Abnormal formation of the glucan network from regenerating protoplasts in Schizosaccharomyces pombe cps8 actin point mutant

To study the close relationship between the actin cytoskeleton and cell wall formation, the process of cell wall formation in reverting protoplasts of the fission yeast, Schizosaccharomyces pombe, cps8 actin point mutant was investigated by ultra-high-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy (TEM). The protoplast of the cps8 mutant began to form a glucan network in a unipolar manner and to secrete alpha-galactomannan. The site of cell wall formation grew in a cylindrical shape in the wild-type protoplast. The alpha-galactomannan did not fill in the intrafibrillar spaces completely, however, and the fibrils were exposed on the cell surface. UHR-LVSEM images indicated that the glucan fibrils were thin and rope-shaped, forming a looser network than the wild-type. TEM images indicated the finest fibrils were approximately 1.5 nm in diameter, the same diameter as the wild-type. These results suggest that the cps8 mutant was insufficient in developing cross-linkage with the glucan fibrils up to the wide ribbon shape as found in the wild-type [Osumi M et al. (1989) J. Electron Microsc. 38: 457-468; Osumi M (1998) Micron 29: 207-233]. These findings appear to indicate that the actin cytoskeleton controls formation of the glucan network and secretion of beta-1,6-glucan, and confirm the close relationship of the actin cytoskeleton and glucan formation.     

Ultrastructure and behavior of actin cytoskeleton during cell wall formation in the fission yeast Schizosaccharomyces pombe

Fluorescence microscopy has shown that F-actin of the fission yeast Schizosaccharomyces pombe forms patch, cable and ring structures. To study the relationship between cell wall formation and the actin cytoskeleton, the process of cell wall regeneration from the protoplast was investigated by transmission electron microscopy (TEM), immunoelectron microscopy (IEM) and three-dimensional reconstruction analysis. During cell wall regeneration from the protoplast, localization of F-actin patches was similar to that of the newly synthesized cell wall materials, as shown by confocal laser scanning microscopy (CLSM). In serial sectioned TEM images, filasomes were spherical, 100-300 nm in diameter and consisted of a single microvesicle (35-70 nm diameter) surrounded by fine filaments. Filasomes were adjacent to the newly formed glucan fibrils in single, cluster or rosary forms. By IEM analysis, we found that colloidal gold particles indicating actin molecules were present in the filamentous area of filasomes. Three-dimensional reconstruction images of serial sections clarified that the distribution of filasomes corresponded to the distribution of F-actin patches revealed by CLSM. Thus, a filasome is one of the F-actin patch structures appearing in the cytoplasm at the site of the initial formation of the cell wall and it may play an important role in this action.     

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.     

百合小孢子母细胞形成和发育过程中细胞间连接形态和结构的变化

超显微结构水平观察发现造孢时期细胞的壁较厚，由中间层，初生和次生壁组成，细胞间的连接为典型的细胞间连丝，减数分裂前间期的细胞壁主要由中间和初生壁组成，典型的胞间连丝消失，但局部位置上的中间层和初生壁组分缺失，构成约２００～８００ｎｍ，宽的“豁口”，进入分裂期后，次生壁组分急剧增加，当整个细胞壁加厚到２４０－５００ｎｍ时，细胞壁出现穿孔，穿孔过程似乎有下列两种方式：１）在细胞壁的一侧或两侧出现电子致     

In situ localization of cell wall alpha-1,3-glucan in the fission yeast Schizosaccharomyces pombe

The yeast cell walls of the budding yeast Saccharomyces cerevisiae are well studied and the results show the existence of a framework composed of beta-1,3-glucan. It is reported that the cell wall of the fission yeast Schizosaccharomyces pombe has different components and our analysis by 13C-nuclear magnetic resonance (NMR) spectroscopy also showed there is alpha-1,3-glucan in its cell wall. To refine our understanding of the architecture of the yeast cell wall, we re-examined the cell wall glucans of S. pombe by NMR spectroscopy and prepared antibody against alpha-1,3-glucan, which is a characteristic component of this yeast. By the competitive enzyme-linked immunosorbent assay (ELISA) system, specificity of the antibody was restricted to alpha-1,3-glucan, which did not take a highly ordered structure. We analysed the localization of the cell wall glucans by immunoelectron microscopy. Transmission electron microscope (TEM) images showed that most of the alpha-1,3-glucan was along the cell membrane and appeared to enclose the cytoplasm, supporting previous reports that this glucan is synthesized on the cell membrane.     

