bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of crystalline phase in cross-section multilayer thin film by High-Resolution Transmission Electron Microscopy
Authors: Nguyen Tai D. , Gronsky Ronald , Kortright Jeffrey B. 
Published: June 2020

High-resolution transmission electron microscopy has proven to be very useful in direct detection of crystalline phases that exist over extremely small volumes, yielding information about structure, orientation, and, under appropriate circumstances, composition. In this paper, we report the detection of a crystalline phase in the tungsten-rich layer of an annealed 7 nm-period tungsten-carbon multilayer produced at the Center for X-Ray Optics at the Lawrence Berkeley Laboratory.The multilayers were prepared by dc magnetron sputtering at floating temperature. The argon sputter gas pressure was 0.0020 torr. Different techniques were employed to produce cross-section and plan-view samples for TEM. For cross-section samples, 70 bilayers of W and C were sputtered on semiconductor-grade Si (111) wafers. For plan-view samples, the substrates on which the multilayer was grown consisted of 3 mm-diameter 300-mesh copper microscope grids, mounted on glass slide with Crystalbond® vacuum adhesive. After a deposition of 4 bilayers of W-C, keeping the same sputtering parameters as those of the Si substrates to guarantee the same layer thicknesses, the glass slide was soaked in acetone to disolve the Crystalbond®, leaving the multilayer spanning the holes of the copper grids. Both the Si-substrate and copper-grid samples were annealed at 500°C for 4 hours under vacuum of 10−6 torr. The annealed Si-substrate sample was then prepared for cross-section by mechanical grinding, and ion milling in a cold stage at 5kV. The cross-section sample was studied in a JEOL JEM 200CX with ultrahigh resolution goniometer, with the eletron beam parallel to the [112] of the Si substrate. The plan-view sample was studied in a Philips 301 operating at 100kV.
Use of ion milling to expose and highlight defects on cross-sectioned integrated circuits
Authors: Martinek K.H. , Benedict J.P. , Klepeis S.J. , Joseph T. , Vandygrift G. 
Published: June 2020

Polishing and cross-sectioning of integrated circuits is standard practice in failure analysis. With most integrated circuit defects, all that is usually needed is mechanical polishing to the cross-sectioned plane of interest. The sample is then ready for examination in the scanning electron microscope (SEM). However, some defects are too small or too subtle to be seen without delineating them in some way. Chemical etching is useful in many situations but sometimes does not highlight the defect and may introduce artifacts. In this experiment, cross-sectioned chips were ion milled for a short period to highlight or uncover defects.After locating failing transistors, samples were cross-sectioned on diamond paper using the open block method (i.e., not encapsulated in epoxy). A special ion mill sample holder was designed to accommodate the polishing fixture. This holder is inserted into the ion mill. (This eliminates the need to transfer the chip from one fixture to another, avoiding damage or contamination.) Samples were milled in a Gatan ion mill for an initial period of two minutes at a 20 degree tilt. A current of ˙5 mA and voltage of 5 kV were used. Samples were positioned so that maximum milling occurred in the area of the failing transistors.
Intracellular structures of rapid frozen biological samples observed by high-resolution low-temperature Scanning Electron Microscopy
Authors: Inoué T. , Koike H. 
Published: June 2020

Low temperature scanning electron microscopy (LTSEM) is useful to avoid artifacts such as deformation and extraction, because specimens are not subjected to chemical fixation, dehydration and critical-point drying. Since Echlin et al developed a LTSEM, many techniques and instruments have been reported for observing frozen materials. However, intracellular structures such as mitochondria and endoplasmic reticulum have been unobservable by the method because of the low resolving power and inadequate specimen preparation methods. Recently, we developed a low temperature SEM that attained high resolutions. In this study, we introduce highly magnified images obtained by the newly developed LTSEM, especially intracellular structures which have been rapidly frozen without chemical fixation.[Specimen preparations] Mouse pancreas and brown adipose tissues (BAT) were used as materials. After the tissues were removed and cut into small pieces, the specimen was placed on a cryo-tip and rapidly frozen in liquid propane using a rapid freezing apparatus (Eiko Engineering Co. Ltd., Japan). After the tips were mounted on the specimen stage of a precooled cryo-holder, the surface of the specimen was manually fractured by a razor blade in liquid nitrogen. The cryo-holder was then inserted into the specimen chamber of the SEM (ISI DS-130), and specimens were observed at the accelerating voltages of 5-8 kV. At first the surface was slightly covered with frost, but intracellular structures were gradually revealed as the frost began to sublimate. Gold was then coated on the specimen surface while tilting the holder at 45-90°. The holder was connected to a liquid nitrogen reservoir by means of a copper braid to maintain low temperature.
Seasonal variation in Golgi dictyosomes in mesophyll cells of Taxus SP.
Authors: Rivera E.R. 
Published: June 2020

