 virus isolate in vitro and the subsequent effect on morphology and activation biology studied. Our results show that dengue 2 virus exposure at doses comparable to natural viremic states in human infections can activate platelets with an increase in P-selectin expression and fibrinogen-binding property. Atomic force, scanning and transmission electron microscopy also showed typical activation-related morphological changes such as altered platelet membrane architecture, degranulation, presence of filopodia and dilatation of the open canalicular system in the dengue 2 virus-exposed platelets but not in the controls. Importantly, Japanese encephalitis virus exposure at the same dose did not activate platelets or show any morphological changes. Our findings suggest that dengue 2 virus may directly interact with and activate platelets - an event that might be important in the origin of dengue-associated thrombocytopenia. Detailed molecular characterization of this effect might provide key knowledge toward better prophylaxis of the hemostatic complications of dengue disease.     

Budding process and maturation of human immunodeficiency virus examined by means of pre- and post-embedding immunocolloidal gold electron microscopy

The techniques of pre- and post-embedding immunocolloidal gold electron microscopy were tested for their usefulness for analyzing the morphogenesis of human immunodeficiency virus (HIV) strain LAV employing U-937 cells persistently infected with the virus (U-937/LAV). By both techniques the following results were obtained. (1) Budding process was restricted to a localized area at the membrane adjacent to the Golgi apparatus. (2) The distribution of viral envelope antigens was restricted to the electron-dense crescent structures on the cell surface. (3) Virions released from the cells could be classified mainly into 2 categories: virions with doughnut-shaped cores or with conical cores at the center of the particles. And p24 proteins localized on the thick electron-dense cores of both types. These findings support the idea that pre- and post-embedding immunogold electron microscopy is very useful for studying the morphogenesis of viruses.     

冷冻电镜单颗粒重构中的病毒三维显示

利用冷冻电镜显微技术和单颗粒三维重建方法获得BmCPV（家蚕质多角体）;CSBV（中蜂囊状幼虫病痛毒）;C6／36DNV（C6／36浓核病毒）的三维结构体数据,用空间低通滤波对体数据矢量场进行处理并进行三维显示,较之原来的显示,提高了信噪比,增强了显示稳定性与显示质量、病毒每个表面细节和轴上突起更加清晰可见。     

Single particle conformations of human serum albumin by electron microscopy

Using single particle images taken by electron microscopy, pH-dependent conformational changes of human serum albumin were investigated. Despite the noisy particle images of negatively stained serum albumin (67 kDa), our novel algorithm for automated particle picking and reference-free classification resulted in the appropriate grouping of the particle images. Iteratively aligned particle images in the same group provided recognizable image features for individual groups. In a pH 7.0 study, monomer images were consistent with an available crystal structure model; the dimer images were separated into different classes. At pH 3.5, the monomer images were similar to those at pH 7.0; slight differences included a small number of elongated conformations and increased population of larger multimers. Our images were also compared with projection images of an atomic model from crystallography, and demonstrated consistency of the molecular conformation both at pH 7.0 and pH 3.5. Our classification method was effective in discriminating monomers from a mixture of different conformations of the protein, enabling the study of the conformational dynamics of small proteins, using the atomic model as a reference.     

基于地基GNSS和COSMIC掩星数据吸收的三维电离层电子密度重构方法

精确给出电离层的实时变化信息对现代通信和卫星导航等系统可靠应用具有非常重要的意义.为提高电离层经验模型电子密度的输出精度，提出了一种基于地基GNSS和COSMIC掩星数据吸收的三维电离层电子密度重构的新方法.以最新版国际参考电离层模型IRI-2016为背景模型，选择IG指数与Rz指数作为驱动量，采用Brent算法分两步实现了地基GNSS和COSMIC掩星数据的吸收.与欧洲区域8个垂测站实测数据的对比表明:数据吸收后模型重构的电离层N_mF₂的绝对平均误差和标准差分别下降了33%和29%；电离层h_mF₂的重构误差则分别下降了约55%和30%.对比结果验证了所提方法的精度和有效性.     

用皮秒激光产生宽度可调的高压电脉冲

本文报道用两个具有可变相对延时的皮秒激光脉冲,先后驱动两个光电子开关,产生前后沿均为亚毫微秒的宽度可调高压电脉冲。     

Efficient and accurate analysis of mitochondrial morphology in a whole cell with a high-voltage electron microscopy

Mitochondria in all eukaryotes are essential organelles responsible for adenosine triphosphate synthesis, calcium homeostasis and steroidogenesis. Because the structure and distribution of mitochondria are highly diverse depending on their function and cellular conditions, it is important to develop a rapid and accurate method to assess their morphology. In this study, we visualize whole mitochondria in cultured cells using high-voltage electron microscopy (HVEM). Compared with conventional transmission electron microscopic approaches, the present method does not require thin sectioning and thus requires less time for image acquisition and processing. Furthermore, compared with fluorescence-based light microscopic approaches, our method provides more accurate size information. Thus, we propose that HVEM is a useful tool for rapid and accurate analysis of mitochondrial morphology and distribution in a cell.     

