bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies of silica sphere growth
Authors: Bogush G. H. , Zukoski C. F.
Published: June 2020

Recipes are available for the preparation of monodisperse oxide powders and these materials are seeing increased use in the preparation of low flaw density ceramics. Despite the growing demand for these single sized powders, little is known about the physico-chemical processes which determine particle morphology. Here we report on studies of the mechanisms of particle growth.

As a model system we choose silica spheres prepared through the hydrolysis of silica alkoxides in basic alcoholic solutions. The rates of particle growth were studied by dipping carbon sputtered formvar coated electron microscope grids into the reaction mixture at different times. The grids were immediately placed on filler paper which rapidly wicked the fluid off the grid leaving the silica particles behind.
Short-range order in amorphous co-sputtered Ta-Al thin films
Authors: Barry J.C. , Timsit R.S. , Landheer D.
Published: June 2020

Tantalum-aluminium thin films have assumed considerable technological importance since the discovery in the late 1960's that the films are useful in the fabrication of thin film resistors and capacitors. It is generally claimed that these films, when prepared by co-sputtering Ta and Al, are amorphous over a range of Ta concentrations extending approximately from 15 to 75 at%, and are crystalline beyond this range. Diffuse electron diffraction patterns and ‘mottle pattern’ transmission electron micrographs are typical characteristics of the amorphous phase. In this present study we have attempted to identify any atomic short range order in the amorphous Ta-Al films and to follow the changes in this order as the Ta concentration increases across the amorphous/crystalline transition. The co-sputtered Ta-Al films of ≈100A thickness were examined in a high resolution 4000EX electron microscope (top entry, ±15°(x,y) tilt, Cs = 1.0mm ) at 400kV.
Noise reduction in digitized images
Authors: Christenson K. K.
Published: June 2020

In order to produce useable images at high scan rates, the detectors on modern electron microscopes often have a very high bandwidth (short response time to signal changes). This bandwidth makes the signal noisey; the signal has large fluctuations about the mean. Because the film integrates the signal this noise is not a problem when photographing an image. But it causes an annoying blurring of the trace In slow y-modulated line scans and can result in large errors if the signal if measured with a fast analog to digital converter (ADC).

We interfaced an EDAX 9100/70 x-ray analyzer with a PV9242 line scan/mapping option to a Philips EM420 STEM. The EDAX unit controls the x-y raster and digitizes the detector signal at each pixel. When reading the raw signal from the standard Philips SED detector and amplifier the images were very noisy. This is because the ADC actually samples the signal for a very small portion of the total conversion time and then holds the sampled value for the actual conversion, effectively reading the signal for 0.3 usee out of 170 usec.
Preparation of integrated circuits for TEM electromigration in situ studies
Authors: Maskowitz J. V. , Rhoden W. E. , Kitchen D. R. , Omlor R. E. , Lloyd P. F.
Published: June 2020

The fabrication of the aluminum bridge test vehicle for use in the crystallographic studies of electromigration involves several photolithographic processes, some common, while others quite unique. It is most important to start with a clean wafer of known orientation. The wafers used are 7 mil thick boron doped silicon. The diameter of the wafer is 1.5 inches with a resistivity of 10-20 ohm-cm. The crystallographic orientation is (111).

Initial attempts were made to both drill and laser holes in the silicon wafers then back fill with photoresist or mounting wax. A diamond tipped dentist burr was used to successfully drill holes in the wafer. This proved unacceptable in that the perimeter of the hole was cracked and chipped. Additionally, the minimum size hole realizable was > 300 μm. The drilled holes could not be arrayed on the wafer to any extent because the wafer would not stand up to the stress of multiple drilling.
Side-entry differentially pumped environmental chamber for the AE1-EM7 HVEM
Authors: Barnard D. , Rexford D. , Tivol W.F. , Turner J.N.
Published: June 2020

A side-entry differentially pumped environmental chamber (SEDPEC) has been designed and constructed for the AEI-EM7 high-voltage electron microscope (HVEM). The SEDPEC has been tested in the same way as previous chambers for the HVEM. In contrast to the lengthy procedures necessary to install previous environmental chambers in the HVEM, the SEDPEC can be installed in about one half hour. Thus a user can install the SEDPEC, use it for a day and return the HVEM to normal operating status without causing delays for other HVEM users. This is particularly important for our facility, which is supported as a national biotechnology resource by the NIH.
Corneal wound healing following excimer laser ablation
Authors: Gelber Tammy A. , Waltz Kathleen , Ghosn Corine R. , Stern Michael E. , Cochener Beatrice , Liaw Lih-Huei L. , Berns Michael W.
Published: June 2020

