The Experts below are selected from a list of 282 Experts worldwide ranked by ideXlab platform
Masashi Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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convenient method for better preservation of fine structures of cultured macrophages and engulfed yeast cells by Freeze Substitution fixation
Journal of Electron Microscopy, 2017Co-Authors: Masashi Yamaguchi, Azusa Takahashinakaguchi, Yuko Aida, Michiyo Satookamoto, Hiroji ChibanaAbstract:Rapid Freeze-Freeze Substitution after glutaraldehyde fixation (CF-FS method) obtained the natural and fine structures of macrophages and engulfed yeast cells. Culturing macrophages on single hole molybdenum grids placed in culture dishes made possible the rapid freezing of cells by the 'open sandwich method'. This method may be convenient when rapid-freezing cannot be performed immediately, or when a rapid-freezing device is not available in the lab.
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structome of saccharomyces cerevisiae determined by Freeze Substitution and serial ultrathin sectioning electron microscopy
Journal of Electron Microscopy, 2011Co-Authors: Masashi Yamaguchi, Hiromitsu Furukawa, Yuichi Namiki, Hitoshi Okada, Yuko Mori, Jinfang Wang, Misako Ohkusu, Susumu KawamotoAbstract:The cell structure has been studied using light and electron microscopies for centuries, and it is assumed that the whole structure is clarified by now. Little quantitative and three-dimensional analysis of cell structure, however, has been undertaken. We have coined a new word, 'structome', by combining 'structure' and '-ome', and defined it as the 'quantitative and three-dimensional structural information of a whole cell at the electron microscopic level'. In the present study, we performed structome analysis of Saccharomyces cerevisiae, one of the most widely researched biological materials, by using Freeze-Substitution and serial ultrathin-sectioning electron microscopy. Our analysis revealed that there were one to three mitochondria, ~220 000 ribosomes in a cell, and 13-28 endoplasmic reticula/Golgi apparatus which do not form networks in the cytoplasm in the G1 phase. Nucleus occupied ~10.5% of the cell volume; cell wall occupied ~17%; vacuole occupied ~5.8%; cytoplasm occupied ~64%; and mitochondria occupied only ~1.7% in the G1 phase. Structome analysis of cells would form a base for the post-genome research.
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improved preservation of fine structure of deep sea microorganisms by Freeze Substitution after glutaraldehyde fixation
Journal of Electron Microscopy, 2011Co-Authors: Masashi Yamaguchi, Yuichi Namiki, Hitoshi Okada, Katsuyuki Uematsu, Akihiro Tame, Tadashi Maruyama, Yoshimichi KozukaAbstract:A method was proposed for improving preservation of ultrastructures of deep-sea microorganisms by using rapid-Freeze Freeze-Substitution after glutaraldehyde fixation. This method produced clear high-resolution images of cells appearing in their natural state, close to the quality of images obtained by rapidly freezing Freeze-substituted specimens of living cells. The method may be useful for observing any microorganism when rapid freezing of living samples is difficult and only glutaraldehyde fixation can be carried out.
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Novel Freeze-Substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying biosafety requirements.
Journal of microbiological methods, 2009Co-Authors: Hiroyuki Yamada, Satoshi Mitarai, Kinuyo Chikamatsu, Kazue Mizuno, Masashi YamaguchiAbstract:Transmission electron microscopy (TEM) of virulent bacteria is usually performed following chemical fixation (CF) with aldehyde fixatives such as glutaraldehyde because of the biosafety problem. However, CF may alter sample ultrastructure. In this study, we used a rapid-Freeze Substitution (RFS) sandwich method without pre-embedding in agar. TEM images obtained using this method were completely different from those of conventional chemically fixed samples; the bacilli cytoplasm of the RFS preparations was filled evenly with numerous ribosomes, and there was no positional variation of electron density that was obvious in those obtained with CF samples. The sandwich method is suitable for microbiological materials without expensive devices and can be easily performed in a biosafety cabinet. In future, this method coupled with novel labeling techniques may help localize structural and functional molecules throughout a bacterial cell.
