A new method for detecting and localizing cell markers endocytosed by fibroblasts in epoxy resin semi-thin sections using scanning electron microscopy combined with energy dispersive X-ray microanalysis after ion-etching

Cell marking is widely used to examine cell development and differentiation in developmental biology. We developed a new method for localizing cell markers in a semi-thin epoxy section with scanning electron microscopy. Cultured fibroblasts ingesting carbon particles were autologously transplanted into a rabbit transparent ear chamber, 6 mm in diameter and 100 microm in depth. Eight days after the transplantation, tissues in the chamber were fixed and embedded in epoxy resin. Semi-thin sections were cut and stained with toluidine blue. Fibroblasts in connective tissues which contained black spots were observed with a light microscope. These sections were subsequently ion-etched with an ion-coater and coated with platinum. The same fibroblasts were then visualized by secondary electron imaging using a scanning electron microscope. A nucleus with nuclear envelope, nuclear pores, a nucleolus and heterochromatin, mitochondria with cristae and rough endoplasmic reticulum were observed in the fibroblasts. The black spots in the fibroblasts were identified as bright bodies with the scanning electron microscope. The bright bodies were found to be a lump of tiny particles less than 100 nm in diameter. In order to analyse such particles with energy dispersive X-ray microanalysis, ion-etched sections were coated with carbon. X-ray energy spectrometry clearly demonstrated that these were carbon particles, which had been endocytosed by the fibroblast. This suggests that scanning electron microscopy combined with energy dispersive X-ray microanalysis is useful for detecting carbon particles in the cytoplasm at an ultrastructural level in semi-thin epoxy sections subsequent to ion etching and that this method may be applicable to other cell markers, such as gold particles to track cells in the field of cell development and cell differentiation.     

透射电镜神经组织制样包埋剂比较

为探讨两种常用包埋剂Epon 812和Eponate 12对神经组织透射电镜样品的影响,本文采用多聚甲醛-戊二醛溶液对wistar大鼠进行全身灌流固定后,取材海马、脊髓及坐骨神经,按常规透射电镜样品技术制作超薄切片.电镜观察显示,经Eponate 12包埋后的样品,在切片的透明度及超微结构的清晰度上均优于Epon 812包埋的超薄切片样品.由于Eponate 12的浸透效果好,有效地避免了有髓神经纤维髓鞘上出现孔洞结构的制样问题.本实验小组的实验结果表明:对于神经组织,尤其是周围神经组织的超薄切片样品,使用Eponate 12作为包埋剂,效果会更好一些.     

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.     

连续切片三维重构技术在细胞超微结构研究中的应用

目的:探究三维重构技术在细胞超微结构研究中的重要性.方法:收集Hela细胞,样品经OTO制样方法处理后进行连续切片,并通过扫描电镜收集序列切片背散射电子信号.通过Amira软件对图片集进行对中、分割处理及三维展示.结果:获得细胞三维重构图,对线粒体长度、宽度和厚度进行了测定和统计,着重对线粒体内质网偶联三维结构进行了观...     

多肽胰岛素正常转运经淋巴而非门脉途径IGMEI胰组织注射SEM观察

探讨胰岛素等多肽激素在细胞外的正常转运途径或规律,对免疫金标记外源性胰岛素（IGMEI）注射的大鼠胰组织冷冻切片,进行了二次电子和背散射电子图像（SEI／BEI）的扫描电镜（SEM）示踪性观察,SEM观察SEI显示胰腺小叶和小叶间结缔组织,血管和淋巴管,胰腺导管和胰岛的结构特点清晰可见,根据管腔内是否存在红细胞和内皮细胞的结构特点,可以区别血管或淋巴管。SEM观察BEI表明,较强的背散射电子出现在胰结缔组织间隙,淋巴管或毛细淋巴管内;然而,胰的血管或毛细血管内,无标记金颗粒的背散射电子或BEI微弱,结果提示,注射或释放入胰组织液中的胰岛素等多肽激素或分泌颗粒,其正常转运途径或规律,可能通过淋巴而非肝门静脉途径转运进入血液。     

En bloc staining available for stereoscopic observation of epoxy resin Quetol 651-embedded thick sections under a high voltage transmission electron microscope.

