bluuebunny/crossref_metadata_2025 huggingface dataset:
On-line alignment and astigmatism correction using a TV and personal computer
Authors: Hosokawa F. , Kondo Y. , Honda T. , Ishida Y. , Kersker M.
Published: June 2020

High-resolution transmission electron microscopy must attain utmost accuracy in the alignment of incident beam direction and in astigmatism correction, and that, in the shortest possible time. As a method to eliminate this troublesome work, an automatic alignment system using the Slow-Scan CCD camera has been introduced recently. In this method, diffractograms of amorphous images are calculated and analyzed to detect misalignment and astigmatism automatically. In the present study, we also examined diffractogram analysis using a personal computer and digitized TV images, and found that TV images provided enough quality for the on-line alignment procedure of high-resolution work in TEM. Fig. 1 shows a block diagram of our system. The averaged image is digitized by a TV board and is transported to a computer memory, then a diffractogram is calculated using an FFT board, and the feedback parameters which are determined by diffractogram analysis are sent to the microscope(JEM- 2010) through the RS232C interface. The on-line correction system has the following three modes.
Analysis of electron-specimen interactions of thick biological specimens in Transmission Electron Microscopy at 200 KEV
Authors: Han Karen F. , Gubbens Alexander J. , Koster Abraham J. , Braunfeld Michael B. , Sedat John W. , Agard David A.
Published: June 2020

The primary project of our laboratory is the investigation of chromatin structure by three dimensional electron microscope tomography. The goal is to understand how 30nm fibers fold into higher order chromatin structures. Three dimensional tomography involves the reconstruction of an object by combining multiple projection views of the object at different tilt angles. Due to the electronspecimen interaction and the characteristics of lens aberration in the electron microscope, however, the image is not always an accurate representation of the projected object mass density. In this abstract, we analyze the various types of electron-specimen interaction for thick biological specimens up to 0.7 microns thickness.

Electron-specimen interactions include single elastic and inelastic, and multiple elastic and inelastic scattering. Of the imaging electrons, the single elastic and the plasmon electrons give rise to image intensities that can be linearly related to the projected object mass density. Multiply scattered elastic electrons contribute to an increase in background intensity. In addition, due to the chromatic aberration of the TEM’s objective lens, multiply scattered inelastic electrons cause a blurring of the image because of an effective broadening of the focus spread.
Image processing to extract line information from micrographs
Authors: Turner John H. , O’Keefe Michael A. , Paciornik Sidnei
Published: June 2020

In processing microscope images, it is sometimes important to be able to extract the lengths and directions of linear features. For example, in characterizing transmission electron microscope images of faceted grain boundaries, the desired information is the length of grain boundary lying along each direction. In this case we require the sum of the lengths of the facets as a function of direction, but not their positions.

The problem of having the computer recognize and extract lines from images is not trivial. In order to measure grain boundary lengths automatically, micrographs must be digitized and the linear features (e.g. the grain boundaries) binarized and reduced to a single pixel width. Then the problem requires finding two simple parameters for each line in the image, its direction and number of pixels. A new approach to this problem is to project the intensities of the source image in all possible directions, and to collect the projection results into a new two-dimensional “image” with one dimension representing the projection direction. This new image is the sinogram of the source image.
Effect of raised temperature on a stainless-steel surface by ambient atomic-force microscopy
Authors: Jung Pan S.
Published: June 2020

We report images from atomic force microscopy (AFM) of a heated metallic surface performed under an ambient environment. The stainless steel surface was heated up to 121C, which is limited only by the capacitive heating element used (100W). We observed that the topography of the surface changes dramatically. It is found that at high temperatures, the surface appears flat compared with the presence of many round bumps observed on the same sample at room temperature when the ambient humidity is high. To the best of our knowledge, this is the first report on ambient AFM performed on a metallic surface above 100C.

Most AFM systems adopt moving sample stages [1]. In this case, it is difficult to implement heavy heating heating elements and a heat reservoir block. For this experiment, the TAK3 from AT Corporation [2] is used. The sample stage is kept stationary and the heating element (the tip of a soldering iron) is fixed firmly on one side of a surface of the block using a screw.
Cross-sectional TEM specimen preparation from magneto-optical disk by ultramicrotomy
Authors: Shaapur F. , Kim M.J. , Lee Seh Kwang , Kim Soon Gwang
Published: June 2020

TEM characterization and microanalysis of the recording media is crucial and complementary to new material system development as well as quality control applications. Due to the type of material generally used for supporting the medium, i.e., a polymer, conventional macro- and microthinning procedures for thin foil preparation are not applicable. Ultramicrotorny (UM) is a viable option and has been employed in previous similar studies. In this work UM has been used for preparation of XTEM samples from a magneto-optical (MO) recording medium in its original production format.