电镜重构病毒的三维显示与三维任意区域分割

利用冷冻电镜单颗粒三维重建方法获得分辨率1.5nm家蚕质多角体（BmCPV）;2.5nm,中蜂囊状幼虫病病毒（CSBV）;1.4nm,伊蚊C6/36细胞浓核病毒（C6/36DNV）的三维结构体数据,对三维结构数据进行了数据归并,并找到对显示细节较为敏感的向量维度,用表面显示方法对病毒体数据进行三维显示,用基于空间的方法完成了任意区域的交互分割。显示时间和信噪比与原有显示方法有了提高。病毒表面细节和轴上突起清晰可见。所分割的部分结构准确完整,并可以对所分割的部分进行量化分析。为冷冻电镜单颗粒重构提供了新的三维可视与分析方法。     

用低温电子显微术研究E.coli SecA的三维结构

新生肽链在细胞质内合成之后要经过复杂的跨膜转运过程被运送到发挥功能的场所，如跨膜蛋白及胞间质蛋白。信号肽依赖性的蛋白转运通路（Sec途径）是原核生物中最主要的新生肽链转运系统。SecA蛋白是Sec系列蛋白中的核心组分，其转过程中的结构变化直接介导了新生肽链的跨膜转运。因此，获得SecA蛋白的结构信息及该结构在转运过程的变化对于我们理解SecA发挥活性的分子机制至关重要。     

互相关法实现距离选通远距离目标的三维重建

针对距离选通激光主动成像二维切片图像无法提供远距离目标全面空间位置信息的问题,提出了采用阈值法截断灰度级曲线与互相关法提取距离信息相结合的算法对远距离目标进行高精度三维重建。首先,利用阈值法剔除实测灰度级曲线中零值点,从而消除对后续互相关法计算距离值的影响;然后,将每一像素点实测截断灰度级曲线与理想灰度级曲线进行互相关,利用各像素点相关峰值对应的延迟时间推算各点距离值,并结合成像系统参数计算每一像素点的三维坐标;最后,对距离1.2 km处凉亭切片图像进行处理,得到了凉亭目标的三维图像。     

植物病毒的电镜诊断

本文介绍了植物病毒新的分类和命名系统，综述了植物病毒电镜诊断的常规技术和以及各个病毒属的形态学特征。在ＩＣＴＶ报告的１１科、４７属共７８９种植物病毒中，花椰菜花叶病毒属、杆状ＤＮＡ病毒属、番茄斑萎病毒属、苜蓿花叶病毒属、双生病毒科、吸肠孤病毒科、弹状病毒科等七个具有典型形态特征的属或科，以及螺旋对称结构的杆状及线状病毒，通过负染色可作出诊断。寄生细胞的病理变化尤其内含体的形态结构也是诊断的重要依据     

肽泡的电子显微镜观察

本文用电子显微镜方法研究肽泡的形态结构及其特性。结果表明，肽泡大小不等，泡壁由厚度不均一且电子致密度较高的丝状物组成，轲与兔红血球碎片自然重组成双分子层结构，肽泡的包裹特性，有可能使之成为一种良好的药物载体。     

Three-dimensional reconstruction of transillumination tomographic images of human breast phantoms by red and infrared lasers

A laser transillumination tomographic system, using red and near infrared lasers, to obtain cross-sectional images of human breast phantoms and human hand is proposed. The scanning assembly is consisting of upward, downward, rotational and pitch movements. The phantoms, made of paraffin wax, agar gel, and milk placed in a glass model, are embedded with abnormalities like blood, water, solid objects, and tissues. By illuminating the phantoms at various heights by either red or infrared laser the projection data are collected. Based on 64 projections the tomogram of each section is constructed. By volume visualization procedure applied to the tomograms the objects of varying composition embedded within the phantoms are detected and their size, shape, and location depth are determined. The cross-sectional image of a human hand obtained by this procedure further shows the possibility of application of this technique for imaging of organs.     

水生动物病毒的电镜和荧光显微镜观察

应用透射电镜和荧光显微镜对三种水生动物病毒粒子形态、病毒感染细胞的超微结构变化及亚细胞定位进行比较和研究。负染电镜观察显示:鳜鱼弹状病毒(SCRV)粒子呈典型的子弹状形态,长约76～118 nm,直径约29～52 nm;鲈鱼呼肠孤病毒(LJRV)粒子呈正二十面体结构,具有双层衣壳,直径约70～80 nm;蛙虹彩病毒(RGV)粒子呈正二十面体结构,具有囊膜,直径大小约150 nm。超薄切片观察表明,宿主细胞的基本结构遭到不同程度的破坏,成熟病毒粒子分布在胞质中或以出芽的方式释放到胞外,且RGV增殖后在胞质中聚集形成晶格状结构。免疫荧光观察进一步显示,RGV感染细胞后能产生多个直径大小不一的包涵体。本研究结果有助于了解和认识水生动物病毒的超微形态特征、复制过程及致病机理。