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.     

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.     

Three-dimensional reconstruction by scanning electron microscopy from serial epoxy resin semi-thin sections after ion-etching

Three-dimensional reconstruction was performed using scanning electron micrographs of serial semi-thin sections of Epon embedded specimens. Connective tissue in a rabbit ear chamber was fixed in glutaraldehyde and osmium tetroxide, and then embedded in Epon. One-microm-thick serial sections were cut with a diamond knife, mounted on glass slides and stained with toluidine blue. After observation with a light microscope, the sections were ion-etched with an ion-spatter coater. Following double staining with uranyl acetate and lead citrate, the consecutive sections were ion-coated with platinum. Each serial section was photographed with a scanning electron microscope. Profiles of a blood vessel and fibroblasts were digitized with a computer and computer reconstruction of the blood vessel was performed. Three-dimensional reconstructions showed that the newly formed blood vessel was a cylinder-like, bare endothelial tube with a rather smooth outer surface. Fibroblasts were situated around the endothelial tube. Several openings were found in the endothelial tube, suggesting the morphological feature of high permeability and fragility in newly formed blood vessels. The availability of three-dimensional reconstruction from scanning electron micrographs of serial semi-thin epoxy resin sections was discussed; structures of interest can be reconstructed (1) quickly and easily, (2) without skilful techniques, and (3) almost at the level of ultrastructure.     

Culture prior to transplantation preserves the ultrastructural integrity of monkey pancreatic islets

Transplantation of pancreatic islets represents a promising way of curing type I diabetes (insulin-dependent diabetes mellitus). Culture enables the survival of endocrine tissue awaiting islet transplantation and reduces islet immunogenicity prior to xenografting. In this study, attempts were made to preserve the monkey islets in culture for 7 days and to study the ultrastructure by electron microscopy. The islets were isolated from monkey pancreas by the collagenase digestion method and were separated from acinar cells by dextran density gradient centrifugation. These islets were preserved in a humidified atmosphere of 5% carbon dioxide and 95% air for 7 days. The culture medium used was CMRL-1066. After 7 days of culture the islets were processed for light and electron microscopic studies, which revealed that the cultured islets were intact and maintained their structural integrity. Semi-thin sections of the cultured islets showed morphology with occasional structural alterations at the periphery. Dithizone staining of the cultured islets showed crimson red colour, proving that the islets were pure and without any exocrine contamination. Electron microscopy showed that the cultured islets had well-preserved alpha-, beta- and delta-cells. Different cell types of the monkey pancreatic islets were identified by the presence of their characteristic secretory granules. The ultrastructural characteristics present in hormone-synthesizing cells, i.e. rough-endoplasmic reticulum, Golgi apparatus, mitochondria and secretory granules, were observed as in native islets.     

Synthesis and characterization of cerium–silver co-doped zinc oxide as a novel sunlight-driven photocatalyst for effective degradation of Reactive Red 120 dye

Cerium–silver (Ce–Ag) co-doped ZnO was synthesized by precipitation–decomposition and tested for the degradation of Reactive Red 120 dye under natural sun light irradiation. Three weight percent Ce co-doped Ag–ZnO was found to be most efficient. Hence, this catalyst (3 wt% Ce–Ag–ZnO) has been characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). XRD and XPS reveal the presence of metallic Ag and tetravalent Ce. Ag and Ce shift the absorption of ZnO to entire visible region. It was found that the Ce–Ag–ZnO exhibited higher degradation efficiency when compared to Ag-ZnO, Ce–ZnO, prepared ZnO, Commercial ZnO, TiO₂, and TiO₂-P25 at neutral pH (=7). Quantum yields of all processes were calculated and compared. Higher activity of Ce–Ag–ZnO in natural sunlight may be due to higher visible light absorption of Ce–Ag–ZnO when compared to native ZnO. The influences of operational parameters such as the amount of photocatalyst, dye concentration, initial pH on photo mineralization of RR 120 have been analyzed. The mineralization of RR 120 dye was confirmed by chemical oxygen demand (COD) measurements. A dual mechanism has been proposed for efficient degradation of RR 120 dye by Ce–Ag–ZnO under solar light at neutral pH. This photocatalyst was found to be reusable up to four runs.     