The as-received material system consisted of a 4-layer, 2100 Å thick medium including a 300 Å TbFeCo layer enveloped by silicon nitride protective layers supported on a 1.2 mm thick × 135 mm (5.25 in.) diameter polycarbonate disk. Recording tracks had an approximate pitch of 1.6 μm separated by 800 Å deep peripheral grooves. Using a Buehler Isomet low-speed diamond saw, 1 mm wide and 20 mm long strips were cut out of the disk along the recording tracks.
Nanosurgery of biological specimens using a focused ion beam
Authors: Mackenzie John M. , Woodward Tracy H. , Griffis Dieter P. , Russell Phillip E.
Published: June 2020

While ion beams have been utilized for sample preparation in Materials Science for many years, this technology has rarely been applied to biological sample preparation. With the advent of computer controlled, finely focused ion beam technology (also widely used for modification and preparation of materials samples, many opportunities exist for nanosurgery of biological specimens at particular locations of interest e.g. many biological interactions, such as parasites/host interactions, occur as rare events at specific binding sites. In order to visualize the binding sites of viruses to their host cells with thin sectioning, cells are commonly infected with a thousand fold more viruses than would be present in the case of a normal infection. Although this technique allows visualization of the interaction site, this is clearly not a natural state and may bias the results. Utilizing a technique such as freeze etching that exposes large surfaces , one can locate rare events such as an isolated viral infection site.
Comparison of high-angle take-off and low-angle take-off EDX detector geometry of the HF-2000 FE-TEM
Authors: Sato Y. , Hashimoto T. , Ichihashi M. , Ueki Y. , Hirose K. , Kamino T.
Published: June 2020

Analytical TEMs have two variations in x-ray detector geometry, high and low angle take off. The high take off angle is advantageous for accuracy of quantitative analysis, because the x rays are less absorbed when they go through the sample. The low take off angle geometry enables better sensitivity because of larger detector solid angle.

Hitachi HF-2000 cold field emission TEM has two versions; high angle take off and low angle take off. The former allows an energy dispersive x-ray detector above the objective lens. The latter allows the detector beside the objective lens. The x-ray take off angle is 68° for the high take off angle with the specimen held at right angles to the beam, and 22° for the low angle take off. The solid angle is 0.037 sr for the high angle take off, and 0.12 sr for the low angle take off, using a 30 mm2detector.
Visualizing the intracellular membrane system of yeast cells using a laser scanning confocal microscope
Authors: McCoy Karen L. , Dillin Andrew G. , McCracken Ardythe A.
Published: June 2020

Progress has been made in developing new preparative techniques for electron microscopic visualization of the intracellular structures of yeast. In addition, development of the laser scanning confocal microscope (LSCM) has provided improved resolution for fluorescent microscopy. We asked whether the LSCM in combination with new preparative techniques could be used for comparable investigative research of the intracellular organizaton of the yeast cell.

To investigate this possibility, a BioRad MRC600 LSCM equipped with a krypton/argon laser and integrated computer imaging capabilities, was used to study various dipliod strains of the yeast Saccharomyces cerevisiae. Cells were treated with the lipophilic, cationic fluorescent dye DiOC6 (3,3’-dihexyloxacarbocyanine iodide), which has been used to visualize intracellular membrane structures, and in particular the endoplasmic reticulum of mammalian cells and living yeast cells. Since one of our interests is the intracellular localization of proteins in the yeast cell, we utilized transformed yeast cells expressing a human gene encoding a protein that inappropriately accumulates in the endoplasmic reticulum (ER).
Loss of membrane Structure and cytoplasmic volume in glutaraldehydefixed neutrophils compared to cryofixed-freeze-substituted neutrophils
Authors: Gilbert Charles S. , Parmley Richard T.
Published: June 2020

Cryofixation instantly preserves cell processes without chemical alteration and freeze-substitution (FS) preserves cell constituents. Investigation has shown that cell membranes and microfilaments appear better preserved in cryofixed-freeze-substituted (CFFS) specimens, including basophils. However, the cells have not been looked at morphometrically nor have neutrophils been looked at specifically.

Peripheral blood was collected in heparin from four male donors by venipuncture. Blood was centrifuged at 500 x g for 45 min to separate the granulocytes using a one-step density gradient approach, and then washed in RPMI 1640. Cells were split and half were resuspended in RPMI with 10% DMSO, cryofixed using the Eiko RF-2 and stored in liquid N2 until FS processing in 4% OsO4 in acetone with embedment in Spurr’s resin. The other half were resuspended and fixed in 3% glutaraldehyde in 0.1M Cacodylate, pH 7.35 for 1 hr at 4-6°C. Cells were dehydrated, embedded in Spurr’s resin, counterstained with UALC and examined using a Zeiss EM109.
Retention of vanadium compounds in cells and tissues during processing for analytical microscopy
Authors: Hatch Victoria , Stearns Rebecca C. , Katler Marshall , Godleski John J.
Published: June 2020

