Intercellular attachments between calcified collagenous tissue forming cells in the rat

Summary Osteoblasts of the young rat cranium, and cementoblasts and odontoblasts of young rat molars were prepared by ethanol freeze-fracture prior to critical point drying for scanning electron microscopy (SEM) as well as conventional transmission electron microscopy (TEM) techniques. Critical point drying causes shrinkage which separates the lateral intercellular contacts between neighbours in the same sheet in the case of cementoblasts and osteoblasts, but not those between odontoblasts. These differences are considered to be of functional significance and need to be taken into consideration when formulating theories of calcium influx into the mineralizable matrix of the respective tissues.     

A correlative transmission and scanning electron microscopic study of the pigeon myocardial cell

Summary A comparative study of the pigeon ventricular myocardial cell has been performed by transmission electron microscopy (TEM) and by scanning electron microscopy (SEM). Three-dimensional access to the cell interior was obtained by cryo-fracturing paraffin-embedded tissue immersed in liquid nitrogen. The TEM studies revealed parallelly arranged myofibrils separated by rows of mitochondria. The sarcoplasmic reticulum is represented by a well-developed network of tubules which, at the Z- and H-band level of the sarcomere, expands to form belt-like cisternae. The cisternae at the Z-band level lie in close proximity to both myofilaments and mitochondria. Transverse tubules are absent and thus only peripheral couplings are present.SEM observations of the fractured tissue revealed the spatial relationship between the different cell organelles, the most important of these being the parallel myofibrils and the mitochondria. The conspicuous ridges transversing the myofibril at the Z-band level consist mainly of expanded Z-bands, but overlying SR-tubules also contribute to these ridges. Traces of the SR can sometimes be seen covering the myofibrils. The close proximity between the SR and the mitochondria was also confirmed in the SEM.Preparation and examination of SEM prepared tissue in the TEM confirmed that no essential damage or reorganization of cell organelles had taken place during the SEM procedure. On the other hand some shrinkage of the tissue, which was probably caused by critical point drying, was noticed.     

Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.     

高水分、富含淀粉植物组织的扫描电镜制备技术优化

应用常规高真空扫描电子显微镜观察生物样品必须经过脱水和干燥处理,但无论采用临界点干燥还是冷冻干燥方法,都存在样品表面不同程度失真的问题。植物高水分、富含淀粉组织样品经处理后,容易出现淀粉流失、细胞壁变形等现象,从而造成扫描图像粗糙,无法获得真实的细胞内部结构。本文通过对CO_2临界点干燥、化学固定样品冷冻干燥和新鲜样品冷冻干燥3种扫描电镜样品制备技术中后期制样进行机械断裂和液氮脆断改进,优化出两种植物高水分、富含淀粉组织的扫描电镜样品制备方法:(1)样品首先进行FAA化学固定,经冷冻干燥后用液氮脆断,对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞结构完整,细胞壁整齐,淀粉粒和蛋白轮廓明确,可用于分析淀粉粒和蛋白颗粒在细胞内的分布。(2)新鲜样品直接进行冷冻干燥,经液氮脆断后对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞壁整齐,淀粉粒轮廓更清晰,并且无蛋白颗粒干扰,用于分析淀粉粒在细胞内的分布更加理想。     

Scanning electron microscopy preparation protocol for differentiated stem cells

The lack of an established protocol for scanning electron microscopy (SEM) studies on stem cells differentiating into adipogenic lineage led us to develop a protocol for the preparation of differentiated adult bone marrow-derived mesenchymal stem cells (BMSC) for SEM. This protocol describes the procedure to maintain and preserve the structural organization of cellular components following differentiation, for morphological and physical characterization. The fixation of the differentiated cells was followed by dehydration using methanol, and vacuum desiccation before microscopy. The use of longer chain alcohols as dehydrating agents was avoided in our method to reduce the dissolution of lipid deposits in cells, thus allowing the maintenance of their structural integrity. The time period for the processing of samples was reduced by avoiding the osmium tetroxide postfixation and critical point drying. Thus, this protocol helps in determining the potential, fate, and degree of stem cell differentiation. This may be useful for SEM analysis of differentiated cells, especially those grown on various scaffolds.     

