High Voltage Electron Microscope Study Research Articles

High voltage electron microscopic studies of endothelial cell tubular structures in the mouse blood-brain barrier following brain trauma

High-voltage electron microscopy was applied to the study of endothelial cell (EC) transport of macromolecules in a murine model of blood-brain barrier injury to study the role of the EC canalicular system following brain insult. Semithick sections from mouse brains subjected to acute (2-3 h) mechanical trauma demonstrated permeation of intravenously injected horseradish peroxidase via tubular structures either (a) in the absence of lysosome-associated structures in close proximity, or (b) in association with lysosomes, dense bodies or multivesicular bodies. Our data suggest a dual-purposed system of tubules, one portion that supplies the metabolic requirements of the cell and another portion, suggested to be more limited, that opens up as a result of brain injury.

High Voltage Electron Microscopic Studies on Mitral, Tufted, and Granule Cells in the Mouse Olfactory Bulb

High-voltage electron microscopic observations have been performed on intraglomerular dendritic branches of mitral and tufted cells and spines of granule cells in the Golgi-impregnated mouse olfactory bulb. The observations revealed that intraglomerular tufts of mitral and tufted cells have a very similar structure consisting of four morphologically distinct parts: (i) thick, smoothly outlined proximal parts; (ii) relatively thick varicose parts; (iii) very thin varicose parts, which form terminal branches; and (iv) spine-like small appendages. Granule cell spines in the external plexiform and mitral body layer have terminal swellings of various shapes and sizes, whose width is between 0.4 and 1.5 microns. Spines in the granule cell layer usually have swellings smaller than 0.4 micron in width. In addition, the density of spines per proximal 10 microns length of a deep dendritic trunk was estimated in 10 granule cells and proved to vary greatly from cell to cell--from 7 to 28 spines.

High Voltage Electron Microscope Study of Incommensurate Phase in Quartz

The modulated structure in the incommensurate phase of quartz was studied by high voltage electron microscopy. The modulation was found to be due to formation of triangular microdomains of the Dauphiné twins. In addition to the well known Dauphiné twin relation we found that at the Dauphiné twin boundaries there exist lattice displacement parallel to the boundaries. The significant relations among the atom arrangements in neighboring domains, the lattice displacement and the orientation of the Dauphiné twin boundary were deduced, which determine a domain configuration in the modulated structure. Our observations indicate that the modulated structure is formed by freezing of the two kinds of atomic displacement which correspond to the B 1 type optic phonon mode and transverse accoustic (TA) phonon mode on the Σ line.

HVEM studies of the effects of hydrogen on the deformation and fracture of AISI type 316 austenitic stainless steel

Abstract The mechanisms of hydrogen embrittlement in AISI type 316 austenitic stainless steel have been investigated by in situ straining in a high-voltage electron microscope (HVEM) equipped with an environmental cell. Hydrogen effects on strain-induced phase transformations, the generation rate and velocity of dislocation, and crack propagation rates were studied. The salient features of the fracture were similar for cracks propagating in vacuum and in hydrogen gas. In each case, e and α′ martensite formed at the crack; the e phase extended ahead of the crack while the α′ phase was restricted to high stress regions near the crack tip. The principal effect of hydrogen was to decrease the stress required for dislocation motion, for phase transformation of the austenite, and for crack propagation.

On the direct observation of the formation of ferrite in steels

Recent contributions to the debate on the detailed nature of austenite decomposition processes in steels have focussed on the character of the Widmanstatten ferrite/austenite transformation interface (1,2) and on the nature of the bainite reaction in steels (3,4). The arguments presented concerning Widmanstatten ferrite formation have been exhaustive, and have served to define rather precisely the opposing positions. In this sense, they have been of considerable value. However, the discussors have not made reference to a body of experimental evidence, derived from in-situ high voltage electron microscopic studies, which has bearing on the subject, and which has the virtue of being somewhat more direct than certain of the observations quoted in the debate. Physical and instrumental factors limit the resolution of the method to a level inferior to that of present day high resolution microscopy (5,6); for the present discussion, however, it has the significant advantage of permitting the imaging of structural defects by diffraction contrast, at temperature. Access to thicker regions of the specimen also alleviates sampling problems inherent in wedge foil studies, and increases the probability that the local phenomena observed are closely similar to those which occur in bulk specimens. The purposes of this letter are two: The first is to review existing HVEM studies of ferrite/austenite interface migration, particularly as they relate to the question of Widmanstatten ferrite growth; the second is to add some new observations of high and intermediate temperature interface migration, which were prompted by a study of the published debate. The new observations of interest duplicate and augment some of the previously published findings. (They were obtained using somewhat different materials.) They also relate to possible differences between high and lower temperature interface migration modes for the Widmanstatten ferrite/austenite interface, a topic which has been the subject of recent suggestion (3).

