bluuebunny/crossref_metadata_2025_split huggingface dataset:
Techniques in preparing TEM specimens of diamond thin films
Authors: Chiou W. A. , Wong M. S. , Chen F. R. , Li D. X. , Chang R. P. H. , Meshii M. 
Published: June 2020

Diamond, with its unique combination of optical, physical, mechanical, and electrical properties, has provided a superior potential for industrial and military applications. It has caught the attention of a growing number of researchers over the past decade. The recent development of low-pressure vapor deposition of diamond film has greatly increased the prospects for the practical utilization of diamond. To use a diamond film, the nature of the interface between diamond film and substrate must be understood. This paper presents three different techniques in preparing diamond thin films specimens for TEM study of the interfacial structure between diamond and substrate as well as the internal structure of diamond crystals.Diamond crystallites and films produced by radio frequency (RF) and microwave (MW) plasma enhanced chemical vapor deposition (PECVD) techniques were deposited on Si (100), polycrystalline Mo, and Cu substrates (925°C) at 40 mbar, with 0.5% methane and 0.2% oxygen in hydrogen gas at a total flow rate of 200 seem for MWPECVD and 2000 seem for RFPECVD. To examine the internal and interfacial structures of the diamond film on different substrates, three techniques were employed: (1) the chemical etching technique, which removes substrates then the substrate-free diamond film was supported on a Cu grid, (2) the conventional epoxy-embedding technique for cross-section study, and (3) the direct deposition of diamond crystallites on metal grids.
Solid-state reaction of Ru and Al in molten Bi
Authors: Garland C. M. , Johnson R. W. 
Published: June 2020

There has been much recent interest in the reactions that occur when two crystalline metals inter-diffuse at low temperatures. Metastable phase formation can occur when structural relaxation into equilibrium crystalline phases is kinetically inhibittedf. These reactions are typically studied in thin—film diffusion couples formed by sequentially depositing two elemental crystalline metals. In this paper we describe a novel low—temperature reaction sequence for the formation of very fine-grain Alx Ru,1-xalloys that allows a further extension of these techniques. This reaction regime uses molten Bi as a medium for the transfer of Al solute atoms to the surface of Ru powder where reaction occurs with the formation Alx Ru1-x,intermetallic phases. The low melting point and ease of handling of Bi, its solvent ability towards Al and Ru, the absence of equilibrium phases of Bi with Al and Ru, and previous experience in our laboratory with equilibrium and metastable phase formation in this system made it attractive as a prototype for this initial study.Ru powder and Al pellets were separatedly prereacted in molten Bi at 500 C for several hours to ensure wetting of the solid surfaces. This Ru and Al and additional excess Bi were then sealed together in a fused silica tube and reacted in a vertical tube furnace. Separate such reactions were carried out at temperatures ranging from 300 to 600 C. After reaction samples were broken free from the fused silica tube and the Bi transfer medium was etched away in concentrated HNO3.
A digital processing method for obtaining dark-field HREM image
Authors: Liu Yang , Huiming Tang 
Published: June 2020

The contrast of images can be increased by use of dark-field HREM. But in comparison with bright-field imaging, the increased exposure times required will result in more serious radiation damage effects on the specimen. This work is to develop a method to obtain computing dark-field images from bright-field images by use of computer processing, which can overcome the shortcome above.For A weak phase object,σϕ≤1, the transmission function can be written as[1].(1)Assuming that the amplitude of an incident wave is unity, the distribution of amplitude on the back focal plane of an ideal lens is(2)Cowley [2] demonstrated that a reasonable approximation to the ideal imaging condition, sinχ = 1, can be achieved for an optimum defocus, then we have(3)The image amplitude can be given by Fourier transform of (3) as the following:(4)
Apical poiarity of glycosyl-phosphatidylinositol anchored proteins in epithelial cells
Authors: Lisanti P. , Le Bivic A. , Saltiel A. , Rodriguez-Boulan E. 
Published: June 2020

