Antibody-decorated dystrophin molecule of murine skeletal myofiber as seen by freeze-etching electron microscopy

Dystrophin is the protein product of Duchenne muscular dystrophy gene which is defective in this genetic disorder. Here we identified ultrastructurally the dystrophin molecule from the various cytoskeletons at the cytoplasmic surface of murine myofiber plasma membrane by using quick-freeze, deep-etch, rotary-shadow replica of anti-dystrophin antibody-decorated muscle samples. The molecule was really cytoskeleton and incorporated in the meshwork of the plasma membrane-associated cytoskeletons. The molecule appeared to connect directly and/or through another cytoskeletal molecule with actin filament.     

Visualization and identification of cytoskeletal filaments in embryonic chick heart by the quick-freeze deep-etch method combined with immunocytochemistry

Cytoskeletal filaments in myocardial cells of chick embryo (stage 18-20, day 3) were studied by immunocytochemical and rapid-freeze deep-etch methods. A three-dimensional network of cytoplasmic filaments surrounding nascent myofibrils was visualized in saponin-treated myocardial cells. The major part of the network was composed of 12 to 14 nm filaments in platinum replicas. To identify the filaments, the myocardial cells were permeabilized with Triton X-100 and treated with myosin subfragment-1 (S1) for actin or immunogold-labeled antibody for desmin. A large number of filaments in myofibrils and a few cytoplasmic filaments were decorated with S1. A loose network surrounding the myofibrils was not decorated with S1 but with gold particles. This finding means that the majority of filaments occupying the interfibrillar space were desmin-containing filaments.     

人精子甘露糖受体的胶体金标记电镜观察

本文首次报道人精子甘露糖受体（ＭＲ）的电镜标记技术和初步观察了，采用甘露糖化牛血清白蛋白（ＤＭＡ，Ｓｉｇｍａ，Ａ８３０３）结合１０ｎｍ胶体金制备探针，标记获能前后精子和甲醇处理或Ａ２３１８７诱导顶体反应（ＡＲ）的精子。结果表明，甲醇处理后ＭＲ表达顶体区质膜表面，活精子在ＡＲ过程中以不同方式表达ＭＲ活性；ＭＲ定位于：１．ＡＲ早期的精子顶体区质膜表面。２．ＡＲ期间精子的顶体小泡表面，顶体内膜及顶体基质     

He─Ne激光辐射对番鸭精子活力的影响

实验表明，Ｈｅ－Ｎｅ激光照射能提高番鸭精子的活力，使它们在体外存活的时间延长２４～３６小时。如果照射精液孵育在６℃冰箱中，可存活时间６０个小时，若用激光间断照射，则可以存活１０８小时。但随着孵育时间的延长，精了畸形比例提高。     

Translocation of sLe(x) on the azurophilic granule membrane to the plasma membrane in activated human neutrophils

Using immunogold electron microscopy, we found that human neutrophilic sialyl Lewis x (sLe(x)), an adhesive ligand for selectins, detectable by a monoclonal antibody, KM-93, is present in the sacculi of the Golgi apparatus as well as on the membranes of large electron-lucent azurophilic granules and the plasma membrane, including surface projections and microvilli. Neutrophilic sLe(x), however, was not detected on the membranes of specific granules. In comparison with the distribution of sLe(x), CD18 was localized on the plasma membrane and specific granule membrane but not on the azurophilic granule membrane. We also found by immunogold electron microscopy and flow cytometry that treatment of neutrophils with sialidase resulted in a loss of sLex on the plasma membrane. In contrast, intracellular sLex on the azurophilic granule membrane was not destroyed by sialidase. When sialidase-treated neutrophils were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory mediator peptide, in the presence of cytochalasin B, we observed by immunogold electron microscopy and flow cytometry that sLe(x) again appeared on the plasma membrane. These results indicate that stimulation by fMLP induces the up-regulation of sLe(x) on the cell surface by promoting translocation of sLe(x) from the azurophilic granule membrane to the plasma membrane in human neutrophils.     