Previous studies from our laboratory have shown that soluble vanadium compounds accumulate in phagolysosomes of alveolar macrophages. In routinely fixed and processed material, the vanadium appears primarily as a precipitate in the phagolysosomes when examined using the Zeiss CEM902. Although macrophages respond to vanadium exposure with a massive respiratory burst within minutes of exposure, vanadium is not detected by Electron Energy Loss Spectroscopy (EELS) or Electron Spectroscopic Imaging (ESI) in these cells prepared by glutaraldehyde and OsO4liquid fixation. However, if the cells are prepared by rapid cryofixation and freeze drying, vanadium can be detected at these very early stages of exposure. Work is now in progress to define the pathways used by vanadium ions in moving from the cell membrane to the phagolysosome. The purpose of the study reported here is to directly compare the retention of radioactive vanadium,48V, in lung macrophages prepared by conventional liquid fixation protocols and by cryomethodology.
Eosinophils in the lamina propria of the rat stomach bind a biotinylated gastrin analog
Authors: Miller Caroline A. , Nichols David H. , Murphy Richard F. , David Smith D.
Published: June 2020

Gastrin is a small peptide known to function as both a stimulator of gastric acid secretion and as a growth factor. Eosinophils are white blood cells which form a part of the body's defenses against invading microorganisms. Douglas et al. have reported staining, using a biotinylated gastrin analog (biotinyl-L-Trp-L-Leu-ß-Ala), of unidentified cells in the lamina propria of the rat fundic stomach. This gastrin analog has been shown to suppress gastric acid secretion in dogs. We here identify the cells binding this analog as eosinophils. We have perfused and fixed small pieces of fundic stomach with 2% paraformaldehyde/0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 1 hour. The tissue was then embedded in 5% agar and 50 ¼m sections were cut on a vibratome. Sections were incubated for 30 min. with 1.63 x 10-6 M biotinylated gastrin analog, rinsed, and exposed to an avidin-biotin-glucose oxidase complex (ABC-GO; Vector).
Distribution of VLA-5 and VLA-4 fibronectin receptors in human monocytes demonstrated by immunofluorescence, immunocytochemistry, and autoradiography
Authors: Williams Robert , Lee Che-Hung , Quella Sara E. , Harlan David M. , Kang Yuan-Hsu
Published: June 2020

Monocyte adherence to endothelial or extracellular matrices plays an important role in triggering monocyte activation in extravascular sites of infection, chronic inflammatory disorders, and tissue damage. Migration of monocytes in the tissues involves the response to a chemoattractant and movement by a series of attachments and detachments to the extracellular matrices which are regulated by expression and distribution of specific receptors for the matrix proteins such as fibronectin (FN). The VSAs (very late antigens or beta integrins), a subfamily of the transmembrane heterodimeric integrin receptors, have been thought to play a major role in monocyte adherence to the extracellular matrices and cells. In this subfamily, VLA-5 and VLA-4 are believed to be the most essential integrins mediating monocyte adherence to FN. In the present report, we have established and compared different procedures for morphological evaluation of the expression and distribution of the FN receptors on human monocytes in order to investigate their response to endotoxin or cytokine stimulation.
Proliferation of microtubules from discrete electron-dense bodies in the ciliary dendritic portion of mechanosensory setae of copepod antennae
Authors: Weatherby Tina M. , Lenz Petra H.
Published: June 2020

Sensory setae on the appendages of most arthropods contain microtubules in their innervating dendrites. Both mechano- and chemoreceptors are of ciliary origin. Mechanoreceptive dendrites typically contain more microtubules than do chemoreceptive dendrites, perhaps reflecting a role for microtubules in stiffening the setae. The presence of microtubules may also increase the sensitivity of mechanotransduction.

The overall arrangement of calanoid copepod mechanoreceptors has been described elsewhere. In calanoid copepods the arrangement of microtubules in the distal ciliary segment of the dendrites is dramatic: as many as 3000 microtubules per dendrite are found in parallel rows (fig. 1,3a). These bundles of microtubules extend some distance into the setae themselves. The basal bodies within these dendrites are especially electron-dense and give rise to a circlet of 9 + 0 microtubules (fig. 2,3f)- Just distal to the circlet electron-dense material begins to form adjacent to the microtubules (fig. 3e), eventually forming elongate electron-dense bodies radiating outward (fig. 3d).
A freeze-fracture-etched study of the physarum polycephalum plasma membrane during early formation of the macroplasmodial stage
Authors: Taylor Randolph W. , Treadwell Henrie
Published: June 2020

The plasma membrane of the Slime Mold,Physarum polycephalum, process unique morphological distinctions at different stages of the life cycle. Investigations of the plasma membrane ofP. polycephalum, particularly, the arrangements of the intramembranous particles has provided useful information concerning possible changes occurring in higher organisms. In this report Freeze-fracture-etched techniques were used to investigate 3 hours post-fusion of the macroplasmodia stage of theP. polycephalumplasma membrane.