The excimer laser is currently being investigated in ophthalmology for refractive correction, including myopia, hyperopia and astigmatism. Laser photo ablation offers a consistent, precisely controllable method of tissue removal that potentially gives the surgeon more accurate control of surgical results than do conventional methods of corneal reshaping (i.e., diamond knives for radial keratotomy). In addition, there is interest in using laser photo ablation to smooth irregular corneal surfaces and to remove corneal stromal opacities. Early clinical studies have elucidated two problems common to refractive surgery. First, the cornea tends to develop a subepithelial haze approximately two weeks post-op in the ablation zone. In most cases this subsides during the ensuing six months. Second, the initial level of refractive correction reverses during the process of healing. This problem tends to be exaggerated in patients whose correction is greater than five diopters requiring a deeper ablation. Depending on the depth of the ablation, the epithelium, Bowman’s layer and the anterior stroma will be removed. The reparative process includes reepithelialization and the restoration of the stroma through the migration of keratocytes to the wound area. This is followed by the secretion of collagenases as well as new collagen and extracellular matrix (GAGs).
Ultrastructure and immunocytochemistry of interstitial cells of the deep muscular plexus in the canine small intestine
Authors: Torihashi Shigeko , Gerthoffer William T. , Sanders Kenton M.
Published: June 2020

In the canine small intestine one of the pacemaker regions that generates electrical activity is located in the deep muscular plexus. Within this region special cells, known as interstitial cells (IC-DMP), are distributed. The ultrastructure and immuno-cytochemistry of IC-DMP were studied to clarify their identification and relationship to smooth muscle cells.

Segments of the ileum were perfused through mesenteric arteries with a physiological buffer solution (pH5.0) for 3 min followed by a fixative (2.5% glutaraldehyde, 1.25mM Ca Cl2 and 3% sucrose in 0.05M cacodylate buffer; pH 7.4), postfixed in 1% OsO4 and embedded in Epon 812 for transmission electron microscopy (TEM). For immunoelectron microscopy, segments were perfused with a fixative solution (0.25% glutaraldehyde, 4% paraformaldehyde, 0.2% picric acid, 0.1% Triton X-100 and 0.1M phosphate buffer pH7.4), dehydrated and embedded in glycol methacrylate (GMA). Sections were stained with monoclonal antibody B4 to smooth muscle actin (ICN 1:200), anti-myosin light chain antibody (1:25), anti desmin- antibody(1:200), and anti-vimentin antibody (1:500) respectively. Immunoreactivities were detected by IgG gold (10nm, 6nm).
Ultrastructural studies of the digestive system of the leafhopper Euscelidius variegatus infected with BEV bacteria
Authors: Cheung W.W.K.
Published: June 2020

The leafhopper Euscelidius variegatus Kirshbaum transmits plant diseases such as aster yellows and X-disease. It also harbours a BEV bacterium as a result of congenital infection . Since the bacterium can reduce the fecundity of the insect it has the potential of being used as a controlling agent for E. variegatus. Presently, little is known about the cytopathology of the host infected with BEV. This study aims to understand the damage done by the bacterium to the digestive system of E. variegatus and the defense reaction of the host to the bacterium.

Digestive systems of BEV infected and non-infected adult females of E. variegatus were dissected in buffered insect saline and fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.2) for 1 hr. Tissues were post-fixed in 1% osmium tetroxide (pH 7.2) and dehydrated in alcohol/acetone series. These were blocked in Spurr resin and cut with a Reichart ultrotome. Thin sections were stained with uranyl acetate and lead citrate and were observed in a Zeiss EM 9S-2 electron microscope.
A microbial model for visualization of surface proteins using cryoimmobilization and high-resolution cryo-SEM
Authors: Erlandsen Stanley L. , Olmsted Stephen , Walther Paul , Chen Ya , Pawley Jim , Dunny Gary , Wells Carol L.
Published: June 2020

Numerous studies on high resolution visualization of surface membrane proteins have utilized colloidal gold markers in conjunction with SEM, TEM, and by freeze-fracture cytochemistry using conventional or fracture flip methods, but in each case only information on the distribution of the marker complex, and not the actual structure of individual surface protein(s) was obtained.

To investigate the detection of individual surface proteins, we have used a microbial model to examine surface proteins involved in conjugation or the transfer of DNA inEnterococcus faecalis. Two surface proteins encoded by the pCF10 plasmid, aggregation substance (Asc10; globular shape) and surface exclusion protein (Sec10; helical shape), mediate the aggregation of donor and recipient cells and prevent aggregation of like donor cells, respectively. Isogenic strains of E. faecalis that constitutively express neither Asc10 or Sec10 (pWM401) or both (pINY1801) were processed by 1) conventional chemical fixation, dehydration, critical point drying with CO2, ion beam sputtering with a discontinuous platinum film (<1 nm), and examination in a Hitachi S-900 FESEM operated at low voltage (<3 keV), or 2) cryoimmobilization of cells by high pressure freezing (Balzers HPM 010), mounting in a Gatan cryoholder followed by etching for 60 minutes at −100°C, cryocoating by planar magnetron sputtering (Balzers MED 010), and examination at −100°C in the S-900 FESEM.
Where’s the fat? Freeze-fracture analysis of ingredient interactions in food systems
Authors: Fincher Jean
Published: June 2020

An important trend in the food industry today is reduction in the amount of fat in manufactured foods. Often fat reduction is accomplished by replacing part of the natural fat with carbohydrates which serve to bind water and increase viscosity. It is in understanding the roles of these two major components of food, fats and carbohydrates, that freeze-fracture is so important. It is well known that conventional fixation procedures are inadequate for many food products, in particular, foods with carbohydrates as a predominant structural feature. For some food science applications the advantages of freeze-fracture preparation procedures include not only the avoidance of chemical fixatives, but also the opportunity to control the temperature of the sample just prior to rapid freezing.