In plants, the Golgi dictyosomes are active in cell wall synthesis. During cell growth and division the dictyosomes are very numerous; many of them are components of the phragmoplast. After the cells mature, the dictyosome population decreases. Mesophyll cells of Taxus canadensis and Taxus cuspidata were observed over a two year period to determine dictyosome population variations in cells that survive for more than one growing season.Leaves of Taxus canadensis and Taxus cuspidata were collected every two weeks starting in May when new buds expanded. Collections were continued over two years in three experiments. The final specimens had gone through two full seasonal cycles. All specimens were fixed with 5% glutaraldehyde in 0.075M PIPES buffer pH 8. They were post fixed in 2% osmium tetroxide and embedded in Spurr's resin. Determination of oxidizable sugar residues was done by the periodic acid-thiosemicarbozide-silver protein method. For this study observations were limited to the mesophyll palisade cells on the upper half of the leaf. Random searches were done in the viewing screen as well as on micrographs.
Microtubules in plant cell division
Authors: Wick S. M. 
Published: June 2020

Immunofluorescence microscopy has proven to be a valuable accompaniment to electron microscopy for study of the cytoskeleton of plant cells. Whereas electron microscopy provides greater resolution and details of the spatial relationships of the cytoskeleton to other cellular components, fluorescence visualization makes it possible to see the three-dimensional organization of cytoskeletal elements without laborious reconstruction of views from serial sections. An area in which immunofluorescence microscopy has been useful is the investigation of how plant cells organize and position the various microtubule arrays that are utilized during mitosis, cytokinesis and cell expansion phases. One of the earliest indications of an impending division in a meristematic plant cell is the formation of a preprophase band of microtubules in the cell cortex, at the site where the new wall will be placed at the subsequent cytokinesis. At its later stages, the band is narrower than when first identifiable. In most cells, preprophase band microtubules have the same general orientation as the preceding interphase microtubules, and so preprophase band formation here could, in theory, be achieved by lateral bundling of microtubules.Cells in which the division site and the preprophase band that marks it are not oriented parallel to interphase microtubules are found in stomatal complexes of grass leaves . Fig. 1 illustrates the arrangement of two such cell types: the guard mother cell, which divides lengthwise to form two guard cells, side-by-side, and the subsidiary mother cell, which undergoes a very asymmetric division to produce one of the pair of lens-shaped subsidiary cells that flank the guard cells. Interphase and preprophase arrangements of microtubules for each cell type are diagrammed in Figs. 2-4. In order to examine how these cell types achieve the reorientation of microtubules that is necessary to progress from interphase to preprophase, sheets of epidermis containing actively dividing stomatal complex cells were examined with immunofluorescence microscopy using antibodies to tubulin. Thin epidermal slices of leaves were fixed and glued down to a slide, whereupon cell walls were enzymatically weakened so that unwanted cell layers could be removed . Because waves of division pass along grass leaves, cells of the same type in a given file tend to be at similar stages, which facilitates deduction of the developmental pattern.
Involvement of plasma membrane complexes in cellulose deposition in desmids
Authors: Giddings T. H. 
Published: June 2020