扫描电镜的最新发展——低电压扫描电镜（LVSEM）和扫描低能电镜（SLEEM）

本文从仪器和电子光学的观点评述了在扫描电镜上的装备场发射枪以后,向低电压扫描电镜和扫描低能电镜发展中的一些重要问题。讨论了低能和极低能时二次电子和背散射电子的特性,低电压扫描电镜作扫描低能电镜能量段的划分,实现低电压扫描电镜和扫抵低能电镜的困难,以及利用阻场透镜的解决办法。讨论了低能情形下信号的探测,杂散电磁场的影响的估计和仪器的分辨率。     

Ultrastructural verification of anchoring role of lamina fibroreticularis of dental basement membrane in odontogenesis

In a previous study of the developing tooth a characteristic fibrillar layer associated with the basement membrane of the inner enamel epithelium was found to be a highly specialized lamina fibroreticularis of the basement membrane which is unusually rich in basotubules, 10 nm wide microfibril-like structures. In this study this layer was further examined in detail in the hope of ultrastructurally elucidating its role in odontogenesis. Tooth germs of the monkey (Macaca fuscata) were processed for thin section observations. Dental papilla cell processes were inserted into the lamina fibroreticularis and their surface was closely associated with numerous parallel basotubules. With high-resolution observations the space between the surface and nearest basotubules as well as the spaces between neighbouring basotubules were bridged by 1.5-3 nm wide filaments running perpendicular to the axis of basotubules. These results indicate that the processes of dental papilla cells are linked to groups of basotubules by means of 1.5-3 nm wide filaments. Immunoperoxidase staining showed the presence of fibronectin along basotubules as well as within the space between the process and basotubule. This result, together with the comparison of these filaments with microfibril-associated 1.2-3 nm wide fibronectin filaments and the reported abundance of fibronectin in the basement membrane area during odontogenesis, indicates that these 1.5-3 nm wide filaments are composed of fibronectin. After immunostaining for amyloid P component, done with the rat tissue because of the nature of an available antiserum, basotubules in the lamina fibroreticularis were positively stained, as has been shown in basotubules/microfibrils in other locations. Microfibrils function as anchoring rods by interlinking connective tissue components to one another and to the cells. Basotubules, thought to be basement membrane-incorporated microfibrils, in the lamina fibroreticularis in this study are also likely to function as an anchoring device to immobilize dental papilla cells along the basement membrane. Such an arrangement of mesenchymal cells is known to be crucial for the successful differentiation of odontoblasts in the developing tooth.     

Development of the Fibrillar and Microfibrillar Structure During Biomimetic Mineralization of Wood

Wood is a hierarchical composite, consisting at its lowest hierarchy level of crystalline cellulose elementary fibrils with diameters of 2–4 nm embedded in a matrix of hemicelluloses and lignin. At the micrometer scale, it has a cellular architecture resembling a honeycomb structure. The transformation of the hierarchical wood structure into a silica replica has been reported recently. Its formation process and structural details are studied in this contribution. First, a silica/biopolymer composite is prepared by wood delignification and cell‐wall modification, followed by silica precursor infiltration and condensation. The calcination process is monitored to gain insight into the structure development upon decomposition of the biopolymers. The material changes its architecture gradually from fibrillar structures of 10–20 nm in diameter with homogeneous electron density, into fibrils of 8–10 nm in diameter with inhomogeneous electron density, exhibiting internal sub‐fibrillar structures of about 2 nm in diameter. The steps of the successful replication of the cellulose elementary fibrils into nanopores of similar diameter and orientation in a fibrillar silica matrix are demonstrated. These nanopore replicas of the original cellulose are wound in a steep helix within the macropore walls. These advanced materials may have lightweight structural applications and the nanopores may be advantageous for molecular separation.     

Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata

Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomyces cerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10?min, filamentous materials covered the entire surface of the protoplast at 1?h, granular materials started filling interspaces of filamentous materials at 2?h and regeneration was completed at 6?h. The filamentous materials consisted of microfibrils of various widths ranging from ≤5 to 40?nm, and composed of β-glucan. Protoplasts of the two chitin synthase mutant strains of Δchs3A and Δchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes.     

Influence of surrounding media on preservation of cell wall ultrastructure of Candida albicans revealed by low temperature scanning electron microscopy.