Clathrin sheets on the protoplasmic surface of ventral membranes of osteoclasts in culture

Physical cell-shearing resulted in various degrees of disruption of the basolateral (upper) membranes, cytoskeletons or cell organelles and exposed the protoplasmic surface of ventral (adhesion) membranes of osteoclasts that were attached to the underlying substratum, such as coverslips, mica or synthetic apatite plates. Freeze-dried replicas of the ventral membranes left behind on the substratum after cell-shearing provided three-dimensional information on the ultrastructure of the protoplasmic membrane surface of cultured osteoclasts. An extensive area of the protoplasmic surface and various amounts of cytoskeletal structures attached to the adherent ventral surface of the plasma membrane were visible. In particular, the most characteristic finding of the present study is that numerous clathrin sheets displaying various sizes, shapes and curvature were revealed on the ventral membrane. The polygon substructures of the clathrin lattices appeared to be composed of hexagons with a few pentagons interspersed. They were seen at the peripheral membranes where they were situated at the sites of close contact with the underlying substratum. In addition, clathrin lattices were never observed on the basolateral (upper) membranes. In favourable stereo views, most cytoskeletons were not in direct contact with the clathrin sheets. However, a few observations indicated possible remnants of cytoskeletons attached to clathrin lattices. Podosomes did not have a direct structural relationship to clathrin lattices. Although it is generally accepted that cytoskeletal podosomes in motile cells, such as osteoclasts, play a major role in cell adhesion, the present study indicates that membrane-associated clathrin might also function during attachment to the substrate. In this regard, clathrin is thought to be required for receptor-mediated endocytosis, but whether it might also function in cell attachment is still a matter for debate. This type of clathrin-related adhesion appears to be a previously unrecognized site of cell/substrate adhesion in osteoclasts. To assess this possible function, we focused on clathrin and related cytoskeletal elements on the ventral membranes of cultured osteoclasts.     

Distal surface of the rat ruffle-ended ameloblasts at the stage of enamel maturation observed by scanning electron microscopy

Distal surface of the rat ruffle-ended ameloblasts was observed by high resolution scanning electron microscopy. Specimens fixed by perfusion with 0.5% formaldehyde and 0.5% glutaraldehyde were decalcified with ethylenediamine tetraacetic acid and freeze-fractured using dimethyl sulfoxide. They were treated with 0.1% osmium tetroxide for 96 hr to remove excess cytoplasmic matrices, dehydrated, and critical-point dried. The present method was useful for observing both surface and intracellular structures simultaneously. The dense lamina lining the distal surface of the ruffle-ended ameloblasts having been dissolved in this preparation, the surface was clearly demonstrated in three dimensions under SEM. The surface was characterized by a complex labyrinth formed by protrusion and invagination of the plasma membrane. At high magnification, two kinds of minute granules are visible: small and larger granules measured as 10-20 nm and 70 nm in diameter, respectively. The former were more numerous than the latter. Furthermore, microfibrils connecting the protrusions of the plasma membrane were observed on the distal surface. The small granules probably connect the dense lamina with the distal surface of the ameloblasts. In addition, a denuded area free from the granules was sometimes recognized on the distal surface. These surface structures of the distal end of the ameloblasts appeared to be concerned with the enamel maturation.     

Multiple-wavelength conversion with gain by a high-repetition-rate pulsed-pump fiber OPA

The authors propose and demonstrate a novel multiple-wavelength converter with gain, based on a pulsed-pump fiber optical parametric amplifier (OPA). It generates multiple replicas of the signal, as well as spectrally inverted versions. The device is modeled by using quasi-steady-state OPA gain equations, as well as by the split-step Fourier method. Predicted conversion gains of up to 20 dB have been confirmed by experiments. A 10-Gb/s nonreturn-to-zero (NRZ) input signal was converted into several replicas, with penalties ranging from 0.26 to 1.24 dB for frequency shifts of /spl plusmn/k/spl times/100 GHz (k=1, 2, 3, 4).     

Reproducibility and applicability of gallium replication as evaluated by biological specimen use

Structures of biological surfaces pressed on to a pure liquid gallium surface were successfully traced on to the gallium surface by quick-freezing below the melting point (28.78 degrees C) in air or water for replication in scanning electron microscopy. Gallium's high surface tension (approximately 700 mN m(-1) at 30 degrees C) deteriorates the spatial resolution of replicas and destroys some types of specimens. Five different biological surfaces were replicated on to gallium surfaces to evaluate spatial resolution and specimen resistance, i.e. reproducibility and applicability. Gallium replication of jewel beetle wing and human hair demonstrated submicron spatial resolution in the horizontal direction at least. Trials of protozoa, bacteria, and culture cell replication showed that protozoa are suited to replication because the cell membrane has characteristic structures with sufficient resistance to the gallium surface.     