Deposition of cellulose microfibrils in oriented patterns in plant cell walls is believed to be one of the mechanisms by which cell shape is controlled. In some desmids, such as Closterium. circumferentially oriented microfibrils appear to help generate an elongated spindle shaped cell. In contrast, the primary cell wall of Micrasterias contains an apparently random meshwork of microfibrils, yet an elaborate multi-lobed semicell is formed. Freeze-etch electron microscopy of unfixed, ultrarapidly frozen desmids was undertaken to identify plasma membrane and cytoskeletal elements that may participate in the deposition and orientation of cellulose microfibrils.Micrasterias denticulata and aClosterium sp. were grown under a light/dark cycle to induce partial synchronization of cell division. Groups of several cells at the desired stage of semicell regeneration were loaded between two modified Balzers gold planchets coated with a slurry of yeast. A slot grid spacer prevented the cells from being crushed. Specimens were frozen in a propane jet freezing device designed by Gilkey and Staehelin. Replicas were produced in a Balzers BA 360 freeze-etch device and examined in a JEOL 100 CX electron microscope.
Ultrastructural characteristics of sporidia of Ustilago
Authors: Hackney Patricia N. 
Published: June 2020

Ustilago hordei and Ustilago violacea are yeast-like basidiomycete pathogens ofHordeum vulgare and Silene alba respectively. The mating type system in both species of Ustilago is bipolar, with alleles, A,a, (U.hordei) and a1, a2 (U.violacea) at a single locus. Haploid sporidia maintain the asexual phase by budding, while the sexual phase is initiated by conjugation tube formation between the mating types during budding and conjugation.For observation of budding, sporidia were prepared by culturing the four types on YEG (yeast extract glucose) broth for 24 hours. After centrifugation at 5000g cells were either left unmated or mated in a1/a2,A/a combinations. The sporidia were then mixed 1:1 with 4% agar and the resulting 1mm cubes fixed in 8% gluteraldehyde and post fixed in osmium tetroxide. After dehydration and embedding cubes were thin sectioned with a LKB ultratome and photographed in a Zeiss 9s transmission electron microscope or in an AE1 electron microscope of MK11 1MEV at the High Voltage Electron Microscopy Center of the University of Wisconsin-Madison.
Hair cell changes after cationic stimulation of corti's organ
Authors: Fermin Cesar D. , Zenner Hans-Peter 
Published: June 2020

Contraction of outer and inner hair cells (OHC&IHC) in the Organ of Corti (OC) of the inner ear is necessary for sound transduction. Getting at HC in vivo preparations is difficult. Thus, isolated HCs have been used to study OHC properties. Even though viability has been shown in isolated (iOHC) preparations by good responses to current and cationic stimulation, the contribution of adjoining cells can not be explained with iOHC preparations. This study was undertaken to examine changes in the OHC after expossure of the OHC to high concentrations of potassium (K) and sodium (Na), by carefully immersing the OC in either artifical endolymph or perilymph. After K and Na exposure, OCs were fixed with 3% glutaraldehyde, post-fixed in osmium, separated into base, middle and apex and embedded in Araldite™. One μm thick sections were prepared for analysis with the light and E.M. Cross sectional areas were measured with Bioquant™ software.Potassium and sodium both cause isolated guinea pig OHC to contract. In vivo high K concentration may cause uncontrolled and sustained contractions that could contribute to Meniere's disease. The behavior of OHC in the vivo setting might be very different from that of iOHC. We show here changes of the cell cytosol and cisterns caused by K and Na to OHC in situs. The table below shows results from cross sectional area measurements of OHC from OC that were exposed to either K or Na. As one would expect, from the anatomical arrangement of the OC, OHC#l that are supported by rigid tissue would probably be displaced (move) less than those OHC located away from the pillar. Surprisingly, cells in the middle turn of the cochlea changed their surface areas more than those at either end of the cochlea. Moreover, changes in surface area do not seem to differ between K and Na treated OCs.
Mass determination of the nuclear pore complex and its sub-complexesl by high-resolution Scanning Transmission Electron Microscopy (Stem)
Authors: Reichelt R. , Holzenburg A. , Engel A. , Aebi U. 
Published: June 2020