This method has been devised for easy en block staining for stereoscopic observation of thick sections under a high voltage transmission electron microscope (HVTEM). It uses carbohydrazide as an osmium bridging agent and both osmium tetroxide and uranyl acetate as electron staining agents. Osmium tetroxide-fixed and en bloc-stained tissue blocks are embedded in a Quetol 651 resin mixture. Thick sections (2-3 microns thick) without double staining are observed at an accelerating potential of 300 kV and a tilt angle of +/- 10 degrees by an H-9000 TEM with a side-entry goniometer. Stereoscopic electron micrographs can be obtained.     

Ultrastructure and behavior of actin cytoskeleton during cell wall formation in the fission yeast Schizosaccharomyces pombe

Fluorescence microscopy has shown that F-actin of the fission yeast Schizosaccharomyces pombe forms patch, cable and ring structures. To study the relationship between cell wall formation and the actin cytoskeleton, the process of cell wall regeneration from the protoplast was investigated by transmission electron microscopy (TEM), immunoelectron microscopy (IEM) and three-dimensional reconstruction analysis. During cell wall regeneration from the protoplast, localization of F-actin patches was similar to that of the newly synthesized cell wall materials, as shown by confocal laser scanning microscopy (CLSM). In serial sectioned TEM images, filasomes were spherical, 100-300 nm in diameter and consisted of a single microvesicle (35-70 nm diameter) surrounded by fine filaments. Filasomes were adjacent to the newly formed glucan fibrils in single, cluster or rosary forms. By IEM analysis, we found that colloidal gold particles indicating actin molecules were present in the filamentous area of filasomes. Three-dimensional reconstruction images of serial sections clarified that the distribution of filasomes corresponded to the distribution of F-actin patches revealed by CLSM. Thus, a filasome is one of the F-actin patch structures appearing in the cytoplasm at the site of the initial formation of the cell wall and it may play an important role in this action.     

The expression of actin mRNA in megakaryocytes demonstrated in paraffin sections, and epoxy resin semi-thin and thin sections by in situ hybridization

Megakaryocytes of dog bone marrow were utilized as target cells for identifying actin mRNA expressing cells on semi-thin and thin sections. After in situ hybridization with radioisotope-labeled probes was performed on paraffin sections, gelatin capsules containing freshly prepared epoxy resin were placed on the sections. The resin was solidified and detached from the slide glass, and semi-thin and thin sections were obtained. The signals showing actin mRNA expression were detected on megakaryocytes in these sections by light and electron microscopy.     

Application of SEM-modified X-ray microscope to entomology and histology, and effects of X-ray coherence in imaging

A projection X-ray microscope has been constructed by modifying a scanning electron microscope (HITACHI S-2500CX). Characteristic internal structures and their changes in an aphid, a fly, an ant, a water bear and a beetle are observed first, non-destructively, by X-ray microscope. Stereo-pair X-ray micrographs of Golgi-stained sections of porcine liver show a network of bile canaliculi and stellate cells. The very clear micrographs presented indicate the usefulness of the microscope in studies of entomology and histology. In some micrographs, very thin whiskers or hairs are visible, which suggests that a phase-contrast effect is present in the imaging. X-ray micrographs of an ant hair taken at various distances between the X-ray source and the specimen, D(s), indicate that the image enhancement due to the phase contrast increases with increasing D(s). Degree of the lateral coherence of X-rays of wave length 0.1 nm is theoretically estimated near the hair, with the result that the coherence increases from about zero (almost complete incoherence) at D(s) = 0.1 mm to about unity (almost complete coherence) at D(s) = 6 mm, in agreement with the observation.     