Epithelial cells are characterized by a polarized distribution of structural features and functions between two plasma membrane domains, apical and basolateral. The molecular mechanisms responsible for this surface polarization are currently under intensive investigation. Studies on the biogenesis of viral envelope glycoproteins in the kidney cell line MDCK have demonstrated that sorting occurs in the distal cisternae of the Golgi apparatus (trans Golgi network) by incorporation into different populations of transport vesicles that fuse with the corresponding plasma membrane domain. Recombinant DNA technology has been utilized to localize the “sorting signal” to a specific glycoprotein domain, namely, extracellular, transmembrane or cytoplasmic. These studies have not been conclusive, but it appears that the exoplasmic domain appears to be sufficient for targeting to the apical surface.In order to understand the mechanisms responsible for sorting, we have recently characterized the polarized distribution of a group of plasma membrane proteins that are linked to the plasma membrane via a glycolipid, glycosyl-phosphatidylinositol (GPI). Proteins belonging to this class are attached via the C-terminal aminoacid to ethanolamine which, in turn is linked to an oligosaccharide chain, linked to the inositol ring of phosphatidylinositol (PI). The precise function of this mode of membrane attachment is still unknown. We explored the possibility that either the protein or the GPI anchor carry the sorting information.
Cryomicroscopy of crotoxin complex crystals at 400 KV
Authors: Brink Jaap , Chiu Wah 
Published: June 2020

The crotoxin complex is a potent neurotoxin composed of a basic subunit (Mr = 12,000) and an acidic subunit (M = 10,000). The basic subunit possesses phospholipase activity whereas the acidic subunit shows no enzymatic activity at all. The complex's toxocity is expressed both pre- and post-synaptically. The crotoxin complex forms thin crystals suitable for electron crystallography. The crystals diffract up to 0.16 nm in the microscope, whereas images show reflections out to 0.39 nm2. Ultimate goal in this study is to obtain a three-dimensional (3D-) structure map of the protein around 0.3 nm resolution. Use of 100 keV electrons in this is limited; the unit cell's height c of 25.6 nm causes problems associated with multiple scattering, radiation damage, limited depth of field and a more pronounced Ewald sphere curvature. In general, they lead to projections of the unit cell, which at the desired resolution, cannot be interpreted following the weak-phase approximation. Circumventing this problem is possible through the use of 400 keV electrons. Although the overall contrast is lowered due to a smaller scattering cross-section, the signal-to-noise ratio of especially higher order reflections will improve due to a smaller contribution of inelastic scattering. We report here our preliminary results demonstrating the feasability of the data collection procedure at 400 kV.Crystals of crotoxin complex were prepared on carbon-covered holey-carbon films, quench frozen in liquid ethane, inserted into a Gatan 626 holder, transferred into a JEOL 4000EX electron microscope equipped with a pair of anticontaminators operating at −184°C and examined under low-dose conditions. Selected area electron diffraction patterns (EDP's) and images of the crystals were recorded at 400 kV and −167°C with dose levels of 5 and 9.5 electrons/Å, respectively.
Imaging sub-micron objects with light microscopy: Visualization of the T-4 bacteriophage
Authors: Brown Malcolm , Delgado Reynolds M. , Fink Michael J. 
Published: June 2020

While light microscopy has been used to image sub-micron objects, numerous problems with diffraction-limitations often preclude extraction of useful information. Using conventional dark-field and phase contrast light microscopy coupled with image processing, we have studied the following objects: (a) polystyrene beads (88nm, 264nm, and 557mn); (b) frustules of the diatom, Pleurosigma angulatum, and the T-4 bacteriophage attached to its host, E. coli or free in the medium. Equivalent images of the same areas of polystyrene beads and T-4 bacteriophages were produced using transmission electron microscopy.For light microscopy, we used a Zeiss universal microscope. For phase contrast observations a 100X Neofluar objective (N.A.=1.3) was applied. With dark-field, a 100X planachromat objective (N.A.=1.25) in combination with an ultra-condenser (N.A.=1.25) was employed. An intermediate magnifier (Optivar) was available to conveniently give magnification settings of 1.25, 1.6, and 2.0. The image was projected onto the back focal plane of a film or television camera with a Carl Zeiss Jena 18X Compens ocular.
Cytotoxic mechanisms and disorders associated with natural killer (NK) cells
Authors: Payne Claire M. , Cromey Douglas W. 
Published: June 2020

