Light and Electron Microscopic Observations on Encystment of Acanthamoeba palestinensis, Reich

SYNOPSIS. The ultrastructural changes occurring during encystment of Acanthamoeba palestinensis have been investigated. The cyst wall consists of endocyst and exocyst, both having the same fine structure. At irregular intervals in the cyst wall ostioles occupied by opercula are present. The nuclear membrane forms bulb-shaped projections and releases vesicles bounded by double membranes into the cytoplasm. Dense nucleolus-like bodies of different sizes and variable numbers are found in the nucleus of every cyst. The importance of the cyst structure as a taxonomic criterion is discussed.     

Light and Electron Microscopic Observations on the Heterotrich Ciliate Climacostomum virens

SYNOPSIS. Alveolar membranes and an epiplasm exist under the cell membrane of the noncontractile heterotrich ciliate Climacostomum virens. Postciliary microtubular ribbons join at the right of each somatic kinety to form a Km fiber. Two transverse microtubular fibers occur per kinetosomal pair. A myonemal network interconnects the kinetosomal bases intrakinetally and interkinetally. Ultrastructural comparisons are made between the contractile and noncontractile heterotrichs.
The buccal cortex consists of an adoral zone of membranelles, a peristomal field, a buccal tube, the apical membranelles, and a haplokinety. The kineties of the peristomal field and buccal tube are rows of paired kinetosomes, with a postciliary ribbon of microtubules arising from the posterior kinetosome of each pair, and a transverse ribbon and an oblique ribbon from the anterior kinetosome. No Km fibers exist in this region. The haplokinety is a collar of paired kinetosomes surrounding the cytostome; a postciliary microtubular ribbon descends from each kinetosomal pair into the cytostomal region. Ultrastructural details of the buccal cortex of C. virens and other heterotrichs are compared. The nemadesmata which lie under the membranelles are implicated in the body bending of C. virens.
Algae endosymbiotic in the cytoplasm of C. virens are described.     

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM). and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.     

Classification and Reconstruction of a Heterogeneous Set of Electron Microscopic Images: A Case Study of GroEL–Substrate Complexes

Image analysis methods were used to separate images of a large macromolecular complex, the chaperonin GroEL, in a preparation in which it is partially liganded to a nonnative protein substrate, glutamine synthetase. The relatively small difference (6%) in size between the chaperonin in its free and complexed forms, and the absence of gross changes in overall conformation, made separation of the two types of particles challenging. Different approaches were evaluated and used for alignment and classification of images, both in two common projections and in three dimensions, yielding 2D averages and a 3D reconstruction. The results of 3D analysis describe the conformational changes effected by binding of this particular protein substrate and demonstrate the utility of 2D analysis as an indicator of structural change in this system.     

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.     

A Study of Criteria Permitting the Use of Plastinated Specimens for Light and Electron Microscopy

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultra-structural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.     

Light and Scanning Electron Microscopy of Greenbug Aphid Damage in Wheat Using the Same Section

A method has been developed to enable correlative light microscopy (LM) and scanning electron microscopy (SEM) on the same section of wheat (Triticum aestivum L.) leaves infested by greenbug aphids (Schizaphis gra-minum Rondani). Segments of infested leaf tissue were fixed, embedded in paraffin, sectioned, and affixed to slides by standard histological techniques. Serial sections were viewed by LM as temporary mounts in xylene. Sections of interest were identified and re-embedded in fingernail polish, affixed to aluminum stubs, freed of polish with ethyl acetate or acetone, and sputter-coated for SEM. SEM of re-embedded leaf sections showed excellent preservation of leaf anatomy. The same aphid tracks and regions of cell damage identified by LM were visible. SEM increased resolution and provided a much clearer sense of the three-dimensional relations involved in the interaction between plant and insect.     

Light and Scanning Electron Microscopy of Greenbug Aphid Damage in Wheat Using the Same Section

A method has been developed to enable correlative light microscopy (LM) and scanning electron microscopy (SEM) on the same section of wheat (Triticum aestivum L.) leaves infested by greenbug aphids (Schizaphis gra-minum Rondani). Segments of infested leaf tissue were fixed, embedded in paraffin, sectioned, and affixed to slides by standard histological techniques. Serial sections were viewed by LM as temporary mounts in xylene. Sections of interest were identified and re-embedded in fingernail polish, affixed to aluminum stubs, freed of polish with ethyl acetate or acetone, and sputter-coated for SEM. SEM of re-embedded leaf sections showed excellent preservation of leaf anatomy. The same aphid tracks and regions of cell damage identified by LM were visible. SEM increased resolution and provided a much clearer sense of the three-dimensional relations involved in the interaction between plant and insect.     

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea

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电子断层成像技术及其在细胞生物学中的应用

电子断层成像技术（electrontomography）是近年来发展起来一项三维成像技术,可以在纳米分辨率（2-10nm）水平上获得生物大分子及其复合物或聚集体、细胞器、细胞以及组织的三维结构,而且可以用于研究生物大分子在细胞中的定位、排列、分布以及相互作用,已逐渐成为细胞生物学领域中的一项重要技术手段。该文针对这项技术及其在细胞生物学中的应用作一简要介绍。     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

冷冻电镜单颗粒三维重构密度掩模的自动生成方法研究

冷冻电镜单颗粒三维重构技术是用来解析生物大分子三维结构的常用方法.然而目前在单颗粒三维重构过程中,溶剂平滑操作还存在一定缺陷:没有一款主流的单颗粒三维重构程序能够自动寻找掩模(mask)三维密度图,使得三维重构过程难免受到噪音统计学模型计算偏差的干扰.为解决这一问题,本研究借鉴X射线晶体学中解析优化相位所广泛采用的溶剂平滑方法,采用高斯滤波、坎尼边缘检测、最小误差阈值处理等方法处理重构所得三维密度图,优化溶剂平滑操作,发展在单颗粒三维重构过程中自动寻找mask三维密度图的方法.运用三维密度图傅里叶壳层相关系数(fourier shell correlation,FSC)曲线图、模拟颗粒数据重构角度误差散点图等指标评估此方法的效果.结果表明,自动寻找mask密度图的方法能够较好地找到涵盖分子结构信号区域的mask密度图,较为明显提高三维重构所得密度图分辨率.     