Influence of surrounding media on the surface structures of the cell wall of Candida albicans was discussed with respect to the preservation of ultrastructure during the specimen preparation for scanning electron microscopy. The fibrillar structure of the cell surface was distinctly identified by the rapid-freezing technique. It was difficult, however, to observe this structure by the conventional specimen preparation technique. The reason for the difference between these two preparation techniques was studied using a low temperature SEM. Through investigating the influence of each step of the conventional technique on the fibrillar structure, it was found that the fibrils were drastically deformed and disappeared during the dehydration step in ethanol above 80% in concentration. In order to study which physicochemical properties participated in this disappearance phenomenon, yeast cells were treated with various media: solutions in different pH ranges and at different salt concentrations, ionic solutions, surfactants, formamide, dimethyl sulfoxide, acetone and Fehling's solution. As a result, the fibrillar structure was found well preserved when the medium had an affinity for the constituent molecules of the fibrils. When without affinity, the fibrils suffered a remarkable deformation. The mechanism of this deformation is discussed in terms of molecular interaction of solute and solvent.     

Application of a quick-freezing and deep-etching method to pathological diagnosis: a case of elastofibroma

A case of elastofibroma in a middle-aged Japanese woman was examined by the quick-freezing and deep-etching (QF-DE) method, as well as by immunohistochemistry and conventional electron microscopy. The slowly growing tumor developed at the right scapular region and was composed of fibrous connective tissue with unique elastic materials called elastofibroma fibers. A normal elastic fiber consists of a central core and peripheral zone, in which the latter has small aggregates of 10 nm microfibrils. By the QF-DE method, globular structures consisting of numerous fibrils (5-20 nm in width) were observed between the collagen bundles. We could confirm that they were microfibril-rich peripheral zones of elastofibroma fibers by comparing the replica membrane and conventional electron microscopy. One of the characteristics of elastofibroma fibers is that they are assumed to contain numerous microfibrils. Immunohistochemically, spindle tumor cells showed positive immunoreaction for vimentin, whereas alpha-smooth muscle actin, desmin, S-100 protein and CD34 showed negative immunoreaction. By conventional electron microscopy, the tumor cell had thin cytoplasmic processes, pinocytotic vesicles and prominent rough endoplasmic reticulum. Abundant intracytoplasmic filaments were observed in some tumor cells. Thick lamina-like structures along with their inner nuclear membrane were often observed in the tumor cell nuclei. The whole image of the tumor cell was considered to be a periosteal-derived cell, which would produce numerous microfibrils in the peripheral zone of elastofibroma fibers. This study indicated that the QF-DE method could be applied to the pathological diagnosis and analysis of pathomechanism, even for surgical specimens obtained from a patient.     

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.     

Electron microscopical evaluation of connective tissue healing to periodontally involved teeth.

The present study was undertaken to examine if our novel root treatment for human periodontally involved teeth can facilitate a new connective tissue attachment. The novel treatment was in two parts: 1) planning and surface decalcification with citric acid on the coronal quarter of the exposed root corresponding to pocket, and 2) curettage of superficial cementum of the apical three quarters of the exposed root. The interface of the root surface and regenerating connective tissue was observed 2 and 3 weeks after surgery by light and electron microscopy. The apical migration of junctional epithelium was effectively prevented. The fibroblasts synthesized new fibrillar materials and collagen fibrils towards the curetted cementum. The results suggested the possibility that this treatment for periodontally involved teeth would provide ideal root surface conditions for the formation of new fibrous attachment.     

Imaging saponin-induced structural changes in neural processes with atomic force microscopy

Temporal changes in the structure of neuronal processes in the presence of saponin were studied by atomic force microscopy in a fluid medium. After saponin treatment, concavities were formed on the surface of some neurites and fibrous structures in other neurites were splayed. The vertical height of these splayed fibrils or fibrillar bundles ranged from 13 to 370 nm, and the horizontal width was less than 500 nm. These findings suggest that formation of concavities and separation of bundled fibrils occurred simultaneously in saponin-treated neurites.     

Scanning electron microscopy of proteoglycans in metaphyseal cartilage.

Alcian blue (AB) was used for scanning electron microscope investigations on metaphyseal cartilage. In the pericellular area three-dimensional network connecting the cell membrane surface to the lacunar wall is evident. The network is formed by very long rod-like filaments about 50 nm thick. The segments may be interpreted as the proteoglycans (PGs) of the pericellular area. In the pericellular area in hypertrophic and degenerative zones, the rod-like segments are closely connected to "chain granules" which are the morphological expression of Ca-P non crystalline compounds. The rod-like segments are not at all evident either in glutaraldehyde-osmium fixed fragments or in predigested-(streptococcal hyaluronidase and chondroitinase) AB stained ones. It is concluded that AB is a good method to detect the three-dimensional spatial disposition of cartilage PGs.     

