Showing papers in "Journal of Electron Microscopy in 1989"

Cell Wall Formation in Regenerating Protoplasts of Schizosaccharomyces pombe: Study by High Resolution, Low Voltage Scanning Electron Microscopy

Abstract: The ultrastructure of regenerating cell wall in Schizosaccharomyces pombe protoplasts was studied with a high resolution, low voltage scanning electron microscope (LVSEM). In contrast to the transmission electron microscopy, the LVSEM images give three-dimensional information on the cell wall regeneration in yeast protoplasts. We found that, after only a few minutes of incubation, the protoplasts began to show protuberances in a unipolar manner, and a fibrilar network was formed asymmetrically which covered the whole surface of the protoplasts after 5 hr. The network consisted of microfibrils about 8 to 10 nm wide, forming flat and wavy bundles of various widths and lengths, up to about 200 nm wide and 1 micron long, mainly made of yeast glucan. Free ends of microfibrils were seldom found. Interfibrillar spaces were progressively filled with granular particles and finally the complete cell wall was formed after 12 hr. The fibrillar network was destroyed by the digestion with beta (1----3)-glucanase. When protoplasts were regenerating in the presence of aculeacin A, the fibrillar networks were not formed, resulting in incomplete cell wall formation. These observations suggest that beta-glucan is the main component of the microfibrils and that it plays an important role in the formation of the cell wall in S. pombe.

Image restoration in coherent imaging system involving spherical aberration

Abstract: This paper describes an image restoration method based on defocus modulation processing, assuming weakly scattered objects in the coherent imaging system involving spherical aberration. The present analysis shows that the effect of the spherical aberration can be completely corrected by the image integration applied to the defocus series, if appropriate bipolar (positive and negative) weighting functions as a function of the defocus value are used in the image integration

Quick-freeze, deep-etch electron microscopy.

Abstract: This chapter briefly introduces the quick-freeze replica studies carried out mainly by Japanese scientists. Focus is on this technique's contribution to revealing three-dimensional organization of the cytoskeleton, and membrane with associated structures.

Three-dimensional studies of cytoskeletal organizations in cultured thyroid cells by quick-freezing and deep-etching method.

Abstract: Isolated porcine thyroid cells were cultured on collagen gels (control group, TSH-stimulated group, and double-layered culture). They were split or cut to remove cytoplasmic soluble proteins for replica preparations. Some specimens were immunostained with anti-actin antibody or decorated with S 1 myosin fragments to identify actin filaments. The basal cell membranes of thyroid cells of monolayer culture were in contact with collagen gels and the apical cell membranes faced the culture medium. Networks of actin filaments were attached to the cytoplasmic sides of the apical cell membranes, while intermediate filaments were localized along the basal ones. The thyroid-stimulating hormone (TSH) treatment induced the formation of microvilli only on the apical cell membranes and the accumulation of actin filaments under the apical cell membranes, indicating the apical-basal polarity of the cells. In double-layered culture, the primitive follicular lumens with microvilli appeared between two adjacent cells. The interaction of cell membranes with collagen gels is a determinant factor in the orientation of apical-basal polarity. Moreover, the TSH treatment and cell-cell contact further intensify the polarization through reorganizing the cytoskeletons

The cytoplasmic surface structures of uncoated vesicles in various tissues of rat as revealed by quick-freeze, deep-etching replicas.

Abstract: We examined the cytoplasmic surface structures of the plasmalemmal vesicles in various tissues using quick-freeze, deep-etching replicas, and identified three types of cytoplasmic surface specializations. In addition to the baskets of clathrin-coated vesicles, stripe-surfaced and bumpy-surfaced vesicles could be identified. The striped patterns were clearly observed on the vesicles in endothelial cells and fibroblasts of various tissues, while in epithelial cells such as hepatocytes, renal proximal tubule cells, and superficial cells of the urinary bladder, instead of the striped patterns, rough-surfaced vesicles could be identified. These differences in 'uncoated' vesicles may suggest that there is the tissue specificity of the cytoplasmic surface structures of plasmalemmal vesicles.

Varied cytochrome oxidase activities of the alveolar type I, type II and type III cells in rat lungs: quantitative cytochemistry.