In conventional foods freeze-fracture has been used most successfully in analysis of milk and milk products. Milk gels depend on interactions between lipid droplets and proteins. Whipped emulsions, either whipped cream or ice cream, involve complex interactions between lipid, protein, air cell surfaces, and added emulsifiers.
Scanning Tunneling Microscopy and low-temperature force microscopy of freeze-fractured samples
Authors: Fisher K. A. , Shattuck M. B. , Gustafsson M. G. L. , Clarke J.
Published: June 2020

Monolayer freeze-fracture combined with scanned probe microscopy (SPM) offers unique advantages for studies of biological structure. The freeze-fracture methodology incorporates rapid freezing approaches for sample preservation and stabilization, and the scanned probe microscopies, especially scanning tunneling microscopy (STM) and atomic force microscopy (AFM), allow high resolution examination of surface features including digital mapping, quantification, and display. In routine biological STM a sharp conductive probe is positioned with piezoelectric transducers close to a conductive surface. When electron tunneling begins, the probe is scanned while electronic feedback maintains constant current. Because the tunneling current is logarithmically sensitive to separation between the tip and the sample, the feedback signal can be calibrated to indicate height with sub-Angstrom sensitivity and precision. In routine biological AFM, the sample is scanned while the force between the tip and the sample is kept constant.

There are two fundamental problems in STM examinations of biological systems. First, biological samples are soft and are often perturbed by the scanning probe; and second, they are not electrically conductive. Coating samples with metal replicas simultaneously circumvents both these difficulties, conferring sample stability and electrical conductivity. The STM can be used to measure sample heights quite accurately, and has been used to measure the thickness and changes in thickness of metal-coated purple membrane and the depth of surface features of freeze-fracture replicas of synthetic phospholipids.
STM of freeze-fracture replicas: Problems and promises
Authors: Woodward J. T. , Zasadzinski J. A. N.
Published: June 2020

The Scanning Tunneling Microscope (STM) offers exciting new ways of imaging surfaces of biological or organic materials with resolution to the sub-molecular scale. Rigid, conductive surfaces can readily be imaged with the STM with atomic resolution. Unfortunately, organic surfaces are neither sufficiently conductive or rigid enough to be examined directly with the STM. At present, nonconductive surfaces can be examined in two ways: 1) Using the AFM, which measures the deflection of a weak spring as it is dragged across the surface, or 2) coating or replicating non-conductive surfaces with metal layers so as to make them conductive, then imaging with the STM. However, we have found that the conventional freeze-fracture technique, while extremely useful for imaging bulk organic materials with STM, must be modified considerably for optimal use in the STM.
New polarized-light microscope for fast and orientation independent measurement of birefringent fine structure
Authors: Oldenbourg Rudolf
Published: June 2020

The polarized light microscope has the unique potential to measure submicroscopic molecular arrangements dynamically and non-destructively in living cells and other specimens. With the traditional pol-scope, however, single images display only those anisotropic structures that have a limited range of orientations with respect to the polarization axes of the microscope. Furthermore, rapid measurements are restricted to a single image point or single area that exhibits uniform birefringence or other form of optical anisotropy, while measurements comparing several image points take an inordinately long time.

We are developing a new kind of polarized light microscope which combines speed and high resolution in its measurement of the specimen anisotropy, irrespective of its orientation. The design of the new pol-scope is based on the traditional polarized light microscope with two essential modifications: circular polarizers replace linear polarizers and two electro-optical modulators replace the traditional compensator. A video camera and computer assisted image analysis provide measurements of specimen anisotropy in rapid succession for all points of the image comprising the field of view.
Laser-feedback microscopy: Fundamental principles and application to high-resolution biological imaging
Authors: Bearden Alan , Wong Terrence L. , Mitchell Morgan W. , Osborne Leslie C. , O’Neill Michael P.
Published: June 2020

As discovered by Abbé, a fundamental limit to the lateral resolution of a conventional optical microscope is produced by “far-field” aperture diffraction at the objective lens. This limitation can be expressed in terms of the Airy disc which relates the obtainable resolution to the wavelength of light used and the objective’s numerical aperture (NA). In scanning confocal microscopy with a laser-produced Gaussian profile illumination beam, the lateral resolution in given by 1.22λ/NA. The axial resolution in this design is improved by the use of a pinhole aperture to pass light only from the objective’s focal plane. Typical best values for lateral (x,y) and axial (z) resolution for visible wavelength scanning confocal microscopy are ~200nm and ~300nm with a NA=1.4 oil immersion objective.

Although optical microscopy displays limited resolution when compared to some forms of electron and scanning-probe microscopies (STM, AFM), the convenience of sample preparation and its ease in performing in vivo studies has given rise to new methods of increased resolution (e.g., “near field” techniques, photon-tunneling, phase interference contrast).
Direct mechanical measurement of single DNA molecules through an optical microscope
Authors: Bustamante Carlos , Smith Steven B.
Published: June 2020

Understanding the mechanical properties of DNA such as their bending and torsional rigidity can provide a great deal of insight about the factors that determine the folding of DNA during the cell cycle. These quantities have been obtained from bulk experiments using light scattering, equilibrium sedimentation, DNA cyclization, etc. Determination of the elastic properties of DNA with these methods is indirect, via a theoretical framework relating the observable quantities to the elastic parameters of the molecules. Moreover, the results represent an ensemble average over all accessible configurations of the molecule, with small contribution from stretched un-likely states.