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smart specimen preparation for Freeze Substitution and serial ultrathin sectioning of yeast cells
Journal of Electron Microscopy, 2009Co-Authors: Masashi Yamaguchi, Hitoshi Okada, Yuichi NamikiAbstract:A smart and efficient method for Freeze Substitution and serial sectioning of yeast cells is described. Yeast cells were placed in a single layer between two copper disks, rapidly frozen, Freeze substituted and embedded in an epoxy resin. The cell layer was re-embedded by the same resin, the surface trimmed leaving 1 mum above the cell layer, and serially sectioned. The sections were collected on the two-slit grids and placed on a Formvar film mounted to cover the holes of an aluminum supporting rack. The grids were removed from the rack, stained together using a silicon tube and observed in a transmission electron microscope. The images of yeast cells observed were clear and natural, and would be useful for a detailed 3D structural analysis such as structome.
Shinichi Ohno - One of the best experts on this subject based on the ideXlab platform.
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application of cryotechniques with Freeze Substitution for the immunohistochemical demonstration of intranuclear pcreb and chromosome territory
Journal of Histochemistry and Cytochemistry, 2005Co-Authors: Nobuhiko Ohno, Nobuo Terada, Shinichi Murata, Ryohei Katoh, Shinichi OhnoAbstract:Intranuclear localization of signal molecules and chromosome territories has become more attractive in relation to postgenomic analyses of cellular functions. Cryotechniques and Freeze-Substitution (CrT-FS) have been generally used for electron microscopic observation to obtain better ultrastructure and immunoreactivity. To investigate benefits of applying the CrT-FS method to immunostaining of intranuclear signal molecules and FISH for chromosome territories, we performed an immunohistochemical study of phosphorylated cAMP-responsive element binding protein (pCREB) in mouse cerebellar tissues and a FISH study of chromosome 18 territory in human thyroid tissues using various cryotechniques. The immunoreactivity of pCREB was more clearly detected without antigen retrieval treatment on sections prepared by the CrT-FS method than those prepared by the conventional dehydration method. In the FISH study, more definite probe labeling of the chromosome territory could be obtained on paraffin sections by the CrT-FS method without microwave treatment, although such labeling was not clear even with microwave treatment on sections prepared by the routine dehydration method. The CrT-FS preserved relatively native morphology by preventing shrinkage of nuclei, and produced better immunoreactivity. Because the reduction of routine pretreatments in the present study might reveal more native morphology, the CrT-FS method would be a useful technique for intranuclear immunostaining and FISH.
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dynamic ultrastructure of mouse pulmonary alveoli revealed by an in vivo cryotechnique in combination with Freeze Substitution
Journal of Anatomy, 2000Co-Authors: Ichiro Takayama, Hideho Ueda, Yasuko Kato, Takeshi Baba, Yasuhisa Fujii, Nobuo Terada, Shinichi OhnoAbstract:A morphological approach to cell dynamics is usually difficult, since routine preparative techniques for electron microscopy always induce artifacts due to cessation of the blood supply into organs. An in vivo cryotechnique followed by the Freeze-Substitution method probably reduces such problems. It was applied for examining the pulmonary alveoli of BALB/c mice in vivo. The following ultrastructural features were revealed. (1) A surfactant layer provided a continuous covering to the alveolar epithelium. (2) Pleural epithelial cells, alveolar cells and endothelial cells contained many small vesicles and pits. In the alveolar epithelium, they were often localised near microtubules. (3) Typical lamellar structures in large alveolar epithelial cells were rarely detected. (4) Circulating erythrocytes with various shapes were observed in branching blood capillaries. (5) A close association between erythrocytes and the endothelium was seen at the peripheral alveolar septum. Such ultrastructural arrangements may be appropriate for the physiological functions of the pulmonary alveoli, such as exchanges of gases or materials in vivo.
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scanning electron microscopic study of the renal glomerulus by an in vivo cryotechnique combined with Freeze Substitution
Journal of Anatomy, 1998Co-Authors: Ying Yu, Nobuo Terada, Chongguang Leng, Shinichi OhnoAbstract:The 3-dimensional ultrastructure of mouse renal glomeruli under normal haemodynamic conditions was studied by scanning electron microscopy using an in vivo cryotechnique followed by Freeze-Substitution, and compared with glomeruli prepared by conventional fixation methods. Mouse kidneys were frozen with a cryoknife apparatus and a liquid isopentane-propane mixture (−193°C). Surface areas of the frozen tissues were Freeze-fractured with a scalpel in liquid nitrogen. The specimens were routinely Freeze-substituted, Freeze-dried, ion-sputtered, and then observed in a scanning electron microscope at an accelerating voltage of 5 kV. Renal glomeruli showed good ultrastructural preservation of the surface tissues. Podocytes with interdigitating foot processes covering capillary loops exhibited smooth surface contours and their cell surfaces were arranged more tightly than those seen by the conventional fixation method. Filtration slits between foot processes were found to be narrow. The internal structure of the glomerular tuft was seen in the Freeze-fracture faces. The capillary lumen with variously shaped erythrocytes was kept open in frozen glomeruli under normal blood circulation conditions. The ultrastructure of renal glomeruli, as revealed by the in vivo cryotechnique with Freeze-Substitution, appears to be closer to that of the living state.