Preparation, characterization, and properties of chitosan-g-poly(vinyl alcohol) copolymer

The graft copolymer chitosan-g-poly(vinyl alcohol), with nontoxicity, biodegradability, and biocompatibility, was prepared by a novel method. The copolymer with porous net structure was observed by scanning electron microscopy (SEM). It is a potential method to combine chitosan with the synthetic polymers. The grafting reactions were conducted with various poly(vinyl alcohol) (PVA)/6-O-succinate-N-phthaloyl-chitosan (PHCSSA) feed ratios to obtain chitosan-g-poly(vinyl alcohol) copolymers with various PVA contents. The chemical structure of the chitosan-g-poly(vinyl alcohol) was characterized by Fourier transform infrared and nuclear magnetic resonance (NMR) spectroscopy. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), and SEM were also detected to characterize the copolymer.     

The Use of Osmium-Thiocarbohydrazide for Structural Stabilization and Enhancement of Secondary Electron Images in Scanning Electron Microscopy of Pollen

Pollen grains stained in a sequence of osmium (O) and thiocarbohydrazide (T) solutions (collectively known as OTOTO) appear structurally stable and undistorted in the scanning electron microscope (SEM), and usually do not require special drying. In fact, OTOTO can be regarded as another special drying method in palynology. This sequential incubation also strikingly increases the electrical conductivity of pollen grains in the SEM. Compared to standard sputter-coating or vacuum evaporative procedures, OTOTO reduces charging and yields secondary electron images with significantly higher resolution.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Studying the Morphology of Lyophilized Protein Solids Using X-ray Micro-CT: Effect of Post-freeze Annealing and Controlled Nucleation

The objective of this study was to determine how different techniques used during the freezing step of lyophilization affect morphology of the dried protein solids. Aqueous solutions containing recombinant human albumin, trehalose, and sodium phosphate buffer were dried after their freezing by shelf-ramp cooling, immersion in liquid nitrogen, or controlled ice nucleation. Some shelf-frozen solutions were heat treated (annealed) before the vacuum drying. We used three-dimensional (3D) X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to study the morphology of solids. The X-ray micro-CT images of the lyophilized microporous solids showed traces of varied size and structure ice crystals that were comparable to corresponding SEM images. A post-freeze heat treatment and a controlled nucleation both induced larger ice crystal ghosts in the solids. The variations in the structure of walls surrounding ice crystals, formed by the different freezing procedures, should affect the water vapor transition during the primary and secondary drying. Some solids also showed higher-density layer in the upper surface. Overall, the simple sample preparation procedures and the ample morphological information make the X-ray micro-CT appropriate for analyzing lyophilized pharmaceuticals.     

A Simple Method For The Preparation Of Sperm Of External Fertilizers For Scanning Electron Microscopy

A simple technique is described for the study of sperm of some external fertilizers (sea urchin, Parechinus angulosus; teleost, Liza dumerili; anurans, Xenopus laevis and Bufo rangeri) by means of scanning electron microscopy. The technique involves: (i) dilution of sperm with water of the natural environment of the animals or artificial media resembling their natural environment; (ii) spreading of sperm suspension on 10-mm square Perspex plates; (iii) fixation of diluted sperm with 2.5 pH 7.4 Millonig phosphate buffered glutaraldehyde (1 hour); (iv) repeated washing in phosphate buffer, air drying and coating with palladium-gold in a vacuum evaporator. Postfixation with 1% osmium tetroxide did not improve the results and dehydration with an alcohol series was not necessary.     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.     

A Comparison of Techniques Useful for Preparing Nematodes for Scanning Electron Microscopy

Second-stage juveniles of Meloidogyne incognita were prepared by several different techniques for scanning electron microscopy (SEM). Sequential fixation in the cold (4-8 C) was superior to rapid fixation at room temperature, glutaraldehyde and glutaraldehyde-formalin were better fixatives than formalin alone, and critical point drying with carbon dioxide or Freon gave similar results that were only slightly better than air drying with Freon. Freeze drying sequentially fixed nematodes from 100% ethanol in liquid propane produced the best preserved specimens with the fewest artifacts. Specimens of various free-living and plant-parasitic nematodes were prepared for SEM by freeze drying. This technique was adequate for most genera but unsatisfactory for a few. Although each genus may require a different procedure for optimum preservation of detail, sequential fixation with glutaraldehyde and freeze drying are comparable and often superior to commonly used techniques for preparing nematodes for SEM.     