Fluorescence and Electron Microscopic Localization of F-actin in the Ependymocytes

The organization of F-actin in the ventricular system has been reported to display pronounced regional differences with respect to shape, size, and development. However, the real roles played by F-actin in these cells cannot be understood unless the precise localization of F-actin is defined. In the present study, we used double-fluorescence labeling to further examine the localization of F-actin in the ependymocytes and its spatial relation to the other two cytoskeletal components, microtubules and intermediate filaments. Then we converted fluorescence signals for F-actin to peroxidase/DAB reaction products by use of a phalloidin-based FITC-anti-FITC system. This detection technique provided an overview of the distribution of F-actin in the ependymocytes at the ultrastructural level, and has been proven to be helpful in correlating light and electron microscopic investigations. (J Histochem Cytochem 57:741–751, 2009)     

Electron Microscopic Examination of Factors Influencing the Expression of Filamentous Surface Structures on Clinical and Environmental Isolates of Aeromonas veronii biotype sobria

Strains of Aeromonas veronii biotype sobria isolated from clinical and environmental sources were examined for their expression of surface structures under a variety of culture conditions. When grown on solid media at 37 C, more than 95% of bacteria from the majority of strains isolated from human diarrheal feces and chicken carcasses were non-piliated or expressed only a few pili of long, flexible morphology per cell. Strains isolated from water or other foods were much more likely to express pili. Heavily piliated strains (all sources) possessed pili of several morphological types, including long, flexible pili of varying widths and rigid pili of varying lengths. Expression of pili was favored by growth at temperatures ca. 20 C and below and growth in liquid medium. Most fecal strains expressed some pili under these conditions. In addition, other surface structures (fibrillar aggregates, fibrillar networks, bundle-forming pili) were seen on some strains from most sources. These were also seen most frequently when bacteria were grown in liquid media at temperatures ca. 20 C and below. Pili expression was not dramatically influenced by growth under anaerobic conditions, or in iron-depleted media, or by combinations of the above conditions. The role of the above surface structures in Aeromonas pathogenicity remains to be elucidated.     

中海拔地区慢性暴露大鼠心肌及肝脏的光镜及电镜观察

目的 探讨中度海拔高度地区慢性低氧大鼠心肌、肝的组织学及超微结构变化。方法 本实验用Wistar大鼠20只,雌雄各半,六日内从海拔5米运至海拔3418米饲养,8周后断头处死大鼠,留取心肝组织作光电镜观察,同时高原暴露前后测定血RBC数及Hb含量。结果 心肝组织学改变主要为细胞水肿,即心肌颗粒变性,肝细胞疏松化,其次为心肌、肝细胞嗜酸性变。心肌组织有少量小灶状坏死,肝组织中未见坏死。超微结构主要有肌浆网扩张,线粒体肿胀,糖元颗粒减少,未见不可逆性损伤如线粒体出现杆状嵴、三膜嵴及核染色质边聚现象。毛细血管内皮细胞多有突起伸向管腔,胞质空泡变性,微饮泡较少。另外,高原暴露后RBC数及Hb含量明显升高。结论 该海拔地区慢性低氧大鼠心肌、肝组织及毛细血管的病变是可逆性的; 左右心室病变程度无显著性差异; 肝组织的病变程度明显轻于心肌组织。     

A Procedure for Preparing Undecalcified and Unembedded Bone Sections for Light Microscopy

We have developed a procedure for light microscopic investigation of undecalcified and unembeddedbone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 μm thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.     

A Simple Procedure for in Situ Immunolabeling, Embedding and Sectioning of Layers of Cultured Endothelial and Smooth Muscle Cells for both Light and Electron Microscopy

We present a simple procedure for in situ immunolabeling, embedding and sectioning of layers of cultured endothelial and smooth muscle cells for both light and electron microscopy. Endothelial and smooth muscle cells were seeded in tissue culture chambers /slides precoated with 30% (w/v) gelatin drops fixed with 0.5% glutaraldehyde. Live endothelial cell layers were labeled with an antibody against the surface membrane protein, anti-CD 13. After labeling, the cell layers were fixed and separated from the chambers/slides by lifting all of the samples with a spatula. Sections (1-2 mm) were cut, embedded and processed further for light or electron microscopy. Because of the delicate cell layers and the importance of preserving maximum integrity, labeling was performed under standard culture conditions and treated in situ during the entire procedure. Moreover, the small chamber size of the tissue culture dishes generated the additional advantages of requiring only a limited number of cells, small volumes of media, and little antibody.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Three-Dimensional Reconstruction of the Mammalian Centriole from Cryoelectron Micrographs: The Use of Common Lines for Orientation and Alignment

The microtubule organizing center of the animal cell (S. D. Fulleret al.,1992,Curr. Opin. Struct. Biol.2,264–274; D. M. Gloveret al.,1993,Sci. Am.268,62–68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar ninefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrandet al.,1992,J. Struct. Biol.108,107–128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of γ tubulin localization.