We have carried a systematic study of the elastic properties of single DNA molecules. Single DNA molecules were chemically attached by one end to a glass surface and by their other end to a magnetic bead. Equilibrium positions of the beads are observed in an optical microscope while the beads are acted on by known magnetic and hydrodynamic forces.
Subcortical excitatory inputs to nigral dopaminergic and non-dopaminergic neurons: A light and Electron Microscopic study
Authors: Damlama Meri , Tepper James M.
Published: June 2020

Midbrain dopaminergic neurons are involved in the symptomotology of motor disorders such as Parkinsonism as well as psychiatric disorders such as schizophrenia. The normal functioning of substantia nigra (SN) dopaminergic neurons is greatly influenced by their afferent inputs as evidenced by significant differences in the physiological characteristics of in vivo vs. in vitro preparations. Although the sources and neurotransmitters of many afferents to the SN are known, because dopaminergic and non-dopaminergic dendrites co-mingle in pars reticulata, the precise postsynaptic targets, particularly those of the presumed excitatory inputs, remain to be determined. In the present study inputs from the subthalamic (STN) and the pedunculopontine (PPN) nuclei (both presumed excitatory) to the SN were investigated by combined anterograde tracing and tyrosine hydroxylase (TH) immunocytochemistry at the light and electron microscopic levels.

The anterograde tracers biocytin or PHA-L were iontophoretically injected into either the STN or the PPN. Following appropriate survival times the animals were perfused transcardially with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde.
Energy filtering and correction for the contrast transfer function (CTF) of frozen hydrated objects as illustrated by a reconstruction of Dictyostelium S1 decorated actin
Authors: Schröder Rasmus R. , Manstein Dietmar , Jahn Werner , Spudich James A.
Published: June 2020

Ice embedded samples can not be simply described as weak phase objects, but as strong interacting specimens with inelastically dominated multiple scattering. Removal of the inelastically scattered electrons allows closer to focus imaging and improves the structural signal to noise ratio, both leading to a higher structural information content. Recent work demonstrated, that for radial density distributions the CTF correction of energy filtered images was necessary and sufficient to obtain good agreement with X-ray data. Here we present the fully CTF-corrected three dimensional structure of F-actin decorated with Dictyostelium myosin head fragment (MHF). The results show aggreement to the known protein structure after CTF correction. In addition to the radial density profile also amplitudes of Bessel terms in the Fourier Transform are changing.

Actin from rabbit muscle was decorated with Dictyostelium myosin head fragment under the usual buffer conditions. Images were taken with a Zeiss EFTEM EM902. Magnification was 50000x, electron energy 80kV, electron energy loss δE=0eV (zero-loss mode), energy width ΔE=20eV, defocus values were in the range of 500-900nm. Vitrified samples were observed at a temperature of 90K. All images were taken at an specimen electron dose of 500 e−/nm2, negative material was Kodak SO163 developed for 12 min in undiluted Kodak D19. For image processing and the helical reconstruction a new set of computer programs was written.
3-D imaging of freeze-dried actin filaments in fibroblast using high-resolution cryo-SEM
Authors: Chen Ya , Verkhovsky Alexander B. , Borisy Gary G.
Published: June 2020

A new approach to image the macromolecular structure of actin filaments from detergent-extracted cultured cells using high resolution low-temperature scanning electron microscopy (cryo-SEM) is described. An advantage of studying the cytoskeleton by SEM is its large depth of focus which makes it easy to reveal the three-dimensional relationship between the cytoskeleton filaments. Recently, procedures have been investigated for high resolution visualization of biological samples by SEM. These studies have demonstrated the importance of cryo preparation techniques for preserving the morphology of the specimen and advanced metal coating techniques for revealing its topography.

Coating for HRSEM requires a metal layer that will not obscure fine details while still yielding adequate SE signal. Magnetron-sputter coating of the samples with an ultrathin layer of metal at low temperature provides finer grain size than coating at ambient temperature. Chromium has many properties that make it suitable as coating metal for cytoskeleton: it has fine grain size, low BSE yield due to its low Z number, and it provides adequate SE signal.
Confocal microscopy, image restoration, and nuclear structure
Authors: Shaw Peter , Beven Alison , Rawlins David , Highett Martin
Published: June 2020

Fluorescentin situhybridization and 3-D confocal microscopy have been used to study the arrangement of genes and transcripts in interphase plant nuclei. Wide-field fluorescence microscopy coupled with computer deconvolution has also been used on the same specimens and gives comparable results to confocal imaging. When image deconvolution is applied to confocal images, these show a significant improvement. A comparison of confocal and wide-field (CCD) images of the same specimen (a plant nucleolus labelled with probe to the rRNA genes) is shown in Figure 1. The deconvoluted wide-field data (Fig 1b) and the raw confocal data (Fig 1c) are remarkably similar; the former is somewhat clearer. The deconvoluted confocal data (Fig 1d) is clearest of all.