Use of ionic liquid in fungal taxonomic study of ultrastructure of basidiospore ornamentation

An ionic liquid (IL) is a kind of salt that stays in a molten state even at room temperature. Since ILs do not vaporize even under vacuum conditions and show high ionic conductivity, they can be used in scanning electron microscopy (SEM) studies. The ultrastructural features of basidiospore ornamentation are considered to be important in the delimitation of taxa for fungi. In the present study, we carried out SEM observations on basidiospores that were subjected to an IL treatment, and evaluated the usefulness of this method in comparison with a conventional preparation method in which dehydrating, drying and platinum (Pt) coating were used. Using the conventional method, a considerable number of basidiospores was lost from the gill tissues; however, using the IL method, the decrease in basidiospores was extremely small. No significant differences in ultrastructural morphology or basidiospore size were found between Pt-coated basidiospores and IL-treated ones. SEM images of Pt-coated basidiospores tended to have higher contrast than those of IL-treated ones. Charging effects were observed with Pt-coated basidiospores, especially at the tips of the ornaments, whereas no such effects occurred for the IL-treated ones. In addition, small crinkles were observed in the Pt-coated basidiospores, but not in the IL-treated ones. These results suggest that the IL method is useful for fungal taxonomic studies.     

A comparison of the size of photoreceptor cells from the retina of the domestic pig as determined by light and scanning electron microscopy

While gathering data on the visual pigments of numerous species, many light micrographs have been taken of the photoreceptor cells containing the visual pigment after spectral recordings have been made from these cells. These micrographs are used to view the cells and obtain measurements of their size. Usually the morphology of the photoreceptor cells is retained adequately so that such measurements can be taken. Occasionally it is necessary to partially fix the retinal tissue which aids in the maintenance of photoreceptor morphology while allowing visual pigment spectral recordings to be made. We have found that primate retinal tissue, as well as some other mammalian tissue, disintegrates rapidly. Although partial fixation allows spectral recordings to be made before the micrographs are taken, the treatment does not always adequately preserve cell morphology for quality micrographs to be obtained. In these cases, visual pigment recordings are made from pieces of unfixed and partially fixed retinal tissue; an additional piece of the same retinal material is well-fixed, embedded and thick-sectioned for light microscopy.Preparing retinal material for sectioning is lengthy and time consuming so an alternative tissue preparation technique was sought. Material can be processed for the scanning electron microscope (SEM) more rapidly than for sectioning, however severe tissue shrinkage occurs during this process. It was found that although shrinkage does occur in the retinal tissue prepared for SEM, the relative proportions of the photoreceptor cells are maintained extremely well. Using the critical point drying method (CPD) pig retinal tissue was prepared for SEM. Scanning electron micrographs of the pig photoreceptors were taken for cell measurement. Since these micrographs could be made at higher magnification than is available by light microscopy, a more detailed view of the pig photoreceptor cells was obtained. Cell measurements made from the light and the scanning electron micrographs indicate that an approximate shrinkage of 50% occurs in the SEM prepared material.     

载阿霉素PLGA纳米微球的制备及性质研究

目的:制备新型癌症化疗制剂载阿霉素(Adriamycin)、聚乳酸-羟基乙酸共聚物(PLGA)纳米微球(ADM-PLGA-NP),研究其性质及体外释药特点。方法:以聚乳酸-羟基乙酸共聚物为包封材料,阿霉素为模型药物,采用复乳蒸发法制备ADM-PLGA-NP,扫描电镜观察微球形态,激光粒度分析仪检测粒径分布,紫外分光光度法计算载药率及包封率,体外药物释放实验考察微球对ADM的缓释作用。结果:ADM-PLGA-NP外观呈球形,平均粒径约(237±12.7)nm,载药量及包封率分别为(6.42±1.67)%和(53.82±8.34)%,药物在体外缓慢释放,5 d累积释放量达85%。结论:通过复乳蒸发法制备的ADM-PLGA-NP性质稳定,具有药物缓释性,有望成为一种新型的药物化疗载体。     