Abstract: Cytochemically demonstrated cytochrome oxidase activities in mitochondria of rat pulmonary alveolar epithelial cells were quantitatively compared. As the standardized procedures, lungs were fixed for 10 min at 4 degrees C with 2% pure glutaraldehyde, and incubated for different times at 37 degrees C in a medium containing 1.0 mg/ml 3,3'-diaminobenzidine-4HCl, 1 mg/ml cytochrome c, 0.1 mg/ml catalase, 7% sucrose, and 0.1 M phosphate buffer, pH 7.4. Electron micrographs were then obtained, and the densities of the reaction deposits in the mitochondrial intermembrane-intracristal space were measured in an image analyzer, and were plotted in terms of time in minutes. The initial velocity (maximal rate) of the activity expressed as deposit accumulation rate (% area/60 min) filling the mitochondrial intermembrane-intracristal space of Type I, Type II and Type III cells was 40, 130 and 9.3, respectively. These results indicated that mitochondria are varied in the intensity of cytochrome oxidase activity among 3 types of pulmonary alveolar epithelial cells, and that the image-analyzing measurement of the accumulation rate of reaction product may be useful for quantitative analysis of enzyme activity, in particular, in the cells which are difficult to isolate from complex tissues.

High Resolution Electron Microscopy of Enamel Crystals in Cases of Human Dental Fluorosis

Abstract: Surface enamel from human subjects with dental fluorosis was studied by means of high-resolution transmission electron microscopy. Immediately below the relatively highly mineralized outermost surface enamel layer was an extensive hypomineralized area. The highly mineralized layer was composed of many large elongated hexagonal crystals and extremely small hexagonal crystals. Frequently the small crystals were attached to the periphery of the large crystals. In the hypomineralized area, large crystals were sparsely arranged; and a few small crystals were seen. Some large crystals showed either perforated centers or defects on their peripheries. These findings suggest that the hypomineralized area undergoes caries-like changes in terms of crystal dissolution and that the highly mineralized surface layer is either formed or modified by remineralization.

Two- and Three-Dimensional Ultrastructure of Endothelium and Pericyte Interdigitations in Capillary of Human Granulation Tissue

Abstract: Two- and three-dimensional electron microscopic observation in the immature capillary of the human granulation tissue revealed cytoplasmic interdigitations (CID) between the endothelium and the pericyte. These were composed of a cytoplasmic projection and indentation, and there was a gap space without basement membrane-like components between the two cell membranes. Plasmalemmal vesicles were frequently found locating beneath and/or attached to the cell membranes at the indentated side of the interdigitation. Two kinds of cytoplasmic interdigitation, each having a characteristic configuration, were demonstrated. The CID which was composed of a cytoplasmic projection from the endothelium to the pericyte had a dull-shaped cytoplasmic projection, while the CID which was composed of a cytoplasmic projection from the pericyte to the endothelium had a slender and long finger-like configuration.

Three-dimensional distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts of actively photosynthesizing cell of Euglena gracilis

Abstract: Euglena gracilis Z cells were synchronized under photoautotrophic conditions using a 14-hr light: 10-hr dark regimen. The most actively photosynthesizing cells obtained after 10 hr in the light were serially sectioned, and these sections were labeled with antibody raised against Euglena ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) followed by protein A-gold. Computer-aided construction of three-dimensional models of cells revealed spatial distribution ofRuBisCO in chloroplasts of the cell. RuBisCO was highly concentrated in the pyreoid region

Exocytosis of bovine chromaffin granules in Ficoll captured by rapid freezing.

Abstract: Exocytosis of cultured bovine adrenal chromaffin cells was examined in a balanced salt solution containing Ficoll using rapid freezing followed by freeze-substitution. In solution without Ficoll, exposure to Ba2+ produced many exocytotic lumina between the cells; in most cases, these lumina were large and empty. When chromaffin cells were exposed to Ba2+ in Ficoll, it was possible to observe a small pore between the plasmalemma and each granule. The granule retained the dense contents when the pore was approximately 20 nm in diameter, and became empty when the pore was widened in 14% Ficoll. The addition of Ficoll made it easy to catch the events occurring during exocytosis of individual bovine chromaffin granules.

Quick-freeze, deep-etch visualization of the nuclear pore complex.

Abstract: We have visualized the nuclear pore complex (NPCx) three-dimensionally by quick-freeze, deep etching and suggest that NPCx is composed of four rings and associated filaments. The rings were located at the cytoplasmic side, at the nucleoplasmic side, and at the pore waist as well. Some of the filaments emanated toward the cytoplasm, some sprawled over the cytoplasmic face of the nuclear membrane, and some intervened the rings. The emanating filaments were considered to be vimentin filaments judging from the replica and thin-sectioned image.

