68例脑胶质瘤透射电镜和扫描电镜观察及其鉴别诊断意义

收集人脑原发性胶质瘤标本68例,其中星形细胞瘤9例,间变性星形细胞瘤22例,胶质母细胞18例,少突胶质细胞瘤3例,间变性光突胶质细胞瘤4例,少突－星形胶质细胞肿瘤12例。另有上皮型和纤维母细胞型脑膜瘤6例。采用透射电镜和扫描电镜观察瘤细胞和间质的超微结构特点。结果显示,各型星形细胞肿瘤瘤细胞共同特点是胞浆及胞突内含有多少不等的胶质细丝;胞膜皱折不平,有多少不一的胞突,这些胞突相互交织在一起。Rosenthal纤维由密集排列的细丝状物围绕不规则的嗜锇性团块构成。脑膜瘤瘤细胞有很多指状突起,细胞间可见桥粒连接,胞膜表面缺乏星形细胞瘤细胞表面的那种皱折。结果表明,透射电镜和扫描电镜可以做为脑胶质瘤病理诊断、鉴别诊断的辅助手段。在普通光镜观察的基础上,结合免疫组化结果,应用扫描电镜还可以比较直观地观察到瘤组织内各种成分之间的相互关系。     

Ultra-high-resolution scanning electron microscopy of vaccinia virus and its recombinant carrying the gag gene of human immunodeficiency virus type 1.

FL cells infected with vaccinia virus or its recombinant carrying the gag gene of human immunodeficiency virus type 1 (HIV-1) were examined by ultra-high-resolution scanning electron microscopy. Virions, whether located extracellularly or intracellularly, had a brick-shaped or watermelon appearance as a whole. Extracellular virions observed on the surface of infected cells had variable surface ultrastructures depending on the manner in which particular virions were wrapped in cell membranes. Most of the intracellular naked virions adherent to the inner face of cell surface membranes clearly exhibited ridgy, rod-shaped or globular surface structures on their surface. HIV-like particles with a diameter of about 100 nm and virions of vaccinia virus were both observed distinctly on the surface of FL cells infected with the recombinant virus.     

动物病毒疫苗研制及其产品的电镜检测

应用电镜技术检查了某些原代和传代细胞,并且跟踪检查了疱疹病毒、痘病毒、腺病毒、细小病毒、冠病毒、RNA反录病毒、呼肠病毒、动脉炎病毒、小RNA病毒、弹状病毒、杯状病毒、黄病毒、双RNA病毒、正粘病毒、副粘病毒等某些成员的驯化、培育及筛选的目的毒。结果发现了部分培养细胞有支原体污染,有的还有细菌污染;同时在某些培养的原毒、基础种毒、生产种毒及其产品中也发现了一些病毒的污染和联苗中互相感染现象。如此证明了电镜技术能够及时发现问题,及时解决问题,使之科研少走弯路,提高产品质量。     

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.     

Three-dimensional observation of SiO2 hollow spheres with a double-shell structure using aberration-corrected scanning confocal electron microscopy

Optical sectioning using scanning confocal electron microscopy (SCEM) is a new three-dimensional (3D) imaging technique which promises improved depth resolution, particularly for laterally extended objects. Using a stage-scanning system to move the specimen in three dimensions, two-dimensional (2D) images sliced from any plane in XYZ space can be obtained in shorter acquisition times than those required for conventional electron tomography. In this paper, a double aberration-corrected SCEM used in annular dark-field mode was used to observe the 3D structure of SiO(2) hollow spheres fabricated by a carbon template method. The double-shell structure of the sample was clearly reflected in both XY- and XZ-sliced images. However, elongation along the optical axis was still evident in the XZ-sliced images even when double aberration correctors were used. Application of a deconvolution technique to the experimental XZ-sliced images reduced the elongated shell thicknesses of the SiO(2) sphere by 40-50% and the selectivity of information at a certain sample depth was also enhanced. Subsequently, 3D reconstruction by stacking the deconvoluted slice images restored the spherical surface of a SiO(2) sphere.     

Backscattered electron imaging by scanning electron microscopy of regenerating peripheral nerve axons immunostained with antineurofilament antibody.