Double-stranded DNA and single-stranded RNA probes to the 45S and 5S RNA genes have been used to investigate the functional organization of the nucleolus in pea root tissue.In situhybridization was carried out on vibratome slices approximately 50μm thick (about 3 cells).
3-D image restoration of fluorescence microscope images by regularization
Authors: Carrington Walter A.
Published: June 2020

By combining optical sections of a fluorescently labelled cell with quantitative calibration of the microscope’s blurring, we are able to computationally reverse the blurring of the microscope. We apply this approach to images taken on a conventional wide field microscope using a cooled CCD camera. Our algorithm is flexible in its use of data, which enables us to obtain good results with a small number of optical sections. We use this flexibility to minimize photodamage and photobleaching, so that we are able to obtain 3-D movies of living cells with high spatial and temporal resolution. We obtain rejection of out of focus light, resolution and accuracy of quantitative fluorescent measurement superior to confocal microscopes with substantially less photobleaching and photodamage to the sample. Recent algorithmic advances have enabled us to achieve transverse resolution as good as 100nm and axial resolution as good as 400nm.

The wide variety of cells and labels examined in 3-D in our lab illustrate the robustness and effectiveness of our image restoration approach. These applications range from samples as sparsely labelled as single copy genes in lymphocytes and fibroblasts labelled by in situ hybridization to densely labelled cells with fluorescently labelled dextran or Fura-2 that completely fill the cell volume.
Microstructure of the avian eggshell
Authors: Xiao S.Q. , Agarwal M. , Heuer A.H.
Published: June 2020

Fresh white and dry brown eggshells were studied by TEM and SEM. For TEM, thin sections of the eggshell were prepared by both ultramicrotomy and ion-beam thinning.

The various regions from the inner to the outer part of the eggshell can be summarized as follows : the shell membrane, the mammillary layer, the palisade layer and the cuticle. The shell membrane is about 80 μm thick and is composed of collagen fibers with a mantle and core structure. The mammillary layer is a mixture of calcite crystallites, organic matrix and collagen fibers; it starts at the outer shell membrane and merges gradually into the palisade layer. The collagen fibers present near the palisade are surrounded by tiny calcite crystals (Fig. 1). In this region, the periphery of fibers are calcified. The SEM study of the etched cross section revealed clusters of fine organic sheets at the junction of the membrane and the palisade layer (Fig. 2). Further etching results in the collapse of these sheets, to form knob-like features on the surface of the membrane.
Morphogenetic machines revealed: Microscopy of living cells in the embryo of the frog,Xenopus laevis
Authors: Keller Ray
Published: June 2020

The amphibian embryo offers advantages of size, availability, and ease of use with both microsurgical and molecular methods in the analysis of fundamental developmental and cell biological problems. However, conventional wisdom holds that the opacity of this embryo limits the use of methods in optical microscopy to resolve the cell motility underlying the major shape-generating processes in early development.

These difficulties have been circumvented by refining and adapting several methods. First, methods of explanting and culturing tissues were developed that expose the deep, nonepithelial cells, as well as the superficial epithelial cells, to the view of the microscope. Second, low angle epi-illumination with video image processing and recording was used to follow patterns of cell movement in large populations of cells. Lastly, cells were labeled with vital, fluorescent dyes, and their behavior recorded, using low-light, fluorescence microscopy and image processing. Using these methods, the details of the cellular protrusive activity that drives the powerful convergence (narrowing)
Applications of ultramicrotomy for materials characterization
Authors: Blackham R.T. , Haugh J.J. , Hughes C.W. , Burke M.G.
Published: June 2020

Essential to the characterization of materials using analytical electron microscopy (AEM) techniques is the specimen itself. Without suitable samples, detailed microstructural analysis is not possible. Ultramicrotomy, or diamond knife sectioning, is a well-known mechanical specimen preparation technique which has been gaining attention in the materials science area. Malis and co-workers and Glanvill have demonstrated the usefulness and applicability of this technique to the study of a wide variety of materials including Al alloys, composites, and semiconductors. Ultramicrotomed specimens have uniform thickness with relatively large electron-transparent areas which are suitable for AEM anaysis.

Interface Analysis in Type 316 Austenitic Stainless Steel: STEM-EDS microanalysis of grain boundaries in austenitic stainless steels provides important information concerning the development of Cr-depleted zones which accompany M23C6precipitation, and documentation of radiation induced segregation (RIS). Conventional methods of TEM sample preparation are suitable for the evaluation of thermally induced segregation, but neutron irradiated samples present a variety of problems in both the preparation and in the AEM analysis, in addition to the handling hazard.
Feature enhancement by non-uniform ion-milling for SEM of integrated circuits
Authors: Shaapur F.
Published: June 2020

A variety of techniques has been developed to enhance the image contrast of EM specimens in both physical and biological sciences. In SEM of materials, feature enhancement is mainly based on augmentation of surface topography by non-uniform material removal from different structural elements and preferential erosion at adjoining interfaces. The enhanced surface topography, in turn, translates into better response to the probing beam resulting in higher image contrast.