Target Materials Suitable for Projection X-Ray Microscope Observation of Biological Samples

Abstract: Trials for improving the contrast of projection X-ray images by finding better target materials than Ti (λ K α: 2.75 A), which has been found to be suitable for many kinds of specimens, were carried out, considering the factors of melting point, thermal conductivity, mechanical strength, chemical stability, absorption of the X-ray, etc. Au, Ta, and Ge were found to be suitable, giving 5-10 A X-rays when low electron beam energies around 10 kV were used. In order to take advantage of the long wavelength X-rays of these targets, we tried to minimize the attenuation of the imaging X-rays in the air or to use a vacuum camera. Even in the air ot to use a vacuum camera. Even in non-stained biological samples such as HeLa cells and Lingual muscle section, their microstructures were visible with sufficient contrast

An electron microscopic study of the steroid hormone receptor in uterine cells by the colloidal gold-labeled steroid hormone.

Abstract: To study the localization of the progesterone and estradiol receptors by electron microscopy, the colloidal gold-labeled steroid hormones, intercalated with bovine serum albumin, were developed in the postembedding method. Among the tissues investigated in this study, receptors specifically distributed in the nuclei of the cells in the uterus and liver but not in the colon. Among the uterine smooth muscle cells in the estrus cycle, the frequency of the progesterone receptors in the nuclei indicated the highest level at a proestrus stage when the serum estradiol is maximum and the lowest level at a metestrus stage when the serum estradiol is minimum. On the other hand, the frequency of the estradiol receptor did not show significant changes among the stages of the estrus cycle. In the nuclei, the receptors specifically distributed on the euchromatin and on the peripheral regions of the heterochromatin, thought to be active regions of the transcription, and among the interchromatin granules. No receptors were observed within the nucleolus, the heterochromatin, the perinuclear chromatin, the nuclear sap, and the perichromatin granules. The colloidal gold-labeled steroids proved to be useful to study the microlocalization of the steroid hormone receptors in various target organs by electron microscopy.

High resolution cryo-electron microscopy for biological macromolecules.

Abstract: The irradiation damage of the crystals of proteins as well as nucleic acid is reduced almost exponentially even below the liquid nitrogen temperature down to 8K provided that the specimen area under illumination is cooled down. A cryo-stage cooled by superfluid helium is designed for the high resolution electron microscope based on the idea of the helium evaporation refrigerator with a capillary which was developed by Delong et al. We also developed a new top-entry-type cryo-transfer system for this superfluid cryo-electron microscope. The images of chlorinated Cu-phthalocyanine are taken to estimate the resolution attainable by this cryo-stage at 1.5 K with accelerating voltage of 400 kV. The optical diffraction confirms the resolution of 0.26 nm. The complexes of rec A proteins and DNA molecule are clearly visible in vitreous ice. A new membrane structure of the influenza virus is observed in the case of influenza A virus.

Isolated Schwann Cells Can Synthesize the Basement Membrane in vivo

Abstract: The purpose of the present study is to examine whether isolated Schwann cells that are not in contact with axons can synthesize the basement membrane in vivo. Schwann cells obtained from sciatic nerve of neonate Wistar rats were prepared in culture, harvested, and mixed with collagen matrix. The silicone tube filled with the collagen matrix containing Schwann cells was closed by a Millipore membrane at both ends to isolate the interior from the outer environment and was implanted in a gap between proximal and distal stumps of transected sciatic nerve of an adult Wistar rat. Ten days later, the implanted silicone tube was prepared for electron microscopic observations. Schwann cells in the tube were elongated longitudinally and lined up parallel to the proximo-distal direction. No regenerating axons penetrated the Millipore membrane into the tube. The basement membrane showing almost normal structure was produced on the surface of the Schwann cells. While the silicone tube was placed side by side to the intact sciatic nerve, Schwann cells in the tube were scattered and no basement membrane was observed. These results suggest that some humoral factors might be released from the transected nerve stumps, which may be responsible for the regular arrangement and the basement membrane formation by Schwann cells without any contact with axons.

Electron microscopic studies on the stretch-induced disordering of the myofilament lattice in tetanized frog skeletal muscle fibers.

Abstract: To study the effect of stretch on the hexagonal myofilament lattice in frog skeletal muscle fibers, the fibers were fixed at rest and during the isometric tetanus with or without stretch, and their cross sections were examined electron microscopically. The degree of disorder of the myofilament lattice as estimated by the Fourier transform and rotation methods in the digital image analysis was found to be largest during the isometeric tetanus with stretch and smallest during the isometeric tetanus without stretch, supporting the view that the stretch-induced force enhancement results from the disordering of the myofilament lattice.