High-voltage electron microscopic study in the syndrome of myalgia and cramps

Muscle pain and cramps are neuromuscular symptoms of diverse etiology. When these symptoms are associated with exercise, a biochemical defect in glycolytic or lipid metabolism, a mitochondrial myopathy, or defect in muscle contraction is frequently suspected. Diagnosis is usually established when such defect is demonstrated histochemically or by NMR. In some neuromuscular disorders, these symptoms may not necessarily be induced by exercise and they are usually part of a wide constellation of symptoms. Some of them are distinct enough to be recognized clinically or electrophysiologically, as in peripheral neuropathies, myotonias, or syndromes of continuous muscle activity. However, despite modern histochemical techniques, a number of muscle disorders remain, characterized primarily by muscle pain and cramps, where light microscopy or electrophysiologic studies show either normal or nonspecific findings.

Polarized pigment granule transport occurs in the absence of microtubules in squirrelfish erythrophores: studies of the effects of estramustine.

We have re-examined the involvement of microtubules in the process of pigment granule transport in squirrelfish erythrophores in situ (i.e. on scales). Light-microscopic studies revealed that following exposure to 5 microM-nocodazole for 1 h at 4 degrees C erythrophores retained an ability to aggregate and disperse their pigment uniformly, though at reduced rates. Serial thick-section stereo high-voltage electron-microscopic studies showed that the entire microtubule population was removed by drug treatment and that the microtubules were not reassembled as a result of pigment translocation processes in the presence of reduced levels of nocodazole (0.4 microM). Immunofluorescence microscopic studies confirmed that nocodazole (0.5-1 microM) produced rapid disassembly of the microtubules. Whole-mount electron-microscopic studies showed that the pigment granules were suspended in a cross-linking network of 3-10 nm filaments, which appeared to support ordered pigment transport in situ in the absence of microtubules. Drug inhibition studies showed that micromolar levels of estramustine, a novel anti-MAPs (microtubule-associated proteins) drug, reversibly inhibited pigment transport. The results suggest that an estramustine-sensitive cytomatrix component might produce polarized pigment transport in intact erythrophores.

A classification of Sharpey's fibers within the alveolar bone of the mouse: a high-voltage electron microscope study.

In the remodeling interdental septum of the mouse, four types of Sharpey's fibers were observed. Classification of these fiber types was based on characteristics of their termination within the septum in relation to the resorption-related reversal line separating old and new bone. "Severed fibers" were located only within old bone and terminated at the reversal line. "Arborized fibers" were located only within new bone and terminated therein. "Adhesive fibers" were located within new bone and terminated within a heavy band of granular material at the reversal line. "Continuous fibers" had components within old and new bone. These components were connected across the reversal line by nonstriated fibrils. Adhesive fibers were the least numerous type; severed and arborized fibers were observed at nearly equal frequency. Continuous fibers were the most numerous type, their numbers being significantly greater than any of the other types (P less than .001). Mean numbers of continuous fibers were greater than the mean total of the three other fiber types (P less than .001). Mean numbers of severed, adhesive, and arborized fibers were not statistically different. The study suggested that continuous fibers could be transalveolar--that is, ones which pass through the septum without interruption joining fibers of the adjacent periodontal ligament. Maintenance of their spatial continuity appeared to require a connecting protein to orient new unit collagen fibrils to old ones in areas of reversal. Thus, transalveolar fiber bundles could be characterized as being composed of old and new segments joined by a connecting protein. As their unit collagen fibrils did not cross resorption-related reversal lines these fibers were spatially continuous but temporally discontinuous.

Rearrangement of the open-canalicular system of the human blood platelet after incorporation of surface-bound ligands. A high-voltage electron-microscopic study.

The three-dimensional configuration of the membrane system in human blood platelets following administration of cationized ferritin (CF) was reconstructed by means of thick serial sectioning in combination with high-voltage electron microscopy (HVEM). Surface-bound CF was rapidly internalized at 37 degrees C by the platelets, and a conglomerate of vacuolar and tubular structures containing CF was formed in their interior. Three-dimensional reconstruction of these incorporated membranous structures revealed that most of the internalized structures were interconnected with each other and that the number of sites at which they opened onto the cell surface was significantly reduced compared with the number found in intact platelets. In some cases, the openings completely disappeared. This may indicate that most of the internalized membranous structures originate from the open canalicular system (OCS), and that, following the incorporation of the ligand, the OCS gradually loses its connections with the surface membrane forming a conglomerate of the ligand-containing membranous structures in the interior of the platelet.