In cross-sectional imaging by SEM (XSEM) of ICs, feature enhancement has been carried out by chemical or mechanical, i.e., ion-milling, surface treatments. Ion-milling has been used to clean surfaces and to enhance their topography. The non-uniform material removal from the surface, in this case, merely relies on an inherent characteristic of the structural elements, i.e., their relative erosion rates during the ion-thinning. An additional factor, i.e., minimization of the “shadowing” effect by adjusting the orientation of ion incidence may also be used to elevate the non-uniform ion-milling even further.
A study of the microvasculature of the normal rabbit bladder with vascular corrosion casting, SEM and TEM
Authors: Hossler Fred E. , Monson Frederick C.
Published: June 2020

The mammalian urinary bladder performs two functions, urine storage and expulsion. Bladder function is dependent upon the delivery of oxygen and nutrients via a rich blood supply, yet studies of the bladder vasculature during distension have concluded that blood flow is restricted during filling. Following surgically induced ischemia, regeneration of the wall and its vasculature have been reported but not described in detail. Beyond the gross level, the functional vascular anatomy of the bladder wall is poorly understood. Preliminary to studies of angiogenesis and mucosal regeneration following ischemia, and vascular accommodation during distension, the present study utilizes routine transmission (TEM) and scanning (SEM) electron microscopy, and vascular corrosion casting (VCC) to describe the normal microvasculature of the rabbit bladder.

Bladders were perfuse fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, for routine TEM and SEM. Thin sections were cut from Araldyte and stained with lead and uranium for TEM, and samples were critical point dried from ethanol and CO2 for SEM.
Three-dimensional transmitted brightfield imaging: pragmatic data collection and preprocessing considerations
Authors: Cooper J.A. , Bhattacharyya S. , Turner J.N. , Holmes T.J.
Published: June 2020

We have been developing algorithms for 3D image reconstruction of biological specimens with absorbing stains. This is important because there are many absorbing stains which are widely used in conjunction with transmitted light brightfield (TLB) microscopy, yet most of the 3D microscopic imaging research has been directed toward fluorescence microscopy. For instance, horseradish peroxidase (HRP) is used widely in the neurosciences for its many advantages as a tracer and intracellular marker. It is readily injected into individual neurons, transported long distances, and fills both the dendritic and axonal fields, while it may double as an electron microscopy stain for correlative analysis. With such advantages, it is clear that absorbing stains will continue to be widely used. Their utility will furthermore broaden with 3D visualization and quantitation.

The main principles behind our methodology are the following. Standard optical serial sectioning data collection is used. The iterative, constrained image reconstruction algorithm is designed to reconstruct the 3D optical density distribution .
Use of a post-embedding, indirect, double-sided labeling procedure to identify cona binding sites within apical vesicles of a filamentous fungus
Authors: Bourett Timothy M. , Howard Richard J.
Published: June 2020

Vesicles are thought to play an important role in the polarized apical extension of fungal hyphae. These vesicles are likely to contain cell wall precursors and other molecules destined for exocytosis at the hyphal apex, including polysaccharides and/or other glycosylated molecules which should be recognized by certain lectins, including Concanavalin A (ConA). Here we describe the presence of ConA binding sites (CABS) within the apical vesicles of Trichoderma viride. In addition, we compare single- and double-sided ConA labeling patterns to assess the impact of these different techniques on the interpretation of results.

Somatic hyphae of T. viride were grown as a cell monolayer over pieces of cellophane on the surface of nutrient agar plates. The cellophane pieces and cells were plungefrozen in a liquid propane/ethane mixture, freeze-substituted in 2% OsO4 in acetone,and subsequently embedded in Quetol resin. Thin sections were picked up with empty, formvar-dipped, single-slot, gold grids.
Projections from the periaqueductal gray (PAG) to the periambigual area: Relation to cholinergic vago-cardiac neurons
Authors: Xu Shuang-jin , Rizvi Tilat A. , Jiang Maorong , Ennis Matthew , Shipley Michael T.
Published: June 2020

Activation of the midbrain periaqueductal gray (PAG) elicits species specific defense reactions and marked autonomic adjustments. In agreement with recent studies by Bandler and colleagues in the cat, we found that activation of rostrocaudally-oriented, longitudinally organized columns in PAG elicit differential cardiovascular and behavioral responses. Activation of dorsolateral/lateral PAG produces aggressive/flight behaviors accompanied by pressor responses while stimulation of ventrolateral PAG produces immobility and depressor responses. The pathways mediating these selective behavioral and autonomic responses are poorly understood.

The projection from PAG to the sympathoexcitatory zone in the rostral ventrolateral medulla is a likely substrate for PAG-evoked pressor responses. However, circuits mediating PAG-evoked depressor responses are not known. The present studies have identified a robust, focal projection from PAG to the nucleus ambiguus (NA), a potent depressor area in the ventral medulla. In addition, we have used tract tracing techniques and immunocytochemistry to examine the organization of PAG projections in relation to cholinergic preganglionic parasympathetic NA neurons that innervate the heart.
Viral proteins are expressed on membranous vesicles in the endoplasmic reticulum of elavivirus-infected cells
Authors: Downs Mary Beth , Summers Peter
Published: June 2020