Scanning Electron Microscopic Study of Periendothelial Cells of the Rat Cerebral Vessels Revealed by a Combined Method of Corrosion Casting and KOH Digestion

Abstract: A combined method of corrosion casting and KOH digestion was used for the scanning electron microscopic (SEM) observation of the external walls of the blood vessels running on the surface and in the parenchyma of the rat cerebral cortex. The plastic resin which was preliminarily injected into the cerebral vessels reinforced the preservation of the vessel walls during the chemical digestion. Using the present method, the walls of blood vessels running on the surface and in the parenchyma of the cerebral cortex were clearly seen with SEM. The arterioles showed a rich arrangement of smooth muscle cells embracing the external surface of the endothelia, and the venules had sparse, flat smooth muscle cells. The capillaries were surrounded by many pericytes which were extremely similar to those seen in the other organs of the animals. There were observed two different kinds of pericyte. One type of the pericytes in the present specimens extended very long cytoplasmic processes parallel to the long axis of the capillaries. Another type of the pericytes had relatively short, broad and flat cytoplasmic processes radiated to embrace the capillary walls. The former was frequently seen in the capillaries on brain surface and in the parenchyma, whereas the latter was relatively rare. The availability of the present method was also discussed in comparison with the previous descriptions.

The location of filamentous hemagglutinin and pertussis toxin antigens of Bordetella pertussis by immunoelectron microscopy.

Abstract: Immunoelectron microscopy using colloidal gold-tagged antibodies was used to detect filamentous hemagglutinin (FHA) and pertussis toxin (PT) antigens on the surface and in the cytoplasm of Bordetella pertussis cells. Both gold-tagged antibodies to FHA and PT labeled the aggregates of filamentous material on the surface of sediment-settled phase I cells under static conditions. FHA and PT antigens were detected also on ultrathin sections made after embedding the phase I cells in Lowicryl K4M resin. On the ultrathin sections, intense label of gold-tagged antibodies to FHA and PT was present on the cell surface and also in the cytoplasm, but not in the nucleoid. The aggregates of filamentous material adhering on the surface of phase I cells were most abundant on culture day 3, the end of the logarithmic growth stage, but most of the aggregates were found detached from the cell surface on culture day 5 or 7, the stationary stage. The aggregates were not found on the surface of phase III cells. The present study suggested that FHA and PT antigens were localized on the same cell structure and that both antigens were synthesized in the cytoplasm and secreted across the cell membrane mainly in the logarithmic growth stage of the phase I cells.

Ultrastructural studies on a cadmium-storing cell in rat pancreatic tissues following cadmium chloride administration.

Abstract: A new type of cell was found in the pancreatic connective tissues of rats following cadmium chloride administration. The cells contained several lipid droplets that were strongly electron-dense after the treatment with oxine, which made cadmium-oxine deposits. This suggested that the lipid droplets stored cadmium or related substances. These cells were named "cadmium-storing cells." Cadmium-storing cells are subdivided into two cell types according to their origins, "fibroblast type" and "pericyte type." The structure and function of the cadmium-storing cell were described and discussed.

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

Abstract: 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.

Ultrahigh resolution scanning electron microscopy of biological materials.

Abstract: In recent years, several ultrahigh resolution scanning electron microscopes (SEM) were successively developed. They were all equipped with a field emission electron gun and an objective lens with a short focal length, and showed a resolution better than 1 nm. With such instrument, not only intracellular structures but also virus, bacteriophages, and biological macromolecules were clearly observed. With improvement of the instrumental resolution, some unexpected problems came to the fore in the area of specimen preparation. The first is the metal coating of the specimen, because coated metal particles are plainly seen as rounded "pebbles" at very high magnifications. For observations at the high magnifications, therefore, uncoated and not conductively stained specimens were used. The second problem is contamination by the electron beam. This problem is complicated and remains unsolved. Although the ultra-high resolution scanning electron microscopy has just begun, it will surely open new research fields in biomedicine.

Immunoelectron Microscopy of Proteus vulgaris by the Plasma Polymerization Metal-Extraction Replica Method--Differential Staining of Flagellar(H)and Somatic(O)Antigens by Colloidal Golds

Abstract: Flagellar (H) and somatic (O) antigens of Proteus vulgaris were differentially stained with antibodies coupled to different sizes of colloidal gold particles, and the distribution of these antigens was visualized by the plasma polymerization metal-extraction replica (PMR) method. The H antigen, labeled with 5 nm colloidal gold, was almost exclusively located on the flagella, whereas the O antigen, labeled with 10 nm colloidal gold, was almost exclusively located on the bacterial body. The marker gold particles were clearly observed as electron-dense particles on the relatively low contrast background of three-dimensional replica image of the flagellated bacteria. Thus, the PMR method may prove to be a useful tool for studying the localization of multiple substances on the cell surface, at a high resolution and in three dimensions. The diameter of the flagella measured by the replica method was about 15 nm, close to the value obtained by negative staining (16 nm). When treated with anti-flagellar (H) factor serum and protein A-gold, the diameter of flagella was significantly increased to about 35 nm. This increase in diameter was presumably caused by binding of immunoglobulins to H antigens of flagella.