Conventional Electron Microscopy and Electron Holography of Organic Solar Cells

Organic solar cells are a promising route towards large‐area and low‐price photovoltaic systems. The devices are composed of at least two layers: the hole‐transport layer and the electron‐transport layer. The light absorption can occur in one or both layers. At the interface of the layers the excitons are separated into charge carriers, and every layer deals with one type of carrier. Higher efficiencies of the separation process can be obtained by using a mixed layer containing both materials to obtain a very high interface area. Although the structure of the mixed layers used plays a crucial role for the device performance, until now the morphologies have not been elucidated. In order to correlate physical and optical findings with structure and morphology for the materials in question, electron microscopy experiments were performed on the single components as well as on the layer systems. The conventional electron microscope is a poor phase microscope. As consequence, weak‐phase objects like organic molecules have to be stained or imaged under strong defocus to produce an observable contrast. Artifacts caused by chemical staining and the appearance of Fresnel diffraction using the defocus technique represent the main problems of conventional microscopy. These artifacts can be avoided using electron holography. Holograms of ultrathin sections of thin layers composed of organic dye molecules were recorded. Subsequently, the phase images were reconstructed. In this manner, we succeeded in obtaining high‐contrast electron micrographs without applying staining or defocus. In addition, holograms of crystalline C₆₀ and zinc phthalocyanine were successfully recorded. Holography has been shown to be a useful tool to image beam‐sensitive and weak‐phase objects without artifacts.     

中枢神经细胞瘤的超微结构观察

应用光镜检查及免疫标记,对2例中枢神经细胞瘤进行电镜观察.结果显示,中枢神经细胞瘤在电镜下具有一定的结构特征：瘤细胞间可查见桥粒样结构;胞质内有神经分泌颗粒和哑铃状致密核心颗粒;胞突内有丰富的微管和囊泡状结构.在外科病理诊断中,中枢神经细胞瘤难以与少突胶质细胞瘤、透明细胞室管膜瘤等肿瘤鉴别.电镜检查对鉴别诊断有比较重要的意义.肿瘤细胞胞突内的微管和囊泡、胞质内的神经内分泌颗粒等超微结构与免疫组织化学标记一致,同时也提示肿瘤起源于神经元细胞.     

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.     

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.     

基于可见光响应的纳米硒半导体器件

不加入任何表面活性剂、利用溶解-重结晶原理合成了一维结构的Se纳米带,并用XRD,SEM及TEM等方法对产品进行了表征。以单根的硒纳米带为光电响应材料、以银浆为接触电极组装成纳米器件,并对光电响应特性以及光谱响应特性进行了测试。结果表明:在可见光(日光灯)照射下,其对开灯的最快响应时间约为30 ms,关灯时最快衰减时间为50 ms。该纳米器件的光电流对温度有依赖作用,低温下有利于光电流的产生;单色光的波长对纳米器件的光电流及响应时间的影响不同,在单色光谱响应范围内,纳米器件在650 nm处产生的光电流最大,而在350 nm处的响应时间最快。     

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.     

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.     

Electron sensitive resists derived from vinylether-maleic anhydride copolymers

A series of polymers derived from vinylether-maleic anhydride copolymers were prepared and their electron sensitivities measured. One of the most sensitive polymers prepared was the allyl half-ester of an octadecylvinylether maleic anhydride copolymer (VL-100). This material was stable, formed excellent films, and was insensitive to UV and visible light with λ > 2000 Å. The sensitivity to electron irradiation was found to be 4 × 10^-8C/cm²at 9 keV compared to 5 × 10^-7for KTFR under identical conditions. The evaluation of this material as an electron resist as well as sensitivity data for numerous derivatives is reported.     

Appearance of electron-dense segments: indication of possible conformational changes of pre-mineralizing collagen fibrils in the osteoid of rat bones

To elucidate precise mechanisms of appositional mineralization of bone, structural features of mineralizing collagen fibrils of the osteoid in normal and hypocalcaemic rats were examined in detail by transmission electron microscopy. Ultrathin sections of the osteoid of various types of bones of the rats fed with regular or normal calcium diet often displayed electrondense segments in the specific regions of the collagen fibrils located immediately adjacent to the mineralization front or to the mineralization nodules. Such dense segments appeared only after Ur-Pb staining and were more distinct in undecalcified specimens. Dense segments were undetectable in ultrathin sections picked up on ethylene glycol instead of water in the trough, even after Ur-Pb staining. Collagen fibrils in the widened osteoid of hypocalcaemic rats fed with calcium-free diet failed to show electron-dense segments. A careful comparison between the hydrously or anhydrously processed adjacent sections of a normal rat bone indicated a drastic dissolution of electron-dense material from the bone matrix near the mineralization front in hydrously processed sections and, thus, implicated the presence of labile mineral-matrix complexes in the recently mineralized bone matrix. Such labile sediments were readily dissociated within the ultrathin sections while the sections were floating on water and immediately adsorbed onto the pre-mineralizing collagen fibrils, where some conformational changes might have occurred. These data indicate that highly electron-dense segments appearing in the osteoidal collagen fibrils are a type of process-induced product, which indirectly represent possible structural alterations in the segmental portions of pre-mineralizing collagen fibrils in the osteoid of rat bones.     