Immunocytochemical studies on co-localization of alpha-granule membrane alphaIIbbeta3 integrin and intragranular fibrinogen of human platelets and their cell-surface expression during the thrombin-induced release reaction

We employed a double-staining method to immunocytochemically study the role of alpha-granule membrane alphaIIbbeta3 integrin in the expression of intragranular fibrinogen (Fbg) and albumin on the surface of thrombin-activated human platelets. In unstimulated platelets, alphaIIbbeta3 was distributed along the alpha-granule membrane as well as on the cell-surface membrane, including the open canalicular system (OCS), while Fbg and albumin were exclusively and evenly detected in the granules. At 30 s after activation by 0.1 U ml(-1) thrombin under non-stirred conditions, a small amount of Fbg in the granules was redistributed in association with alpha-granule membrane alphaIIbbeta3, suggesting that co-localization between alphaIIbbeta3and Fbg occurs at this period during platelet activation. At 1-5 min, the shape of the platelets changed from discoid to spheroid with pseudopodia and intact alpha-granules were no longer observed in these cells. Fibrinogen associated with alphaIIbbeta3 became increasingly expressed on the membranes of both the swollen OCS and the cell surface. Nevertheless, abundant Fbg still remained in the lumen of the swollen OCS, the membrane of which had already been depleted of alphaIIbbeta3. By western blot analysis using anti-human Fbg antibody, we detected no Fbg in the surrounding medium of the thrombin-stimulated platelets for 5 min. Unlike Fbg, abundant albumin in the alpha-granules was released into the surrounding medium without association with the membrane of either the alpha-granules or the cell surface. These results suggest that a small portion of the intragranular Fbg may bind to an Fbg binding site expressed on the activated aIIbbeta3 on the alpha-granule membrane, resulting in the formation of an alphaIIbbeta3-Fbg complex, which is then moved through the OCS membrane and expressed on the cell-surface membrane. Thus, alpha-granule membrane aIIbbeta3 may act as a carrier of intragranular Fbg.     

A Photoluminescence Study of Plasma Reactive Ion Etching-Induced Damage in GaN

Ga N films with reactive ion etching(RIE) induced damage were analyzed using photoluminescence(PL).We observed band-edge as well as donor-acceptor peaks with associated phonon replicas,all in agreement with previous studies.While both the control and damaged samples have their band-edge peak location change with temperature following the Varshni formula,its intensity however decreases with damage while the D–A peak increases considerably.Nitrogen post-etch plasma was shown to improve the band edge peak and decrease the D–A peak.This suggests that the N2 plasma has helped reduce the number of trapped carriers that were participating in the D–A transition and made the D°X transition more active,which reaffirms the N2post-etch plasma treatment as a good technique to heal the Ga N surface,most likely by filling the nitrogen vacancies previously created by etch damage.     

Structural analysis of red blood cell membrane with an atomic force microscope

To evaluate the usefulness of the atomic force microscope (AFM) for structural analysis of biomedical samples and to determine suitable sample preparation methods for AFM observation, the membrane of human erythrocytes prepared by various methods for electron microscopy was examined by the AFM. Strand-like elevations with 20-50 nm in width, 30-80 nm in length and 3-5 nm in height were observed, which formed networks composed of squares, pentagons and hexagons on the cytoplasmic or back surface of the erythrocyte membrane. Using colloidal gold labelled antibody, this network was found to contain spectrin molecules. Therefore it was very likely that the undercoat molecules of the plasma membrane were imaged by AFM. A large number of gentle elevations 300-400 nm in diameter and 2 nm in height were found to be distributed uniformly on the extracellular or true surface of intact erythrocyte, presumably reflecting the presence of undercoat membrane skeleton on the cytoplasmic surface. However, no structure that seemed to be derived from glycocalyces was discernible on the true surface. Structure corresponding to the unit membrane or lipid bilayer structure observable by electron microscopy was not demonstrated in the cross-section of the membrane. In freeze-fractured samples, a large number of small particles that corresponded to the intramembranous particle were also demonstrated on the membrane halves. Since AFM allows depiction of the fine structures of biological samples with very simple sample processing at a resolution comparable to or exceeding that of SEM, imaging technology using AFM can be applied to obtain biomedical information. However, several problems have to be solved in future development of the equipment.     

Ultrastructural study of isolated rat hepatocyte suspensions incubated in incomplete or complete culture medium.