Natural killer (NK) cells represent a first line of defense in the immune system, and are capable of spontaneous cytotoxicity against a variety of malignant cells, virally-infected cells and certain microorganisms. NK cytotoxicity is not dependent on prior sensitization by antigens, since target killing is not restricted to recognition of the major histocompatibility complex (MHC), in contrast to the need for MHC recognition by cytotoxic T-lymphocytes (CTLs). The mechanisms of target cell recognition by NK cells are largely unknown, although the glycolipid asialo GM2, the transferrin receptor and the laminin receptor, known to be present on sensitive target surfaces, have been implicated. NK cells share surface markers with T-lymphocytes and monocytes, and may represent a third mononuclear cell type with a distinct cell lineage.Previous work from our laboratory indicated that, like monocytes, NK cells bear an Fc-receptor for cytophilic antibody , and are capable of phagocytosis. NK cells, however, morphologically resemble lymphoid cells, since the cell surface is covered with microvilli, not surface folds. NK cells are also responsible (after monocyte depletion) for the majority of antibody-dependent cytotoxicity (K cells), since enrichment for K cells also enriches for functional NK cells, and vice-versa. NK cells have the morphologic appearance of large granular lymphocytes (LGLs). The cytoplasmic granules are azurophilic using Wright’s stain, have a substructure that has the appearance of parallel tubular arrays (PTAs) at the ultra-structural level , and biochemically contain a 70 kD protein called cytolysin (similar to the perforin lytic molecules of CTL granules).
Correlative histochemical and immunocytochemical techniques for the study of classical transmitters and neuropeptides
Authors: Pelto-Huikko M.T. 
Published: June 2020

A growing number of small biologically active peptides has been discovered in the nervous system and the endocrine organs of different mammalian species. These so-called neuro- or regulatory peptides may function as neurotransmitters, hormones or trophic factors. In many cases neuropeptides are colocalized in the same neurons and endocrine cells with classical transmitters and hormones. To understand the functions of neuropeptides it is important to determine the exact localization of neuropeptides in tissues and to clarify their relations to classical transmitters. In this paper a method for correlative visualization of epinephrine and norepinephrine with formaldehyde-glutaraldehyde (Faglu) fluorescence and subsequent demonstration of neuropeptides at light and electron microscopic level with pre-embedding immunocytochemistry in the same Vibratome section is described. A method for correlative demonstration of acetylcholinesterase (AChE) and neuropeptides at light microscopic level is also presented.Adrenal glands were fixed with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer, pH = 7.3. The adrenals of small rodents such as rat and hamster were fixed by immersion fixation for 6 h. The adrenals of larger species like cats and guinea pigs were fixed by perfusion for 20 min and further fixed by immersion for 1 hr. After fixation the tissues were rinsed several times in phosphate buffer and 7- to 10-um-thick sections were cut with a Vibratome. Sections are embedded in 0.5M Tris-saline buffer, pH = 7.6, which contained 20% glycerol and the catecholamine fluorescence was photographed (Fig. 1). After photography the coverslip was removed and the sections were processed free-floating through the steps of the pre-embedding modification of the PAP-method. After immunocytochemical staining the sections were post-fixed with 2% aqueous osmiumtetroxide for 1 h and dehydrated in graded ethanol series and flat-embedded in Epon under plastic coverslip. The areas photographed for catecholamine fluorescence were identified and photographed (Fig. 2). Desired areas were processed for electron microscopic examination. Human sympathetic ganglia were obtained from surgery and fixed with 4% paraformaldehyde for 6 h. Tissues were cryoprotected with 10% sucrose and 5 um frozen sections were cut on glass slides. Immunocytochemistry was carried out with indirect immunofluorescence method. After immunocytochemistry the sections were reacted for AChE according to the method of Karnovsky and Roots. Rhodamine-fluorescence and AChE were photographed using appropriate filters.
Immunolabelling of glomerular deposits in kidney biopsies routinely fixed in glutaraldehyde
Authors: Lhoták Ś , Alexopoulou I. , Simon G. T. 
Published: June 2020