热氧化磁控溅射金属锌膜制备ZnO纳米棒

利用射频磁控溅射技术在Si(111)衬底上制备金属锌膜 ,在空气中退火热氧化合成了一维ZnO纳米棒。用X射线衍射 (XRD) ,扫描电子显微镜 (SEM) ,透射电子显微镜 (TEM)和光致发光谱 (PL)对样品进行了结构、形貌及光学特性分析。结果表明 :ZnO纳米棒为六方纤锌矿结构单晶相 ,直径在 30～ 6 0nm左右 ,其长度可达5～ 8μm左右。在 2 80nm波长光激发下 ,有很强的 372nm带边紫外光发射和较微弱的 5 16nm深能级绿光发射 ,说明合成的单晶ZnO纳米棒的质量较高     

Enhanced detection efficiency of genetically encoded tag allows the visualization of monomeric proteins by electron microscopy

A cadmium-binding, genetically encoded protein tag, consisting of three repeats of metallothionein (3MT), can be used in electron microscopy for the visualization of multimeric- but not monomeric-tagged proteins due to insufficient electron density in monomeric proteins. Here, we present a technique for detecting monomeric 3MT-tagged green fluorescent protein (GFP-3MT) using a platinum compound to intensify the electron density. Substitution of cadmium by platinum as a result of incubating purified cadmium-binding 3MT-tagged GFP (GFP-Cd-3MT) with cis-diamminedichloroplatinum(II) (cisDDP) was assessed by a UV absorption band centered at 284?nm thereby indicating platinum-sulfhydryl bonds. The incubation time and the concentration of cisDDP to reach maximal absorption were 2?h and 36-fold molar equivalent of cisDDP, respectively. GFP-Pt-3MT isolated by gel filtration chromatography contained 29 platinum atoms per single GFP-3MT molecule. Electron-dense particles were observed in a GFP-Pt-3MT sample by electron microscopy without negative staining. Further image processing and image analysis demonstrated that particles with higher density relative to their surroundings were detectable in both GFP-Cd-3MT and GFP-Pt-3MT samples. These results demonstrate that replacement of cadmium with platinum, together with proper image analyses, improve detection efficiency and enable the visualization of 3MT-tagged monomeric protein by electron microscopy.     

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.     

New versatile staining reagents for biological transmission electron microscopy that substitute for uranyl acetate

Aqueous uranyl acetate has been extensively used as a superb staining reagent for transmission electron microscopy of biological materials. However, recent regulation of nuclear fuel material severely restricts its use even for purely scientific purposes. Since uranyl salts are hazardous due to biological toxicity and remaining radioactivity, development of safe and non-radioactive substitutes is greatly anticipated. We examined two lanthanide salts, samarium triacetate and gadolinium triacetate, and found that 1-10% solution of these reagents was safe but still possess excellent capability for staining thin sections of plastic-embedded materials of animal and plant origin. Although post-fixation with osmium tetroxide was essential for high-contrast staining, post-staining with lead citrate could be eliminated if a slow-scan CCD camera is available for observation. These lanthanide salts can also be utilized as good negative-staining reagents to study supramolecular architecture of biological macromolecules. They were not as effective as a fixative of protein assembly, reflecting the non-hazardous nature of the reagents.     

透射电镜电子染色方法的改良

为提高透射电镜生物样品超薄切片的染色效率,在传统染色方法基础上进行改良,使用自制染色装置,在较短时间内完成大批生物样品超薄切片的电子染色。与传统方法相比较省时省药,减少污染机率,电镜观察的超微结构清晰,反差较好。     

介孔ZnO微球的制备及其在染料敏化太阳能电池中的应用

制备了一种新型的染料敏化太阳电池的光阳极,该电极由溶剂合成的具有高比表面积和良好光散射特性的ZnO介孔微球组成。采用X射线衍射、扫描电子显微镜、能谱仪及N2吸附脱附等手段,分析了介孔ZnO微球的结构和形貌。所得介孔微球尺寸在亚微米范围,比表面积约为50m2.g-1。将ZnO介孔微球成功应用到染料敏化太阳电池中,当光阳极为3μm时,组成的原型器件的短路电流密度约为4.5mA.cm-2,开路电压约为602mV,转换效率可达1.28%。研究结果表明,ZnO介孔微球是一种优异的染料敏化太阳电池的光阳极材料。