Japanese encephalitis virus is a member of the family Flaviviridae, a group that includes yellow fever virus and dengue virus. Flaviviruses contain a central nucleocapsid made of a capsid protein (C) and a single-stranded RNA genome surrounded by a membranous envelope with two associated proteins: the surface envelope glycoprotein (E), which forms projecting spikes, and the non-glycosylated membrane protein (M). Infected cells also synthesize seven non-structural viral proteins, including the NS1 protein which is found on the cell surface and is secreted in particulate form. Replication of flaviviruses appears to involve the endoplasmic reticulum (ER) as proliferation of ER membranes and formation of intracisternal vesicles are characteristic of flavivirus infection, essentially all viral proteins are membrane associated, and mature virions are first seen in the lumen of the ER.
Differentiation of malignant mesothelioma from adenocarcinoma
Authors: Garancis John , Cafaro John , Cafaro Anthony , Venezia Raymond
Published: June 2020

Malignant mesothelioma (MM) is a rare tumor arising predominantly from either the visceral or parietal pleura or peritoneal surfaces. It primarily effects individuals between 45 and 75 years. of age and asbestos is felt to be the single most important etiologic agent. Studies suggest that the incidence of MM is increasing perhaps at a rate of as high as 13% per year. The diagnosis of MM can be difficult and is often complicated by the tumors similarity to metastatic adenocarcinoma, both in it's microscopic appearance and to some extent clinical course.

In this particular study the immunohistochemical properties of 22 malignant mesotheliomas (20 pleural and two peritoneal) are compared with 20 pulmonary adenocarcinomas. Each tissue was examined using monoclonal antibodies to wide-spectrum cytokeratin, CEA, Leu-M1 and B72.3. Extensive asbestos exposure was identified in all but 1 of the mesothelioma cases. The cases of peritoneal MM were found to have a history of chrysctile exposure only (Fig.1). Distant metastases were present in four cases of MM.
Transport pathways of low-density lipoproteins by arterial endothelium of hypercholesterolemic rats
Authors: Kao C.H. , Yang V. C. , Chen J.K. , Kuo J.S.
Published: June 2020

Atherosclerosis is more likely to occur at the branched regions of the large or mediumsized arteries. Physiological evidences show that changes in endothelial permeability at the branched regions can cause low density lipoproteins (LDL) to filtrate and accumulate in the intima. However, the LDL transport pathways across the arterial endothelium at the ultrastructural level still remain uncertain. The purpose of this experiment is utilizing fluorescein 1,1' -dioctadecyl-3,3,3',3' -tetramethylindocarbocyanine perchlorate (Dil) and colloidal gold as the tracer substances to investigate the transport pathways of LDL in the branched and the unbranched regions of arteries in the diet-induced hypereholesterolemic rats.

Rat or human LDL was coupled to Dil or to 10-15nm colloidal gold particles according to the standard procedures. Male Srague-Dawley rats, weighing approximately 250 gm were fed high-cholesterol diets over a period of 12 months. At 6 and 12 months after feeding, animals were anesthetized by intraperitoneal injections of sodium pentobarbitol. The vascular bed was cleared of blood by perfusion with oxygenated PBS supplemented with 0.25% glucose and 1 mM CaCl2 at 37°C through the left ventricle. After the blood washed out, the right and the left femoral arteries and the left ventricle, the thoracic and the abdominal aorta were cannulated with polyethylene tubings. The tubings between the left ventricle and the thoracic aorta and the tubings between the abdominal aorta and the femoral arteries were connected with peristaltic pump to form two closed circuits.
Cell injury and calcium accumulation following sulfur mustard exposure
Authors: Petrali J.P. , Hamilton T.A. , Mills K.R. , Ray R.
Published: June 2020

Cellular injury, irrespective of causative agents, is associated with a change in the transmembrane potential for calcium with subsequent accumulations of calcium to intracellular domains. At this time however, the association of sulfur mustard (HD)-induced cytotoxicity and calcium metabolism remains at controversy. This study correlates the ultrastructural cytopathology, cytochemical localization and x-ray microanalysis of calcium in cultured cells exposed to HD in vitro.

NG108-15 and HeLa cells, exposed to 300 and 500 ¼M HD respectively and harvested at 1 to 24h post exposure, were fixed in 1.6% formaldehyde and 2.5% glutaraldehyde for 3 h at 4°C and processed for routine ultrastructural evaluation. Replicate populations were cytochemically processed for precipitation of calcium by incubating in a medium of 2% potassium pyroantimonate and 1% osmium tetroxide for 90 min in the dark at 20°C. Cells were then placed in a secondary medium of 0.5% tannic acid for 15 min, rinsed in 1% sodium sulfate, dehydrated in graded ethanols and embedded in epoxy resin.
Peanut agglutinin receptors in the epididymai spermatozoa of the palm squirrel (Funambulus penanti)
Authors: Bawa S. R. , Bawa R. , Bains H. K.
Published: June 2020

Physiological ripening of the mammalian spermatozoa during their sojourn in the epididymal duct is also mirrored on the morphological plane by lectin-labeling studies. In this report peanut agglutinin (PNA), which specifically labels methyl α-D-galactopyranoside, has been employed to monitor the maturation process of the epididymal spermatozoa of the Indian palm squirrel (Funambulus penanti) employing fluorescence microscopy and transmission electror microscopy (TEM in toto).