Paracrystalline arrays in human muscle: HVEM study

Paracrystalline arrays, most often found beneath the sarcolemma membrane in the region of the nucleus, have been observed in postmortem human muscle tissue. Periodic arrangements of glycogen characterize the arrays, and similar arrays have been reported in muscle of patients with nemaline myopathy. When the glycogen is dissolved a fibrillar network with the same periodicities remains. Individual arrays show a variety of morphologies and symmetries. This work optimises the observation of the fibrillar arrays in semi-thick sections observed in the highvoltage electron microscope (HVEM).

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

Anin situ HVEM study of dislocation generation at Al/SiC interfaces in metal matrix composites

ANNEALED aluminum/silicon carbide (Al/SiC) composites exhibit a relatively high density of dislocations, which are frequently decorated with fine precipitates, in the Al matrix. This high dislocation density is the major reason for the unexpected strength of these composite materials. The large difference (10:1) between the coefficients of thermal expansion (CTE) of Al and SiC results in sufficient stress to generate dislocations at the Al/SiC interface during cooling. In thisin situ investigation, we observed this dislocation generation process during cooling from annealing temperatures using a High Voltage Electron Microscope (HVEM) equipped with a double tilt heating stage. Two types of bulk annealed composites were examined: one with SiC of discontinuous whisker morphology and one of platelet morphology. In addition, control samples with zero volume percent were examined. Both types of composites showed the generation of dislocations at the Al/SiC interface resulting in densities of at least 1013 m-2. One sample viewed end-on to the whiskers showed only a rearrangement of dislocations, whereas, the same material when sectioned so that the lengths of whiskers were in the plane of the foil, showed the generation of dislocations at the ends of the whiskers on cooling. The control samples did not show the generation of dislocations on cooling except at a few large precipitate particles. The results support the hypothesis that the high dislocation density observed in annealed composite materials is a result of differential thermal contraction of Al and SiC. The SiC particles act as dislocation sources during cooling from annealing temperatures resulting in high dislocation densities which strengthen the material.

A high voltage electron microscopy study of hydrogen-induced damage in a low alloy, medium carbon steel

A low alloy, medium carbon steel was heat treated to form different microstructures and cathodically hydrogen charged at room temperature in the absence of external stress. The hydrogen-induced microstructural damage was studied using high voltage electron microscopy (HVEM). Direct HVEM studies of three very different microstructures showed that the introduction of hydrogen results in the formation of dislocations, dislocation cells, small-angle subgrain boundaries and microbubbles in heavily charged fully spheroidized air-cooled-and-annealed (AA) and partially spheroidized (LS) samples. The hydrogen-induced dislocations are frequently tangled or piled up at grain boundaries and carbide particles. Hydrogen-induced subgrain boundaries are believed to be formed by a dynamic recovery process. Unlike AA or LS samples, the microstructure of quenched-and-annealed samples is not significantly affected by the charging treatment except for the formation of hydrogen-induced microbubbles. These hydrogen-induced microbubbles are observed to nucleate preferentially at internal interfaces such as grain boundaries and carbideferrite interphase boundaries. Locally high dislocation densities are usually observed to be generated around the microbubbles, indicating large localized strains accompanying bubble nucleation and growth. In the highly hydrogen-charged LS microstructure, the microbubbles are observed to nucleate predominantly at the already spheroidized carbide particles and at the tips of the partially spheroidized carbide lamellae. The more coherent carbide lamellar broad faces are apparently ineffective bubble nucleation sites.

The neuronal endomembrane system. I. Direct links between rough endoplasmic reticulum and the cis element of the Golgi apparatus

This is the first of three papers describing new components and structural relationships within the neuronal endomembrane system. This system includes: the nuclear envelope, rough endoplasmic reticulum, the Golgi apparatus, lysosomes, axoplasmic reticulum, and discrete cytoplasmic compartments such as vesicles, multivesicular bodies, and so on. Previous high voltage electron microscope studies of osmium-impregnated Golgi apparatus have shown that small varicose tubules often arise from the cis element. In bullfrog spinal ganglia, these tubules have been seen to extend into cytoplasmic domains occupied by rough endoplasmic reticulum (RER). This study was undertaken to determine whether varicose tubules form direct connections with RER and to measure cytoplasmic density of varicose tubules. Spinal ganglia from Rana catesbeiana were fixed by osmium impregnation and, in some cases, were then stained en bloc with lead aspartate. Sixty- to 3000-nm-thick sections of this tissue, as well as of ganglia prepared by conventional protocol, were studied using standard and high voltage electron microscopy. Quantitative analysis revealed that 9% of the varicose tubules connected with the Golgi cis element. To determine whether continuities existed between RER and any of these tubules, more than 50 tubules were fully reconstructed from images of serial sections. The "ends" of these reconstructed tubules were then studied by high magnification stereoscopy. This was facilitated by the en bloc lead staining which allowed visualization of unimpregnated RER elements along with the impregnated tubules. Small varicose tubules as well as larger smooth tubules were found to have formed confluent bridges between the cis element and RER. In addition, the varicose tubules were found to bridge widely separated elements of RER. Finally, numerous examples of varicose and smooth tubules were seen to extend from RER and from cis elements to eventually form blind endings. These findings raise the possibility that the tubules form highly dynamic transitory connections between RER and the Golgi apparatus as well as between separated elements of RER. Those between RER and Golgi apparatus are ideally positioned to play a major role in the transfer of protein or lipid components first assembled in RER to the Golgi apparatus for terminal synthetic modification.