Various kidney diseases are characterized by the presence of dense deposits in the glomeruli. The type(s) of immunoglobulins (Igs) present in the dense deposits are characteristic of the disease. The accurate Identification of the deposits is therefore of utmost diagnostic and prognostic importance. Immunofluorescence (IF) used routinely at the light microscopical level is unable to detect and characterize small deposits found in early stages of glomerulonephritis. Although conventional TEM is able to localize such deposits, it is not capable of determining their nature. It was therefore attempted to immunolabel at EM level IgG, IgA IgM, C3, fibrinogen and kappa and lambda Ig light chains commonly found in glomerular deposits on routinely fixed ( 2% glutaraldehyde (GA) in 0.1M cacodylate buffer) kidney biopsies.The unosmicated tissue was embedded in LR White resin polymerized by UV light at -10°C. A postembedding immunogold technique was employed
Digestion techniques for exposure of smooth muscle cells in cerebral arterioles
Authors: Ghoneim S. , Baumbach G. 
Published: June 2020

Scanning electron microscopy has proven to be a useful tool for the quantitation of three- dimensional characteristics of vascular smooth muscle. A major impediment to obtaining useful preparations of cerebral blood vessels for scanning electron microscopy is that smooth muscle in the tunica media is covered by a thin layer of basement membrane and by arachnoid tissue which contains collagen. The goal of this study was to develop a reliable, reproducible method for removing basement membrane and arachnoid tissue from cerebral arterioles, while at the same time leaving the smooth muscle intact.We compared several methods of tissue digestion in pial arterioles in adult Sprague-Dawley rats. The cerebral circulation was perfused with glutaraldehyde (2.5%) in cacodylate buffer (0.1 M) via the ascending aorta and then reinfused with packed red blood cells. In one group of rats, whole brains were immersed in osmium tetroxide (2%) for 2 hours (1 hr at 5°C and 1 hr at 40°C), followed by immersion in HC1 (8 N at 60°C) for 20-25 minutes. In another group, whole brains were dipped briefly in HC1 (8 N at 60°C) and then immersed in collagenase type II (2 mg/ml at 37° C; Sigma) for 12 hours. In a third group of rats, individual arterioles were dissected from the brain with a microsurgical knife, dipped briefly in HC1 (5-10 seconds) or KOH (5M at 60°C for 2-3minutes), and immersed in collagenase type II alone (12 hours), or in combination with collagenase type IV (2 mg/ml; 6-12 hours) and/or pepsin (2 mg/ml; 6-12 hours).
Ultrastructure of superficial neuromasts in the mottled sculpin, Cottus bairdi
Authors: Jones W.R. , Coombs S. , Janssen J. 
Published: June 2020

The lateral line system of the mottled sculpin, like that of most bony fish, has both canal (CNM) and superficial (SNM) sensory end organs, neuromasts, which are distributed on the head and trunk in discrete, readily identifiable groupings (Fig. 1). CNM and SNM differ grossly in location and in overall size and shape. The former are located in subdermal canals and are larger and asymmetric in shape, The latter are located directly on the surface of the skin and are much smaller and more symmetrical It has been suggested that the two may differ at a more fundamental level in such functionally related parameters as extent of myelination of innervating fibers and the absence of efferent innervation in SNM. The present study addresses the validity of these last two features as distinguishing criteria by examining the structure of those SNM populations indicated in Fig. 1 at both the light and electron microscopic levels.All of the populations of SNM examined conform in general to previously published descriptions, consisting of a neuroepithelium composed of sensory hair cells, support cells and mantle cells, Several significant differences from these accounts have, however, emerged. Firstly, the structural composition of the innervating fibers is heterogeneous with respect to the extent of myelination. All SNM groups, with the possible exception of the TRrs and CFLs, possess both myelinated and unmyelinated fibers within the neuroepithelium proper (Fig. 2), just as do CNM. The extent of myelina- tion is quite variable, with some fibers sheath terminating just before crossing the neuroepithelial basal lamina, some just after and a few retaining their myelination all the way to the base of the hair cells in the upper third of the neuroepithelium. Secondly, all SNMs possess fibers that may, on the basis of ultrastructural criteria, be identified as efferent. Such fibers contained numerous cytoplasmic vesicles, both clear and with dense cores. In regions where such fibers closely apposed hair cells, subsynaptic cisternae were observed in the hair cell (Fig. 3).
Ultrastructural analysis of “Sensory” surface nerve endings and “putative” neurotransmitter sites in Schistosoma mansoni Miracidia
Authors: Jones Betty Ruth , Pan Steve Chi-Tang 
Published: June 2020

