Measurement of the repeat period of myelin sheath using ultrathin frozen sections

The myelin sheath of peripheral nerves was observed by transmission electron microscopy (TEM) using plastic-embedded sections and ultrathin frozen sections. Repeat distances of myelin sheaths were measured in high-powered electron micrographs. The ultrathin frozen sections showed a longer repeat distance than the plastic-embedded sections. The ultrathin frozen sections were thought to contain fewer artefacts, as they had not been subject to dehydration and embedding. It is known that broken myelin sheaths are often observed under conventional TEM. It is thought that these procedures cause contraction and partial destruction of the myelin sheath.     

激光共聚焦显微镜样品制备方法（二）——组织切片样品

应用激光共聚焦显微镜技术对荧光标记的组织切片样品进行三维观察成像是生物学研究的常规手段。本文主要介绍实验室制备用于激光共聚焦显微镜成像的冷冻组织切片及免疫荧光标记过程。     

人舌鳞癌组织超薄切片的AFM成像和切割

利用一种基于电镜超薄切片法改进的制样方法，将人舌鳞状细胞癌病理组织以环氧树脂包埋并切片后，将薄片平整地贴附在云母上，用原子力显微镜(AFM)对切片表面进行研究，可以得到高分辨率的细胞超微结构图像，局部的亚细胞水平的形态结构可以与电镜下得到的图像相比拟。在此基础上，利用AFM针尖对肿瘤细胞核内特定区域进行切割和操纵，形成生物分子的堆积，从而为拾取(pjck—up)和进一步用分子生物学手段在亚细胞基因水平研究人舌鳞癌的病理学奠定了基础。     

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.     

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.     

Role of matrix metalloproteinase-7 in angiogenesis associated with age-related macular degeneration

To investigate the possible role of matrix metalloproteinase-7 in choroidal neovascularization associated with age-related macular degeneration, immunoelectron microscopy using ultrathin frozen sections and conventional transmission electron microscopy were performed in subfoveal fibrovascular membranes from patients with age-related macular degeneration. immunoelectron microscopy revealed that matrix metalloproteinase-7 was expressed within basal laminar deposits and amorphous materials around the retinal pigment epithelial cells. The results support a role for matrix metalloproteinase-7 in the development of choroidal neovascularization in age-related macular degeneration.     

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium: an attempt to reactivate the 5'-nucleotidase activity with Mg++ ion supply

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium was performed on cryostat sections using the 5'-nucleotidase (5'-Nase) staining method by light microscopy and backscattered electron imaging of scanning electron microscopy. The inhibition of the 5'-Nase activity by decalcification was cancelled by the Mg++ ion supply. The reaction products of 5'-Nase activity were produced on the lymphatic endothelial cells and the tubular structures of lymphatic vessels were seen as highlights by backscattered electron imaging. The invasion of 5'-Nase-positive lymphatic vessels into the alveolar bone from the periodontium was found in the present study.     

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.     

心脏移植受体的超微结构改变

目的：观察心脏移植标本的超微结构变化。方法：通过移植后心脏的心内膜活检，经固定、常规则超薄切片制备，JEM－2000EX透射电镜检查。结果：移植后的早期阶段，未见明显移植排斥反应的心脏心内膜活检显示间质水肿，偶尔可见毛细血管内皮细胞的肿胀。心肌细胞和内皮细胞内溶酶体明显增多。在早期细胞排斥反应阶段，电镜检查可发现显著的心肌细胞坏死和变性，并可确定浸润炎细胞的性质。在血管排斥反应阶段，辨认内皮细胞增生、内皮细胞损伤和基底膜的改变，在患有移植性冠状动脉病的病人，其缺血性心脏具有显著特点：肌动蛋白丝的缺失比肌球蛋白丝更明显，肌微丝相对较粗大。结论：这些改变对于进一步确诊心脏移植受体的排斥和局部缺血是有用的形态学特征。     

The location of filamentous hemagglutinin and pertussis toxin antigens of Bordetella pertussis by immunoelectron microscopy

Immunoelectron microscopy using colloidal gold-tagged antibodies was used to detect filamentous hemagglutinin (FHA) and pertussis toxin (PT) antigens on the surface and in the cytoplasm of Bordetella pertussis cells. Both gold-tagged antibodies to FHA and PT labeled the aggregates of filamentous material on the surface of sediment-settled phase I cells under static conditions. FHA and PT antigens were detected also on ultrathin sections made after embedding the phase I cells in Lowicryl K4M resin. On the ultrathin sections, intense label of gold-tagged antibodies to FHA and PT was present on the cell surface and also in the cytoplasm, but not in the nucleoid. The aggregates of filamentous material adhering on the surface of phase I cells were most abundant on culture day 3, the end of the logarithmic growth stage, but most of the aggregates were found detached from the cell surface on culture day 5 or 7, the stationary stage. The aggregates were not found on the surface of phase III cells. The present study suggested that FHA and PT antigens were localized on the same cell structure and that both antigens were synthesized in the cytoplasm and secreted across the cell membrane mainly in the logarithmic growth stage of the phase I cells.     