用气溶胶法和摇床法建立铜绿假单胞菌生物膜体外模型

目的模拟体内环境,体外建立细菌生物膜模型,为进一步深入研究细菌生物膜生物学特点提供基础。方法将粘附载体置于气溶胶法和摇床法模拟体内细菌生物膜形成的微环境中,将铜绿假单胞菌株培养3d后,取出标本分别进行通过FITC—ConA染色及SYT09／PI染色,然后分别进行荧光显微镜检测及激光共聚焦检测,观察细菌生物膜的形成情况;进行电子显微镜扫描观察形成的细菌生物膜的形态特点。结果在气溶胶的微环境下,FITC—ConA染色后在荧光显微镜观察到明亮成片状的细菌生物膜;SYT09／PI染色后在激光共聚焦检测,观察到片状,层叠如积云状,棉絮样的细菌生物膜;在电子显微镜扫描观察到大量细菌成团聚集,团状丛生突出表面,具有立体结构的细菌生物膜。在摇床法的微环境下,用3种检测方法都观察到成流线状的细菌生物膜。结论运用气溶胶法、摇床法可成功建立分别模拟体内呼吸系统及循环、泌尿系统的微环境下生物膜形成模型。     

巴西固氮螺菌Yu62在玉米根的定植

将GFPmut2质粒中的gfp基因(编码绿色荧光蛋白)克隆到载体pVK100中,构建成重组质粒pVK1001。将pVK1001通过电转化方法导入到联合固氮菌巴西固氮螺菌Yu62中,获得GFP)标记的巴西固氮螺菌Yu62菌株。用标记菌株接种限菌培养条件下生长的玉米(农大3318)幼苗,在接种后8d、12d,用激光共聚焦扫描显微镜进行观测,结果表明巴西固氮螺菌Yu62菌株能定植于玉米根部皮层的薄壁细胞间隙。用扫描电镜和超薄切片电镜观察表明,大多数细菌主要定植于根表,少数菌可进入玉米根组织内。     

Conventional and microwave-assisted synthesis of ZnO nanorods and effects of PEG400 as a surfactant on the morphology

Conventional and microwave-assisted synthesis of ZnO nanorods have been performed with and without using PEG400. ZnO nanorods were synthesized with 50-250 nm of diameter which depends on the used surfactant and methods. Surfactant effects of PEG400 on the size and morphology of ZnO nanorods were investigated. The microwave method was compared to the conventional heating method. Morphologies were investigated by using scanning electron microscopy (SEM).     

Effects of gel processing on the properties of humic acid isolated from the alga Pilayella littoralis: A humic acid aerogel

Humic acid in the live alga Pilayellalittoralis was isolated as an aqueous gel by a standard sequential extraction method augmented with removal of alginic acid. Portions of the aqueous gel were (1) vacuum oven dried at 40 °C, (2) freeze freeze dried after cooling in liquid N₂, and (3) dried with supercritical fluid CO₂ after substitution of water in the gel with acetone. This paper compares the analytical and spectral properties of the products with compost derived HA and reports significant differences in their surface areas, packing densities, water retention, solute sorption and metal binding properties. The results are discussed in terms of different product morphologies determined by scanning electron microscopy. The aerogel obtained by supercritical fluid CO2 drying of an HA gel from Pilayella has the highest surface area (188 m² g^-1) reported for a humic acid. This revised version was published online in August 2006 with corrections to the Cover Date.     

用于研究植物质外体空间三维结构的树脂铸型技术

本文介绍了用于研究植物体质外体空间三维结构的树脂铸型技术。植物包含许多重要的质外体空间,如木质部管状分子的腔隙、与气孔相连的叶肉细胞间的通气系统、分泌腔等等。这种空间的三维结构可借助于扫描电子显微镜（ＳＥＭ）研究。但问题是,用ＳＥＭ直接观察不到一个组织或细胞切面内部的图像,因此,不能观察它们的全貌。通过运用树脂铸型技术,可以获得完整的组织或腔隙内部空间的铸型。反映管壁结构的各种形象被印在铸型的表面