煤岩样品TEM超薄切片制备方法研究

本文作者以泥炭、褐煤、烟煤及无烟煤为例,探讨了超薄切片制备方法对不同煤阶样品的适用性。在采用包埋切片法制备煤岩样品TEM超薄切片时,首先通过浸解离析方法和HCl、HF逐级化学浸蚀方法使煤中自然共生组合的有机显微组分离析及脱除无机矿物质;利用光学显微镜(透射/反射模式)镜检离析显微组分后,使用SPI812树脂对挑出的单一显微组分块体渗透及包埋聚合。显微组分块体形态学及嵌布矿物成分分析使用扫描电子显微镜的低真空二次电子像模式和EDS面分布分析模式;利用透射电子显微镜检查超薄切片效果。实验表明采用上述方法制备煤岩TEM超薄切片样品的成功率较高,并且能够比较真实的再现显微组分的微观结构。     

颅内血管内栓塞治疗的超微结构电镜观察

应用扫描电镜及透射电镜对三种不同颅内血管栓塞材料进行颅内动静脉畸形（AVM）和动脉瘤治疗后的手术标术进行了超微结构的观察和比较。研究结果显示：可控性钨制电解式微弹簧圈栓塞效果理想，血栓形成完全；丝线栓因栓形成较差，而氰基丙烯酸正丁酯（NBCA）则对血管组织可引起慢性炎症及异物吞噬反应。本文对颅内AVM和动脉瘤等血管内栓塞治疗选择栓塞材料提供一定参考价值。     

细胞质多角体病毒的高分辨成像及其三维重构的初步结果

使用装有液氦冷却的冷冻样品支架的300kV场发射冷冻电子显微镜拍摄了细胞质多角体病毒（CPV），对这些电镜照片的频谱分析显示图像分辨率优于0．6nm。我们的三维重构结果使用了最新设计的图象管理和二十面体重构系统（IMIRS）软件包，这是第一个基于Microsoft Windows平台上运行的冷冻电镜三维重构软件包。从得到的一个初步的CPV三维结构，可以看出CPV是具有T＝1的病毒壳体，它的核心半径为28．5nm，在二十面体的顶点有十二个具有五次对称的突起，使它的半径达到36nm。     

实验性慢性缺氧与肺血管结构的重建

目的：主要研究慢性缺氧与肺血管结构重建的关系,利用常压缺氧舱建立大鼠缺氧性肺动脉高压模型。实验动物分为3组：缺氧3天组、7天组、14天组。通过免疫组化的方法检测一氧化氮合成酶(NOS)的表达和含量,部分标本通过电镜观察。结果：在缺氧过程中,肺血管的胶原纤维明显增生,超微结构观察显示血管在早期可见内皮细胞损伤并伴有血小板性血栓形成;到后期出现肌纤维母细胞和胶原纤维增生。实验显示随缺氧时间的延长内皮细胞NOS的含量逐渐升高,而肺血管内源性的舒张反应反而降低,管腔狭窄,血流阻力加大。文中讨论了慢性缺氧与肺血管结构重建的关系,肺缺氧性肺动脉高压的机理,内皮细胞形态及功能的改变在肺动脉高压形成中的意义。     

大鼠急性出血坏死性胰腺炎时肾损害的超微结构研究

胰胆管内逆行注射5%牛磺胆酸钠建立大鼠急性出血坏死性胰腺炎（AHNP）动物模型,用光镜和电镜动态观察肾脏在不同时期的病理形态学变化,从形态学的角度探讨AHNP所引起的肾脏损伤机制;结果发现AHNP时肾脏的病理形态学改变表现为不同程度的肾小球毛细血管腔内红细胞堆积以及血小板和纤维素的析出粘集,毛细血管基底膜增厚,足细胞的足突融合,肾间质血管内新鲜血栓形成,以及肾小管上皮细胞变性、坏死,小管内出现蛋白管型或红细胞管型等.本实验结果提示在AHNP时可伴有明显的肾脏微循环损伤,微循环障碍可能是引起或加重AHNP相关肾损害的重要原因之一.     

Three-dimensional analysis of protein aggregate body in Saccharomyces cerevisiae cells.