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morphometric study of glomerular slit diaphragms fixed by rapid freezing and Freeze Substitution
Kidney International, 1991Co-Authors: Takeshi Furukawa, Shinichi Ohno, Hisao Oguchi, Kumiko Hora, Shinichi Tokunaga, Seiichi FurutaAbstract:Morphometric study of glomerular slit diaphragms fixed by rapid-freezing and Freeze-Substitution. The widths of slit diaphragms in rat glomeruli were quantitatively measured on electron micrographs obtained by the rapid-freezing and Freeze-Substitution (FS) fixation method and compared to those prepared by conventional chemical fixation procedures, such as tannic acid, glutaraldehyde and osmium tetroxide (TGO) or glutaraldehyde and osmium tetroxide (GO) fixation. When fixed by FS using 2% osmium tetroxide, glomerular slit diaphragms had widths of 33.8 ± 3.6 nm, but when fixed by TGO or GO they had widths of 47.0 ± 4.3 and 48.7 ± 5.5 nm, respectively. The width of slit diaphragms was significantly narrower after cryofixation than after conventional TGO or GO fixation (P
Terry J Beveridge - One of the best experts on this subject based on the ideXlab platform.
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high resolution visualization of pseudomonas aeruginosa pao1 biofilms by Freeze Substitution transmission electron microscopy
Journal of Bacteriology, 2005Co-Authors: R. C. Hunter, Terry J BeveridgeAbstract:High-pressure Freeze-Substitution and transmission electron microscopy have been used for high-resolution imaging of the natural structure of a gram-negative biofilm. Unlike more conventional embedding techniques, this method confirms many of the observations seen by confocal microscopy but with finer structural detail. It further reveals that there is a structural complexity to biofilms at both the cellular and extracellular matrix levels that has not been seen before. Different domains of healthy and lysed cells exist randomly dispersed within a single biofilm as well as different structural organizations of exopolymers. Particulate matter is suspended within this network of fibers and appears to be an integral part of the exopolymeric substance (EPS). O-side chains extending from the outer membrane are integrated into EPS polymers so as to form a continuum. Together, the results support the concept of physical microenvironments within biofilms and show a complexity that was hitherto unknown.
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preservation of surface lipids and determination of ultrastructure of mycobacterium kansasii by Freeze Substitution
Infection and Immunity, 1994Co-Authors: T R Paul, Terry J BeveridgeAbstract:The cell wall architecture of a slowly growing mycobacterium, Mycobacterium kansasii, was examined by Freeze-Substitution following growth in vitro. Freeze-substituted bacteria were marked by the presence of an electron-translucent space (or electron-transparent zone [ETZ] described by previous workers [T. Yamamoto, M. Nishiura, N. Harada, and T. Imaeda, Int. J. Lepr. 26:111-114, 1958]) surrounding the majority of cells. At least two morphotypes of mycobacteria were revealed by Freeze-Substitution. In the first, a relatively thin (11 +/- 2.3 to 3.5 +/- 3.1 nm), uniform ETZ surrounded intact cells which contained cytoplasm filled with well-stained ribosomes and a DNA nucleoid distributed throughout the cell. The second morphotype consisted of a small proportion of organisms that were distorted in shape and were surrounded by a much thicker (59 +/- 2.6 to 198 +/- 2.5 nm) ETZ in areas of the cell which appeared to have retracted from the space it had originally occupied, leaving depressions in the ETZ. The lipid nature of the ETZ was demonstrated because cells were devoid of an ETZ when organisms were Freeze-substituted in the absence of osmium tetroxide in the Substitution medium or treated with neutral lipid solvents (acetone or ethanol) before Freeze-Substitution. Moreover, thin-layer chromatography of acetone or ethanol extracts obtained from solvent-treated cells identified a lipid component which corresponded to the M. kansasii-specific phenolic glycolipid. In contrast, negligible amounts of glycolipids were detected in extracts obtained from control HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer-treated cells, and these cells retained an ETZ. These results demonstrate that species-specific phenolic glycolipids are essential components in the architecture of the M. kansasii ETZ. Furthermore, we show that Freeze-Substitution is a reliable technique for the retention and precise preservation of lipid-containing polymers in the mycobacterial cell wall.