Ultrastructural changes in isolated rat hepatocytes (IHC) in suspension were examined after 1, 3, 5 and 10 hr incubation, and the influence of Krebs-Henseleit (K-H) buffer as an incomplete medium and GIT culture medium (Nihon Pharmaceutical Co.) as a complete culture medium were investigated. The viability was approximately 60-75% after 1, 3 and 5 hr incubation, and was slightly higher in GIT medium than K-H buffer. Immediately after isolation, IHC showed a cuboidal shape, and after 3 and 5 hr incubation, a round shape with numerous surface microvilli. Cytoplasmic organelles appeared almost normal. After 10 hr incubation, cells reaggregated and the number of autophagic vacuoles increased. Cellular changes, such as slight dilatation of the endosplasmic reticulum and Golgi apparatus, and bleb formation in the cell surface, were more often observed in IHC incubated in K-H buffer than in GIT medium. IHC incubated in GIT medium contained glycogen particles in the cytoplasm, and lipoprotein-like particles were observed in the cisternae of Golgi apparatus and smooth endoplasmic reticulum. The lipoprotein-like particles were found in the intercellular spaces after 10 hr incubation. These results suggest that GIT medium is superior to K-H buffer in its ability to preserve normal structure and function of IHC.     

福氏2a痢疾杆菌膜抗原的免疫电镜定位

本文报告应用抗福氏2a痢疾杆菌膜成份McAb及胶体金探针对细菌膜抗原进行了定位。结果显示4种抗脂多糖McAb(3A6、2E6、4F1、2A3)可不均一的识别位于细菌壁表面的抗原,而另外一种抗LPS的McAb种(1G8)和一种抗膜蛋白McAb(1A1)识别的抗原未暴露于细菌表面。应用细菌膜碎片包埋前染色成功定位了抗膜蛋白McAb(1A1)位于细菌内膜的抗原。与McAb的生物活性比较表明阳性标记细菌表面抗原的McAb的抗原与其阻段志贺氏菌接触性溶血试验和对小鼠的被动保护力密切相关。提示免疫电镜标记技术确定LPS抗原表位在细菌表面的可及性和拷贝数对构建和筛选痢疾工程菌苗具有重要意义。     

Cleaning and passivation of the Si(100) surface by low temperature remote hydrogen plasma treatment for Si epitaxy

This paper presents the results of a study of the hydrogen-passivated Si(100) surface prepared by a remote hydrogen plasma treatment which serves the dual purpose of cleaning and passivating the Si(100) surface prior to low temperature Si epitaxy by Remote Plasma-enhanced Chemical Vapor Deposition (RPCVD). The remote hydrogen plasma treatment was optimized for the purposes of cleaning and passivation, respectively. To achieve a clean, defect-free substrate surface, the remote hydrogen plasma process was first optimized using Transmission Electron Microscopy (TEM) and Auger Electron Spectroscopy (AES). For hydrogen passivation, the substrate temperature was varied from room temperature to 250° C in order to investigate the degree of passivation as a function of substrate temperature by examining the amount of oxygen readsorbed on the substrate surface after air exposure. Low temperature Si expitaxy was subsequently performed on the air-exposed substrates without further cleaning to evaluate the effectiveness of the hydrogen passivation. It was found that better Si surface passivation is achieved at lower substrate temperatures as evidenced by the fact that less oxygen is observed on the surface using AES and Secondary Ion Mass Spectroscopy (SIMS) analyses. The amount of readsorbed oxygen on the H-passivated Si surface after a two hour air exposure was found to be as low as 0.1 monolayer from SIMS analysis. Using Reflection High Energy Electron Diffraction (RHEED) analysis, different surface reconstructions ((3 × 1) and (1 × 1)) were observed for H-passivated Si surfaces passivated at various temperatures, which was correlated to the results of AES and SIMS analyses. Epitaxial growth of Si films at 305° C was achieved on the air-exposed Si substrates, indicating a chemically inert Si surface as a result of hydrogen passivation. A novel electron-beam-induced-oxygen-adsorptiom phenomena was observed on the Hpassivated Si surface. Scanning Auger Microscopy (SAM) analysis was performed to study the reaction kinetics as well as the nature of Si—H bonds on the H-passivated Si surface. Preliminary results show that there is a two-step mechanism involved, and oxygen adsorption on the H-passivated Si surface due to electron beam irradiation may be due to the formation of O-H groups rather than the creation of Si—O bonds.     

Ultracytochemical demonstration of Ca2(+)-ATPase activity in the rat saphenous artery and its innervated nerve terminal

The localization of Ca2(+)-ATPase activity was ultracytochemically investigated in the rat saphenous artery and nerve terminals innervating the saphenous artery using a lead citrate method devised by Ando et al. (1981). Intense reaction products in the saphenous arterial endothelial cells were observed inside the caveolae and vesicles along the luminal and abluminal sides. In addition, Ca2(+)-ATPase activity was observed on the external side of the luminal, abluminal and lateral plasma membrane, and the outer membrane of mitochondria. In the smooth muscle cells, intense Ca2(+)-ATPase activity on the inside of caveolae and vesicles was observed, comparing in intensity with that on the plasma membrane of smooth muscle cells. In the nerve terminals innervating the saphenous artery, Ca2(+)-ATPase activity was demonstrated on the plasma membrane of the nerve terminal-Schwann cell interface, the axolemma of unmyelinated axons and the plasma membrane of Schwann cells. It is suggested from the above ultracytochemical results that Ca2(+)-ATPase activity plays an important role in the contraction and relaxation of the saphenous artery, and in the neurotransmitter release.     