Modified genes of peroxisomal isocitrate lyase of Candida tropicalis (CT-ICL) were constructed and expressed in Saccharomyces cerevisiae cells. We observed subcellular localization of expressed products of the mutant CT-ICL genes by immunoelectron microscopy. An unknown structure termed a protein aggregate body (PAB) storing the expressed product was observed in cytoplasm in various mutants (Kamasawa et al. (1996) J. Electron Microsc. 45: 491-497). We chose two typical cells harbouring the mutant ICL genes delta 550 and delta 237-339 to analyse the ultrastructure and three-dimensional (3D) structure of PABs. The PABs had a homogeneous matrix with a wavy periphery in the cell image using a high-pressure freezing fixation method. Although PABs could not be separated from the cytoplasm or mitochondria under a confocal fluorescence microscope, 3D reconstruction of serial electron micrographs clearly showed the PAB was an independent structure of varying size and had the shape of an incomplete sphere. A cell was sometimes observed to have multiple PABs.     

Etched profiles of SiO2 Layer

Etched-edge profile equations of SiO2 in a double layer of fast- and slow-etching SiO2 layer have been derived using Fermat's principle of least time. Etched profiles obtained from the SEM (scanning electron microscope) micrographs for experimental samples show reasonable agreement with calculated profiles.     

基于先验信息的二值血管锥束迭代重建

基于旋转DSA(Digital Subtraction Angiography)的血管三维重建是当前医学图像处理领域的一个新的研究热点,具有广阔的应用前景.本文在SART(Simultaneous Algebraic Reconstruction Technique)算法的基础上,根据穿过每个体素的锥束射线误差的加权平均值,构造了二值体素状态转移的概率函数,实现了一种适用于二值三维图像的迭代重建算法.针对二值三维血管的特点,本文采用最大均匀性准则作为重建目标的先验信息对迭代过程进行约束,使得迭代过程具有很好的体积聚类功能,大大提高了三维图像的重建质量.以Defrise模型和冠状动脉模型作为研究对象,试验结果表明,本文的重建算法在抑制噪声保持目标结构信息等方面优于经典的Feldkamp算法.     

Correlative light microscopy, scanning electron microscopy, and transmission electron microscopy of osmium-macerated biological tissues

A method facilitating correlation of light microscopic (LM), scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images was developed. Rat kidney and heart were initially subjected to the osmium maceration procedure and then embedded in acrylic resin. Semithin sections of the tissue blocks were first provided for LM and then examined by SEM after resin removal. Furthermore, the ultrathin sections adjacent to the semithin sections were observed by TEM. The three-dimensional images of intracellular organelles provided an informative adjunct to LM and TEM.     

In-situ sem observation of electromigration in thin metal films at accelerated stress conditions

With so-called in-situ SEM experiments, electromigration experiments are performed in a SEM (scanning electron microscope) equipped with a heating stage. BSE (back scattered electron) images are taken continuously over the entire length of a metal line submitted to high current and temperature stress, monitoring in detail the microstructure. Comparing the electrical resistance curves with the corresponding SEM micrographs and with ex-situ AFM measurements leads to detailed qualitative and quantitative information about the occurring electromigration and precipitation / dissolution effects in the metal lines.     

Fabrication and Electrical and Mechanical Properties of Carbon Nanotube Interconnections

Individual carbon nanotubes (CNTs) have been cut, manipulated, and soldered via electron‐beam‐induced deposition of amorphous carbon (a‐C) and using a scanning tunneling microscope inside a transmission electron microscope. All CNT structures, including simple tube–tube connections, crossed junctions, T‐junctions, zigzag structures, and even nanotube networks, have been successfully constructed with a high degree of control, and their electrical and mechanical properties have been measured in situ inside the transmission electron microscope. It is found that multiple CNTs may be readily soldered together with moderate junction resistance and excellent mechanical resilience and strength, and the junction resistance may be further reduced by current‐induced graphitization of the deposited a‐C on the junction.