Association of cytoskeletons with the Golgi apparatus: three-dimensional observation and computer-graphic reconstruction

The association of cytoskeletons with the Golgi apparatus (GA) in cultured 3Y1 cells was investigated by stereo electron microscopy of thick sections and computer-graphic reconstruction of serial thin sections. The 3-dimensional analysis has demonstrated that: 1) both microtubules (MT) and vimentin intermediate filaments (IF) were abundantly present in close vicinity to GA, and some of them were closely associated with GA; and 2) such GA-associated cytoskeletons were attached to GA either at their termini or at their lateral side. The present observations suggest that both MT and vimentin IF are responsible for maintaining the structural integrity of GA.     

小鼠骨髓瘤细胞系(Sp2/0—Ag14)及鼠—鼠淋巴细胞杂交瘤细胞系的形态定量比较研究

小鼠骨髓瘤细胞系(Sp2/0—Ag14)是不分泌型的浆细胞样细胞。我们用Sp2/0—Ag14细胞与免疫小鼠的脾细胞融合,建立了抗本周氏蛋白单克隆抗体的杂交瘤细胞系并采用立体计量方法对亲本及杂种细胞进行了形态定量测量。以便探讨细胞形态结构的变化与分泌抗体的关系。从半薄切片上细胞的结构数据,计算出平均细胞的各种三维空间结构参数。在超薄切片上得到了胞浆内结构成分的三维超微结构参数。结果表明:杂种与亲本细胞相比,杂种细胞的体积明显增大;粗面内质网扩张显著;高尔基体小泡的数量有增多的趋势;线粒体平均体积增大。揭示:杂种细胞的分泌能力较强。     

激光共聚焦扫描显微系统在心血管支架表面内皮化分析研究中的应用

目的:观察内皮细胞(ECs)在植入的心血管支架表面的分布与生长状态,探讨支架材料表面的内皮化情况,并对支架表面内皮化程度的影响因素进行了初步探究.方法:在对内皮细胞不同结构进行特异性染色标记的情况下,利用激光共聚焦扫描显微系统连续扫描观察荧光标记的内皮细胞在植入体内一个月后的支架材料表面的表达程度.结果:在无任何促内皮...     

He-Ne激光对兔正畸牙周组织血管改建影响的实验研究

采用CD34抗体标记正畸牙周组织的血管内皮细胞，探讨He-Ne激光照射对兔正畸牙周组织血管改建的影响。35只大耳白兔随机分未加力组及加力组，对实验标本进行CD34免疫组化染色，用计算机图像分析系统测定正畸牙周组织的微血管密度(MVD)与微血管面积(MVA)，应用SPSS统计软件进行统计学分析。经He-Ne激光照射侧的微血管密度与微血管面积均普遍大于对照侧。除加力1d组外，其余各加力组照射侧与对照侧之间的微血管密度、微血管面积的差异均有统计学意义(P＜0．05，P＜0．01)。     

A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy

Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.     

激光扫描共聚焦显微镜在人胆管癌组织免疫荧光染色切片观察中的应用

目的：探讨利用激光扫描共聚焦显微镜鉴定单克隆抗体特异性。方法：正常人胆管组织、正常人肝组织及人胆管癌组织切片经免疫荧光染色后置于普通荧光显微镜及激光扫描共聚焦显微镜进行观察,比较二者效果。结果：与普通荧光显微镜相比,激光 聚焦显微镜能清晰地显示染色组织的荧光染色部位及强弱,能较好地显示染色组织的形态,显示出良好的准确性及精确性。结论：激光扫描共聚焦显微镜在免疫荧光染色切片的检测中具有准确、特异、清晰的特点,在单克隆抗体的鉴定中具有良好的应用前景。     

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。     

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。     

微波辐射超薄切片快速染色法

本文介绍一个微波辐射超薄切片快速染色法。该方法与常规染色方法相比具有以下突出优点:染色速度快且操作简便,整个染色过程可在数分钟内完成;经微波辐射染色处理的动植物组织切片染色均匀,污染少,反差比常规染色法稍强。动植物组织切片微波辐射染色的最佳辐射时间有所不同,铀染色动植物组织切片最佳辐射时间均为30秒;铅染色动物组织切片最佳辐射时间为30秒,而植物组织切片为20秒。染色液经微波辐射后的温度应控制在20～40℃。     

A support film of plasma-polymerized naphthalene for electron microscopy: method of preparation and application.

Methods of preparing of a new support film by plasma polymerization in a glow discharge, and its application were described. The recently developed plasma polymerization replica technique was used to prepare ultrathin films from naphthalene monomer gas on the surface of a newly cleaved sodium chloride crystal or on cover glass. The film was floated off on water or hydrofluoric acid solution to free it from the base. The plasma-polymerized naphthalene support film (PNS) prepared in this way was shown to have excellent properties by its application to negative staining of virus antigen particles and by the observation of whole ultrathin sections of animal tissue mounted on a 2 x 1 mm single slot grid. Features of the new support film are (1) its ease of preparation as a clean, very thin film of less than 10 nm, (2) its amorphous texture and high transparency to electrons, (3) its mechanical strength, resistance to heat and chemicals, and electron bombardment, and (4) its smooth and slightly hydrophilic surface. The PNS would be useful for both routine and high-resolution electron microscopy.