INTRODUCTION: Schistosomiasis has been described as “one of the most devastating diseases of mankind, second only to malaria in its deleterious effects on the social and economic development of populations in many warm areas of the world.” The disease is worldwide and is probably spreading faster and becoming more intense than the overall research efforts designed to provide the basis for countering it. Moreover, there are indications that the development of water resources and the demands for increasing cultivation and food in developing countries may prevent adequate control of the disease and thus the number of infections are increasing.Our knowledge of the basic biology of the parasites causing the disease is far from adequate. Such knowledge is essential if we are to develop a rational approach to the effective control of human schistosomiasis. The miracidium is the first infective stage in the complex life cycle of schistosomes. The future of the entire life cycle depends on the capacity and ability of this organism to locate and enter a suitable snail host for further development, Little is known about the nervous system of the miracidium of Schistosoma mansoni and of other trematodes. Studies indicate that miracidia contain a well developed and complex nervous system that may aid the larvae in locating and entering a susceptible snail host (Wilson, 1970; Brooker, 1972; Chernin, 1974; Pan, 1980; Mehlhorn, 1988; and Jones, 1987-1988).
Morphology of sensilla of adult Pachypsylla celtids-mamma (Homoptera: Psyllidae) and of the gall formed on leaves of Celtis occidentalis (Urticales: Ulmaceae)
Authors: Beisler J. M. 
Published: June 2020

In comparison to other insects, very little Homoptera, and available information mainly location and type of sensory sens ilia of the psyllid Pachypsy1la celtidis-mamma and the morphology of the gall it forms on leaves of Celtis occidentalis were characterized by conventional scanning electron microscopy.Adult insects were fixed in buffered 4% glutaraldehyde (pH 7.2), postfixed in OsO4, dehydrated in ethanol, critical-point dried with CO2, and sputter-coated with goId-paHadium. For light microscopy (LM) insects were silver stained to identify porous chemosensilla. For SEM, whole or broken galls were fixed in 2.5% glutaraldehyde and processed as described above, except after dehydration whole galls were cryofractured in LN2. For LM, frozen sections of fresh galls were stained for lipids with Sudan Black B; PAS stain for carbohydrates was used on resin embedded galIs. SEM specimens were examined at 20 kV with a Hitachi HHS-2R or a JEOL 35CF.
Combined high-voltage and scanning electron microscopy on the same brain tissue samples for the study of blood-brain barrier injury
Authors: Lossinsky A.S. , Song M. J. , Pluta R. , Moretz R. C. , Wisniewski H. M. 
Published: June 2020

The application of combined techniques utilizing both scanning electron microscopy (SEM) with conventional transmission electron microscopy (CTEM) has a decided advantage because it permits analysis of identical tissue areas. If one is interested in analyzing of endothelial cell (EC) tight junctions, for example, although SEM offers unlimited topographical information, it is of critical importance to concomitantly assess EC junctions by CTEM from adjacent tissue blocks in order to visualize whether or not the junctional complexes are open only at the surface, for example, while deeper regions of this junctional complex may actually be occluded. Generally, three dimensional reconstruction is required employing either serial thin section analysis or high-voltage electron microscopic (HVEM) analysis of thick (0.25-1.0 μm) sections in order to obtain this information.Micro blood vessels (veins and venules) from the thalamus of SJL mice subjected to chronic relapsing experimental allergic encephalomyelitis were used in the present studies for examining EC junctional complexes and perivascular infiltration of inflammatory cells. Animals were perfused, fixed with standards buffered aldehydes, prepared for SEM, and blood vessels were identified and analyzed with an ISI-40 SEM. The same tissue blocks were trimmed to the targeted micro blood vessels, rinsed three times in propylene oxide (p.o.) for 20 min. each and flat-embedded in Spurr plastic following 1 hr. in 1:1 p.o.:Spurr. Thick (0.5 μm) sections were collected on Formvar-coated slot grids, stained with uranyl and lead salts and examined with an AEl HVEM (Albany, NY) at 1000 KV.