To demonstrate three-dimensional architecture of regenerating axons growing through basal lamina tubes in cryoinjured nerve graft, backscattered electron (BSE) imaging in a scanning electron microscope (SEM) was used to visualize immunostained axons. Regenerating axons immunostained with an antibody against the 200 kD neurofilament protein (RT97) were clearly visualized in BSE images as bright components pursuing an irregular, often spiral course within the basal lamina tubes, and commonly branching within the tubes. The morphology of these structures corresponded closely to that of putative regenerating axons in SEM preparations following application of the potassium hydroxide-collagenase digestion method. The present approach, however, is a considerable improvement on the latter, providing three-dimensional information together with the identification of regenerating axons.     

叔丁醇干燥微波超快扫描电镜样品的制备方法

缩短电镜生物样品制备时间，以适应临床工作的需要一直是电镜工作者努力的方向，微波辐射应用于扫描电镜样品的制备，可缩短制样的时间，我们参考有关文献，并在此基础上加以改进，用微波辐射，叔丁醇脱水干燥，不仅可以缩短制样时间，还可以减少对细胞样品表面结构的损伤。     

High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy

Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component alpha-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.     

A re-examination of the cellular reticulum of fibroblast-like cells in the rat small intestine by scanning electron microscopy

The organization and arrangement of fibroblast-like cells in the rat small intestine were re-examined by scanning electron microscopy after removal of the epithelium and underlying connective tissue components by HCl hydrolysis. In the villi, the fibroblast-like cells had numerous slender processes, and formed a dense and elaborate network like a sieve. It consisted of a large number of circular structures (circles) with various diameters ranging from 0.3 microm to 5 microm formed by the twining of slender processes. In the upper area of villi, numerous fragmental protrusions which were considered to be mainly parts of immune-related cells such as lymphocytes, eosinophils and macrophages extended from the circles. The cells around each tubular gland enclosed it like a basket. These findings suggest that in addition to the function as a skeleton for the villi and glands, the fibroblast-like cells in the upper area of villi may play an important role in regulating the migration of the immune-related cells between the epithelial layer and the underlying lamina propria by their cellular sieve-like structure and contractile ability.     

趾甲镰刀菌病真菌的扫描电镜观察和特殊制样方法

本文经反复摸索对比,总结出一套简便、完整、快捷的真菌扫描电镜（SEM）样品制备方法,并对分离培养趾甲镰刀菌病的镰刀菌（Fusarium,F）进行了表面超微结构观察。在SEM下可清晰地分辨出真菌的菌丝和大、小分生孢子。茵丝粗细均匀,形态饱满,表面有较规律且微微隆起的分隔,其上偶有分支,彼此连通。大、小分生孢子则以孢尖或孢体依附贴合于菌丝上,其中大分生抱子较稀疏,呈镰刀形或纺锤形,两端较尖,孢体稍弯曲,背腹明显,有2～5个环状竹节样膨隆,其中小分生孢子较密集,呈卵圆形或梨形,无或偶见一隔膜,且两种孢子均有顶端和侧壁出芽。SEM观察结果为真菌的病原学分类和抗真菌药物筛选等方面提供了形态学依据。     

SEM images of DNA double helix and nucleosomes observed by ultrahigh-resolution scanning electron microscopy.

We observed DNA double helix and nucleosomes in the chromatin of chicken erythrocytes by ultrahigh-resolution scanning electron microscopy. Specimens were prepared according to a modified microspreading technique in combination with the carbon plate method and observed without metal coating. A part of the DNA fibers without nucleosomes showed "left-handed" double-strands with twisting appearances and regular periodicities of the helix. Linker DNA between the nucleosomes showed "right-handed" appearances. Most of the nucleosome particles appeared as prolate ellipsoidal shapes of various sizes. DNA appeared to enter and exit the nucleosome particles on opposite sides winding around the histone core.     

Detection of glycoconjugates by lectin gold labelling, silver enhancement, and scanning electron microscopy

A method for detecting glycoconjugates on cell surfaces in scanning electron microscopy is described. Terminal saccharides were specifically recognized by a lectin conjugated to biotin, and, after incubation with an anti-biotin antibody conjugated to colloidal gold, silver enhancement was used to produce deposits large enough to be detected in standard scanning electron microscopes. Secondary electron images revealed the ultrastructure of the tissue investigated, while backscattered electron images showed the distribution of lectin binding sites. Using digital recording and processing, the two channels were combined in colour-encoded images. The new method brings together lectin histochemistry and scanning electron microscopy and thus allows the three-dimensional distribution of glycoconjugates to be analysed at an ultrastructural level.