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ultrastructural examination of the lipopolysaccharides of pseudomonas aeruginosa strains and their isogenic rough mutants by Freeze Substitution
Journal of Bacteriology, 1992Co-Authors: Joseph S Lam, L L Graham, J Lightfoot, T Dasgupta, Terry J BeveridgeAbstract:The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by Freeze-Substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that Freeze-Substitution electron microscopy is effective in the visualization of LPS morphotypes. Images
Satoshi Mitarai - One of the best experts on this subject based on the ideXlab platform.
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Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and Freeze-Substitution with osmium-acetone at ultralow temperature.
Journal of Microbiological Methods, 2013Co-Authors: Hiroyuki Yamada, Kinuyo Chikamatsu, Akio Aono, Satoshi MitaraiAbstract:Abstract Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the biosafety area. Freeze-Substitution with osmium-acetone (OA) at ultralow temperature (− 85 °C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for Freeze-Substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel Freeze-Substitution preparation method that can be performed using a portable device in a biosafety cabinet at biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid Freeze-Substitution (RFS) after fixation with glutaraldehyde (GA > RFS) are of comparable quality to those obtained using standard RFS. GA > RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA > RFS was not significantly different (6987.8 ± 2181.0 and 6888.9 ± 1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7 ± 2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS.
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Novel Freeze-Substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying biosafety requirements.
Journal of microbiological methods, 2009Co-Authors: Hiroyuki Yamada, Satoshi Mitarai, Kinuyo Chikamatsu, Kazue Mizuno, Masashi YamaguchiAbstract:Transmission electron microscopy (TEM) of virulent bacteria is usually performed following chemical fixation (CF) with aldehyde fixatives such as glutaraldehyde because of the biosafety problem. However, CF may alter sample ultrastructure. In this study, we used a rapid-Freeze Substitution (RFS) sandwich method without pre-embedding in agar. TEM images obtained using this method were completely different from those of conventional chemically fixed samples; the bacilli cytoplasm of the RFS preparations was filled evenly with numerous ribosomes, and there was no positional variation of electron density that was obvious in those obtained with CF samples. The sandwich method is suitable for microbiological materials without expensive devices and can be easily performed in a biosafety cabinet. In future, this method coupled with novel labeling techniques may help localize structural and functional molecules throughout a bacterial cell.
Hiroyuki Yamada - One of the best experts on this subject based on the ideXlab platform.
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Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and Freeze-Substitution with osmium-acetone at ultralow temperature.
Journal of Microbiological Methods, 2013Co-Authors: Hiroyuki Yamada, Kinuyo Chikamatsu, Akio Aono, Satoshi MitaraiAbstract:Abstract Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the biosafety area. Freeze-Substitution with osmium-acetone (OA) at ultralow temperature (− 85 °C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for Freeze-Substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel Freeze-Substitution preparation method that can be performed using a portable device in a biosafety cabinet at biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid Freeze-Substitution (RFS) after fixation with glutaraldehyde (GA > RFS) are of comparable quality to those obtained using standard RFS. GA > RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA > RFS was not significantly different (6987.8 ± 2181.0 and 6888.9 ± 1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7 ± 2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS.
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Novel Freeze-Substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying biosafety requirements.
Journal of microbiological methods, 2009Co-Authors: Hiroyuki Yamada, Satoshi Mitarai, Kinuyo Chikamatsu, Kazue Mizuno, Masashi YamaguchiAbstract:Transmission electron microscopy (TEM) of virulent bacteria is usually performed following chemical fixation (CF) with aldehyde fixatives such as glutaraldehyde because of the biosafety problem. However, CF may alter sample ultrastructure. In this study, we used a rapid-Freeze Substitution (RFS) sandwich method without pre-embedding in agar. TEM images obtained using this method were completely different from those of conventional chemically fixed samples; the bacilli cytoplasm of the RFS preparations was filled evenly with numerous ribosomes, and there was no positional variation of electron density that was obvious in those obtained with CF samples. The sandwich method is suitable for microbiological materials without expensive devices and can be easily performed in a biosafety cabinet. In future, this method coupled with novel labeling techniques may help localize structural and functional molecules throughout a bacterial cell.