Spermatozoa were released from the epididymis by gently mincing the tissues in phosphate-buffered saline (pH 7.4). For fluorescence microscopy, sperm suspensions were smeared onto poly-L-lysine coated coverslips and labeled with 10 ¼l of fluorescein isothiocyanate-linked PNA (100 ¼l/L). For TEM in toto sperm suspensions were adsorbed onto poly-L-lysine and Formvar coated grids and subsequently labeled with colloidal gold-linked PNA. After washing in buffer the grids were examined with a JEOL 1200 EX transmission electror microscope. Controls were prepared by incubating sperm samples in 0.2 M α-D galactopyranoside.
A new cytochemical method: Combined enzyme cytochemistry and immunocytochemistry on ultrathin cryosections
Authors: Takizawa Toshihiro , Robinson John M.
Published: June 2020

Cryo-ultramicrotomy has been a useful method for immunocytochemistry because it can preserve the ultrastructure of cells and also display antigenic sites in cells. Recently, cryo-ultramicrotomy has been successfully applied to enzyme cytochemistry for the demonstration of some phosphatases with lead as the capture metal. However, this application has some problems. The lead reaction product is unstable with uranyl acetate staining, thus leading to poor ultrastructural detail. The purpose of this study is to develop the new method of enzyme cytochemistry using cerium as the capture metal on ultrathin cryosections labeled with immunogold probes. This method should be of importance for demonstrating the ultrastructure localization of certain enzyme and the simultaneous localization of antigens in a variety of cell types.

Freshly isolated human neutrophils were fixed with paraformaldehyde and/or glutaraldehyd in 0.1 M cacodylate buffer (pH 7.2) with 5 % sucrose, then pelleted into 10 % gelatin and subsequently infiltrated with 2.3 M sucrose.
Atypical basal bodies Found in ciliated cells of an asthmatic patient: An acquired ultrastructural alteration or evidence of a disorder of ciliogenesis?
Authors: Gunning W.T. , Judkins R.F. , Akpunonu B.E.
Published: June 2020

Numerous reports of abnormal cilia in human respiratory epithelium have been published within the last ten years; most of these studies have described alterations of the axoneme and attributed these changes to a dysfunction of the ciliary apparatus, otherwise labeled immotile cilia syndrome or primary ciliary dyskinesia. In general, dysfunctional or ineffective mucociliary clearing of the respiratory tract is a prime factor contributing to chronic respiratory tract infections, and while patients with chronic respiratory tract infections do not necessarily have an immotile cilia syndrome, this history is the norm for patients suffering from the congenital disorder. More than 20 alterations of the cilia axoneme have been observed. The most common is the loss of dynein arms and other frequently seen abnormalities include loss of radial spokes and transposition of ciliary microtubules. Approximately one half of the patients found to have ciliary abnormalities also havesitus inversus, bronchiectasis, and chronic sinusitis, a triad of manifestations known as Kartagener's syndrome.
Undeveloped fibers: A source of dyeing problems in cotton textiles
Authors: Goynes W. R. , Ingber B. F. , Thibodeaux D. P.
Published: June 2020

In the growth and development process of cotton fibers, during approximately the first 17 days only elongation of the primary wall structure occurs. As the elongation process is being completed, secondary layers of cellulose fibrils begin to be deposited within the primary wall to form the main body of the fiber. This developing wall provides the fibers with a dimensional rigidity. When the boll opens and the fibers dry, they are able to maintain a dimensional structure, as illustrated in figure 1. Samples of harvested cotton fibers normally contain fibers whose walls have completely developed, as well as some whose walls are thin because of a short development time. Fully developed fibers are referred to as “mature” and incompletely developed fibers are referred to as “immature.” There may also be fibers that are essentially undeveloped beyond the primary wall stage. Undeveloped cotton fibers are produced by seed whose development was interrupted before maturity (motes).
Female sex steroid induced gallbladder epithelium changes and gallstone deposits in female hamsters: TEM and SEM aspects
Authors: Karkare S. , Gilloteaux J. , Kelly T. R.
Published: June 2020

Approximately 1 million people in the United States alone develop gallstones each year. The incidence is higher in women than in men and the ratio being 4 ≥ 1. A correlation has also been suggested between oral contraceptives and cholelithiasis. In addition, postmenopausal or cancer estrogen therapy has been reported to be a factor responsible for gallstone formation. Female sex hormone receptors have been detected not only in the gallbladder musculature, but also in its epithelium. As a follow up to experiments effectuated in the male and the ovariectomized Syrian hamster, this report shows that, a combination of a low cholesterol diet with female sex steroid treatment contributes to the formation of gallstone-like deposits, while modifying the surface epithelium morphology. Syrian hamsters (F1B strain, BioBreeders, Watertown MA) were housed under 12h light: 12 h dark cycle, at 20 °C, fed Purina chow and water ad libitum. Several duration/treatment groups were studied, but this report will focus on data obtained with the group injected weekly with estradiol valerate (E weekly, s.c. 8-10 μg/100 g.b.w., in corn oil) and with i.m. medroxyprogesterone acetate (DepoProvera Upjohn Co., Kalamazoo, MI; 8-10 mg/100 g.b.w.) for a 3-month period. Other parameters (blood and bile) were also studied but not reported here.