The nuclear pore complex (NPC) is an elaborate membrane-bound molecular machine residing in the nuclear envelope of all eukaryotic cells and acting as a passageway for molecular transport between the nucleus and the cytoplasm. Complementary to transmission electron microscopic studies of negatively stained and frozen hydrated NPCs yielding detailed information about their overall size, shape and substructure, the high-resolution scanning transmission electron microscope (STEM) was used to measure the mass, to map the radial mass distribution, and to evaluate the rotational symmetry of unstained native NPCs and of several distinct sub-complexes. “Native” NPCs were prepared for STEM as follows: the nucleus of Xenopus laevis oocytes was placed on a 4 nm carbon film supported by a holey film mounted on a copper mesh grid, and its nuclear envelope ruptured and spread out using fine glass needles. The grid was washed with low salt buffer to remove macroscopic contaminants, and excess liquid was withdrawn with filter paper. Next, tobacco mosaic virus (TMV; serves as internal mass standard) was co-adsorbed, before the grid was washed for 10 sec each on 3 drops of bi-destilled H2O. To disintegrate the NPC into distinct sub-complexes, nuclei were placed on a 4-nm thick carbon film supported by a thick holey carbon foil on a copper grid, ruptured and spread as above, and then detergent extracted. After washing, the samples were frozen in liquid N2, transferred to a VG HB-5 STEM and freeze-dried at 153 K. Low-dose annular dark field (AD) images (50 to 300 e/nm2) were acquired digitally, corrected for nonlinearities in the AD signal due to high mass thickness, and stored on tape for further processing.Native NPCs, with and without plug, are displayed in Fig. 1. As shown in Fig. 2, two types of rings can be distinguished after detergent extraction: bright rings (“outer” rings; thick arrow) and dimmer rings (“inner” rings; thin arrow). In the same preparations particle “pairs” consisting of a ring (Fig. 3, thin arrow) and a plug-spoke complex (Fig. 3, thick arrow) can often be depicted. From these data averaged and 8-fold symmetrized mass maps of the native NPC with plug (Fig. 4; ˜50 NPCs) and without plug (Fig. 5; ˜50 NPCs), the outer ring (Fig. 6; ˜50 rings) and the inner ring (Fig. 7; ˜15 rings) were computed.
Corresponding radial mass distributions are displayed at the same scale for comparison (Figs. 8 to 11). The average masses of the different structures are summarized in Table 1 together with a tentative model. These measurements bring speculations about the mass of the NPC to an end which have stated values ranging from 20 MDa to over 100 MDa, and they suggest that the NPC could easily be made of the order of 100 different polypeptides.
Colder specimens give a sharper impression: Prospects for higher resolution Electron Microscopy at liquid helium temperature
Authors: Lamvik M.K. 
Published: June 2020

Designers of electron microscopes started putting liquid helium into their plans with the hopes of improving instrumental resolution. Liquid helium can be used to cool specimen stages within conventional lenses, but it can also be used to make some materials superconducting. Once started, current flows continuously in a superconducting coil because it faces negligible electrical resistance. Superconducting lenses were introduced in electron microscopy to take advantage of the high magnetic field strengths that could be generated as well as the stability of the persistent current in the superconducting coils. As time passed, it became clear that electronic regulation of power supplies could produce more than adequate current regulation to supercede the need for the persistent current maintained in a superconducting lens. Meanwhile, the realization grew that low temperatures might give another kind of advantage to electron microscopy of radiation-sensitive materials. The very low temperature might help protect sensitive specimens from damage.Several methods are available for detecting damage to specimens in electron microscopes. The loss of mass from specimens is one sign of radiation damage. A mass loss measurement with a liquid helium cooled stage seemed to show a significant reduction in mass loss; a different measurement that included a superconducting lens also indicated reduced damage. Another indicator of damage is the rate of fading of diffraction patterns of ordered specimens, and in this case it has been established that liquid helium temperature causes a moderate reduction of damage.
Three-Dimensional reconstructions of ’light’ and ’intermediate’ capsids of equine herpes virus
Authors: Baker T. S. , Newcomb W. W. , Booy F. P. , Brown J. C. , Steven A. C. 
Published: June 2020