High‐Density Hotspots Engineered by Naturally Piled‐Up Subwavelength Structures in Three‐Dimensional Copper Butterfly Wing Scales for Surface‐Enhanced Raman Scattering Detection

Very recently, wing scales of natural Lepidopterans (butterflies and moths) manifested themselves in providing excellent three dimensional (3D) hierarchical structures for surface‐enhanced Raman scattering (SERS) detection. But the origin of the observed enormous Raman enhancement of the analytes on 3D metallic replicas of butterfly wing scales has not been clarified yet, hindering a full utilization of this huge natural wealth with more than 175 000 3D morphologies. Herein, the 3D sub‐micrometer Cu structures replicated from butterfly wing scales are successfully tuned by modifying the Cu deposition time. An optimized Cu plating process (10 min in Cu deposition) yields replicas with the best conformal morphologies of original wing scales and in turn the best SERS performance. Simulation results show that the so‐called “rib‐structures” in Cu butterfly wing scales present naturally piled‐up hotspots where electromagnetic fields are substantially amplified, giving rise to a much higher hotspot density than in plain 2D Cu structures. Such a mechanism is further verified in several Cu replicas of scales from various butterfly species. This finding paves the way to the optimal scale candidates out of ca. 175 000 Lepidopteran species as bio‐templates to replicate for SERS applications, and thus helps bring affordable SERS substrates as consumables with high sensitivity, high reproducibility, and low cost to ordinary laboratories across the world.     

7A9--Density and temperature of a laser induced plasma

The plasma "blow-off vapor," produced when a high power laser beam strikes a solid surface, has been studied experimentally and theoretically. Electron densities and plasma optical thicknesses have been measured both in time and position resolved manners for plasmas produced from carbon. The ruby laser system consisted of a "Q-switched" oscillator and one amplifier, both 7 inches long and 0.6 inch in diameter. The system energy output was between 8 and 13 joules in a 0.055 μ-second pulse width at half height. The electron density was measured using a gas laser as a probe light source and a Mach-Zender interferometer to measure the phase shifts due to plasma refractivity. The experiments were performed with incident laser energies of 10 to 1000 joules/cm²(by focusing). Electron densities as high as 10¹⁹cm^-3were observed, with correspondingly high plasma optical thicknesses. There were strong indications of heating by the incident laser beam of the front edge of the plasma, which was not expected on the basis of plasma heating by free-free and bound-free absorption due to the low electron density existing there. In general, a theoretical treatment of plasma heating by the bound-free and free-free absorption agreed well with experiments.     

水稻花后衰退珠心和胚乳发育初期Ca2+的超微细胞化学定位

应用焦锑酸盐沉淀技术对水稻花后衰退珠心和胚乳发育初期进行了Ca^2 的超微细胞化学定位。结果显示在初始衰退的珠心细胞中Ca^2 主要分布于液泡膜上和核内；在衰退中期的珠心细胞中，Ca^ 主要分布在核膜、液泡膜及质膜上；在严重衰退的珠心细胞，Ca^2 仅存在于液泡中。珠心降解的Ca^2 跨过胚囊壁，通过质外体向胚乳内运输；发育初期的胚乳细胞，Ca^2 主要位于胞间隙，线粒体和液泡中也有少量分布。讨论了Ca^2 与珠心PCD的关系。     

基于结构光投影的薄膜振动模式分析

为了验证薄膜振动模式的有效性,采用正弦条纹投影和傅里叶条纹分析的方法进行薄膜振动模式分析和振幅重建,并进行了理论分析和实验研究。在基于结构光的主动3维传感技术中,傅里叶变换轮廓术具有单帧获取、高分辨率、全场可实时测量等优势,成为可测量动态3维面形的一种实用方法。正弦光栅条纹被投影到振动中的薄膜表面,采用低帧频的CCD相机采集由薄膜振动导致条纹局部模糊的一系列变形条纹图。通过傅里叶变换轮廓术方法进行处理,最终得到不同频率下实际测量的薄膜振动模式结果。给出了理论计算结果与实测结果的验证比较。结果表明,该方法测量的振动模式结果准确反映了薄膜振动情况。3维面形测量结果和实验验证了该方法的可行性。