烟草单个花粉原生质体透射电镜样品制备及其超微结构观察

本文介绍了烟草花粉原生质体作试验材料，用自制微吸管手工预先挑选单个花粉原生质体，戊二醛微滴固定，然后用低熔点琼脂糖或明胶预包埋，小塑料管顶扣包埋单个花粉原生质体，进行透射电镜样品制备，初步建立了单个原生质体电镜制样的技术流程，通过电镜观察，与群体制样法比较，结果是今人满意的。此项技术的建立，为今后植物性细胞超微结构的研究，提供了一项精细的操作方法。     

Electrostatic correction of the chromatic and of the spherical aberration of charged-particle lenses (part I).

A feasible electrostatic corrector (ECO) is outlined and the principle of the electrostatic correction is elucidated by means of a light-optical analogue. The ECO compensates for the chromatic and the spherical aberration of charged-particle lenses and reduces the resolution limit of a special LVSEM (low-voltage scanning electron microscope) from 6 nm to 1.4 nm. The geometry of the electrodes of the corrector is optimized with respect to the chromatic correction, the maximum strength of the electric field, and the residual higher-order aberrations which limit the resolution. In addition the stability criteria of the electric power supplies are discussed in detail.     

Quantitative three-dimensional structural analysis of Exophiala dermatitidis yeast cells by freeze-substitution and serial ultrathin sectioning

The morphologies, numbers, sizes and volumes of all organelles and cell components identified on ultrathin sections of aerobically grown exponential phase yeast cells of Exophiala dermatitidis in G1 phase were examined by freeze-substitution fixation and serial ultrathin sectioning. The cell wall consisted of three layers and occupied approximately 22% of the cell volume. The nucleus was approximately 1.8 microm in diameter and occupied approximately 7% of the cell volume. There was only one nucleolus in the nucleus and it occupied approximately 16% of the nuclear volume. There were 17-52 mitochondria per cell, occupying 7-12% of the cell volume. Five to ten endoplasmic reticula were present per cell; these occupied only 0.2% of the cell volume and did not form a network. There were 1-4 vacuoles per cell and they occupied 4-10% of the cell volume. Storage material was round and electron transparent and occupied 4-11% of the cell volume. The cytosol occupied 43-53% of the cell volume. The Golgi apparatus, spindle pole body, autophagosomes, multivesicular bodies, lipid bodies, microtubules and microfilaments occupied approximately 1% of the cell volume in total. About 200,000 ribosome particles, 1000 glycogen granules and several tens of microtubules (average length 0.78 microm) were present per yeast cell. The membranes of this yeast could be classified into three groups by their appearance and thickness. This is the first report, to our knowledge, that analysed all the components in the yeast cell quantitatively and in three dimensions, and provides fundamental information for understanding various aspects of cell biology.     

Application of a FIB-STEM system for 3D observation of a resin-embedded yeast cell

The combination of a focused ion beam (FIB) system and a scanning transmission electron microscope (STEM) has been applied to the three-dimensional (3D) observation of a resin-embedded yeast cell. Using a FIB microsampling technique, a sample with a thickness of tens of micrometres was extracted from a resin-embedded block sample. The extracted sample was transferred to a FIB-STEM-compatible specimen rotation holder and trimmed by FIB milling for 3D STEM observation. Although the FIB milling was carried out at an operating voltage of 40 KV, the sample was cross sectioned without forming a harmful damage layer on its surface. Cell structures, such as cell wall, cell membrane, mitochondria, peroxisomes, endoplasmic reticulum and vacuoles, were observed clearly in a pillar-shaped sample of 20 microm long, 4 microm wide and 3 microm deep.     

Investigation of the effect of growth interruption on the formation of InAs/GaAs quantum dot superlattice near the InAs critical thickness

Two kinds of superlattices (i) with and (ii) without growth interrupt (GI) after deposition of 1.77 monolayers (ML) of InAs on GaAs (0 0 1) were grown by solid-source molecular beam epitaxy (MBE) and assessed by transmission electron microscopy (TEM) techniques, double crystal X-ray diffraction (DCXRD) and photoluminescence (PL) measurements in order to gain an understanding of the structural and compositional properties. In case (i) formation of coherent dislocation free self-organized quantum dots (SOQDs) with 2.8-3.2 nm height and 13-16 nm lateral size was observed, whereas in case (ii) no quantum dots had formed. In order to better understand the implication of growth interruption for the formation mechanism, highly localised assessment of the composition of the QD was carried out via atomic resolution Z-contrast imaging and electron energy loss spectroscopy (EELS).