Equine herpes virus type 1 (EHV-1) belongs to an extensive family of large, genetically complex, and medically important animal viruses. The virion consists of an icosahedral nucleocapsid (T=16) separated from the viral envelope by a proteinaceous tegument layer. Assembly occurs in the nucleus of infected cells where capsids assemble, are packaged with DNA, then bud through the nuclear membrane. Two morphological species of EHV-1 capsids have been distinguished: “lights” which are abortive particles incapable of packaging DNA and “intermediates” which are precursors in the assembly of mature virions. Purified “intermediates” contain an additional proteinP22 (46 kDa), which is not present in “lights” and accounts for ˜10% of the total particle mass. In order to characterize the capsid structures of the two particle types and explore differences between them, we have applied three-dimensional reconstruction techniques to electron micrographs of frozen-hydrated specimens.The Kentucky A strain of EHV-1 was propagated in L-929 cells, and the two types of capsids were obtained as separate fractions from Renografin-76 density gradients. Neither fraction contained any significant amount of DNA. Cryo-electron microscopy of capsids on carbon film substrates was performed as described earlier. Micrographs were recorded at a nominal magnification of x36,000 at 2-3μm underfocus and digitized with a 50/μn step size (˜1.38nm sampling at the specimen). Three-dimensional reconstructions of the two particle types were computed to 4.5nm resolution using modified “common-lines” procedures. Images of 37 “light” and 39 “intermediate” particles were separately combined to compute three-dimensional density distributions.
Morphological polarity of intercalating deep mesodermal cells in the organzer ofXenopus laevisgastrulae
Authors: Keller Ray , Shih John , Wilson Paul 
Published: June 2020

The dorsal lip of the blastopore constitutes the “organizer” of the amphibian body plan, both in terms of its tissue interactions and its morphogenetic movements of convergence and extension during gastrulation. This tissue autonomously narrows (converges) and lengthens (extends) during early development, functioning prominently in the morphogenetic movements of both gastrulation and neurulationXenopus laevis. High resolution time-lapse recording of cell behavior in cultured explants and cell labelling studies have shown that the movements of convergence and extension are produced by radial intercalation of cells, in which several layers rearrange to produce fewer layers of greater area, and by mediolateral intercalation of cells, in which several rows of cells rearrange to produce a narrower, longer array. By labelling individual cells with the fluorescent compound, DiI, and making low light level recordings, we found that cells of the notochord intercalate mediolaterally using polarized protrusive activity at their internal medial or lateral ends. Thus polarized protrusive activity appears to play a major role in mediolateral cell intercalation after the boundary between the notochord and somites forms in the late gastrula stage.We examine further the morphology of the deep mesodermal cells with scanning electron microscopy at earlier stages, to search for morphological manifestations of a similar polarity of protrusive activity. The dorsal deep mesodermal cells of early gastrulae were exposed by rapidly pulling the. epithelial endoderm off the deep cells with forceps, in Danilchik's solution, and fixing the embryo within 15 seconds in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) as described previously. The dorsal deep cells adjacent to the epithelium are elongate and aligned parallel to one another and to the circumference of the glastopore (Fig. 1). The medial and lateral ends bear broad, lamelliform protrusions (large pointers, Fig. 1, 2) whereas the anterior and posterior ends bear numerous small filiform protrusions (small pointers, Fig. 1, 2). This characteristic morphology is found only in the dorsal marginal zone, which undergoes convergence and extension by mediolateral intercalation.
New approaches to the diagnosis of childhood leukemias
Authors: Mierau G.W. 
Published: June 2020

Electron microscopy probably is the single most precise method for classifying acute leukemias. However, because it is a relatively expensive technique it must be used very selectively. Ultrastructural studies are of genuine value and fully cost-justifiable in circumstances where: (1) routine cytochemical studies produce conflicting, confusing, or equivocal results; (2) insufficient or aberrant leukemic cell differentiation causes uncertainty; or (3) an unusual or uncommon diagnosis is under consideration. Such cases comprise only about five percent of the total. Establishing an accurate specific diagnosis in the problem case generally will require the use of one or more “special” ultrastructural techniques.The electron microscopist is faced with a challenging set of practical problems when presented with a specimen from a patient suspected of having leukemia, especially when that patient is a child — options are to be preserved, costs minimized, the sample very small, the time element critical, and so on. This presentation will attempt to demonstrate how we deal with these problems. The methodological details for the techniques used in our laboratory have been presented elsewhere.