Morphology, perfection and growth of natural pyrite whiskers and thin platelets

Whiskers and thin platelets of pyrite have been found growing coherently on the face of small cubic pyrite crystals from the Strashimir and Gradishte hydrothermal lead-zinc deposits. The host crystals formed framboids and spheroids of macroscopic size inside large crystals of chalcopyrite from which they are separated by concentric gaps. The whiskers, one to several μm in width, are elongated along [001] and bounded by {100}, partly by {210} and {111} faces. Scanning electron microscopic (SEM) observation revealed longitudinal steps and grooves, flat rectangular pits, drop-like and elongated bulges on their side faces. High-voltage electron microscopic (HVEM) studies showed that the thin whiskers are perfect and dislocation-free, although some ribbon-like whiskers and platelets contain internal longitudinal channels and small isometric fluid inclusions.

Bidirectional organelle transport can occur in cell processes that contain single microtubules.

Intracellular organelle transport was studied in a new model system, the giant freshwater ameba Reticulomyxa. The ameba extends a large reticulate network of cytoplasmic strands in which various phase-dense organelles can be seen to move at a rate of up to 25 microns/s. This combined light and high voltage electron microscopic study shows that organelles move bidirectionally in even the finest network strands that contain only a single microtubule. In terms of microtubule-associated intracellular transport, this observation defines a minimum set of conditions necessary for such movement. The implications of this finding for possible models of force generation are discussed.

Shape changes and polarization of cells migrating through tissue. A high-voltage electron microscope and computer graphics study of serial thick sections

Structural changes of carcinoma cells and fibroblasts migrating through small spaces in the elastic-collagen reticulum of mouse peritoneum have been studied by high-voltage electron microscopy of serial thick sections and by computer graphics reconstruction of cell profiles. The change of shape profile of an individual cell, between serial sections is large and the distribution of organelles is very non-uniform and changes markedly between sections. Conclusions about adhesion, intercell contact, cell shape and polarization of cytoplasmic organelles could only be reached by assessing a complete set of serial sections. Our preliminary results suggest that interesting structural changes occur in both carcinoma cells and fibroblasts when migrating through this tissue.

Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages.

Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0 degree C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37 degrees C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.3 approximately 3 micron thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 200 approximately 1,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0 degree C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37 degrees C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stereomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.

In Situ Hvem Studies of Electron-Beam-Driven Composition Changes in Thin Film Alloys: Displacement-Rate Gradient Effects

AbstractIn situ high-voltage electron microscopy (HVEM) observations have shown that the highly-focused electron beams normally employed for HVEM irradiation experiments can cause large chemical composition changes in the irradiated zone of thin film alloys during elevated temperature irradiations. The driving force for the process comes primarily from the radial gradients in displacement rates generated by the beam. Hence, the rate of change in composition exhibits a strong dependence not only on the temperature and beam intensity, but also on the spatial characteristics of the beam profile. This dependence on beam shape and size poses previously unrecognized problems, particularly for HVEM studies of the effects of dose-rate on radiation-induced phenomena that are sensitive to alloy composition. Moreover, composition changes driven by radial gradients in the displacement- rate occur at increasingly rapid rates as the beam diameter is reduced. Hence, at higher voltages, beam-induced composition changes occurring during analysis may become a serious problem, even at relatively low temperatures, for microchemical analysis techniques, such as EDX and EELS, which employ far smaller diameter electron beams than those used for irradiation purposes in the HVEM.

High voltage electron microscope study of defects in silicon

In-situ observation of defect formation in Fz and Cz silicon with (111) orientation by electron irradiation has been performed at temperatures between -100°C and 550°C using a 2 MeV high voltage electron microscope. Two kinds of interstitial type faulted dislocation loops are formed with and without an incubation period, respectively. The former appears in the region localized at the electron entrance surface, and the latter mainly localized at the electron exit surface. Linear defects, which appear near the electron exit surface, are also observed. Annealing experiments of these secondary defects were carried out around 800°C, and the point defect species which contribute to the shrinkage of the loops were identified as the vacancy, and the mean activation energy of 3.5 eV was obtained for self diffusion.