Microplasmodia ofPhysarum polycephalum(M3C), axenically maintained, were collected in mid-expotential growth phase by centrifugation. Aliquots of microplasmodia were spread in 3 cm circles with a wide mouth pipette onto sterile filter paper which was supported on a wire screen contained in a petri dish. The cells were starved for 2 hrs at 24°C. After starvation, the cells were feed semidefined medium supplemented with hemin and incubated at 24°C. Three hours after incubation, samples were collected randomly from the petri plates, placed in plancettes and frozen with a propane-nitrogen jet freezer.
Collagen ratios in exercise-trained and pressure-overloaded rat hearts
Authors: Burgess ML , Price RL , Abel FL , Jones GP , Buggy J , Terracio L , Borg TK
Published: June 2020

The cardiac extracellular matrix (ECM) is composed of components known to affect cardiac function. Specifically, collagens (types III and I) provide a network integral to cardiac myocyte metabolism and function. Collagens promote the recoil action following contraction and maintain vascular patency during contraction. Chronic pressure-overload adversely affects cardiac compliance and function, and ultimately leads to heart failure. Fibrosis is common in pressure-overloaded hearts, partially accounting for the ventricular “stiffness” and reduced function. Chronic exercise training, however, is identified by enhanced cardiac function. Currently, little is known about ECM cellular biology and of exercised hearts and its relationship to cardiac function (1). This study examined the morphology, biochemistry and function of chronically exercised versus pressure-overloaded rat hearts to further delineate the role of the ECM in cardiac function.

Male Sprague-Dawley rats were adapted to treadmill running and randomly divided into four groups: coarctation of abdominal aorta (Coarc) to induce pressure-overload, sham operated and sedentary controls (Cont) and exercise-trained (XT).
Immunoelectron microscope detection of C-type natriuretic peptide in normal human atrial tissue
Authors: Wei Chi-Ming , Hukee Margaret , McGregor Christopher G.A. , Burnett John C.
Published: June 2020

C-type natriuretic peptide (CNP) is a newly identified peptide that is structurally related to atrial (ANP) and brain natriuretic peptide (BNP). CNP exists as a 22-amino acid peptide and like ANP and BNP has a 17-amino acid ring formed by a disulfide bond. Unlike these two previously identified cardiac peptides, CNP lacks the COOH-terminal amino acid extension from the ring structure. ANP, BNP and CNP decrease cardiac preload, but unlike ANP and BNP, CNP is not natriuretic. While ANP and BNP have been localized to the heart, recent investigations have failed to detect CNP mRNA in the myocardium although small concentrations of CNP are detectable in the porcine myocardium. While originally localized to the brain, recent investigations have localized CNP to endothelial cells consistent with a paracrine role for CNP in the control of vascular tone. While CNP has been detected in cardiac tissue by radioimmunoassay, no studies have demonstrated CNP localization in normal human heart by immunoelectron microscopy.
Lobular and cellular distribution and content of glycogen synthase in periportal and pericentral hepatocytes of the rat liver
Authors: Giffin Bruce F. , Morris Randal E. , Drake Richard L. , Cardell Robert R.
Published: June 2020

Glycogen and the enzymes involved in hepatic carbohydrate metabolism have a heterogeneous distribution throughout the parenchyma of the liver. Although the precise lobular localization for many of these enzymes has been determined, information on the distribution and content of glycogen synthase (GS) has not been published. These studies were undertaken to determine: (1) the lobular and cellular distribution of GS in the fed rat; (2) any relationship between glycogen patterns and the distribution of GS.

Livers of fed male Spraque-Dawley rats were perfused with cold 30% sucrose and 10 um frozen sections collected on Vectabond treated slides. After fixation for 5 min in 4% paraformaldehyde at room temperature, tissue sections were incubated in polyethylene slide boxes with blocking buffer (PBS, 0.5% Triton X-100, 5% fetal calf serum, 2% bovine serum albumin, 0.95% fish gelatin, pH 7.5) overnight at 4°C. Tissue sections were incubated for 6 hours at room temperature with goat anti-rat liver GS diluted in blocking buffer (1:1000) followed by 5 nm gold conjugated rabbit anti-goat IgG overnight at 4°C and 3 hours with 5 nm gold conjugated goat anti-rabbit IgG at room temperature.
Peroxisome proliferation: Organelles or activity as an endpoint?
Authors: Taylor Catherine A. , Jarnot Bruce M.
Published: June 2020

Peroxisome induction can be expressed as an increase in peroxisome area (proliferation) or as an increase in peroxisomal fatty acid oxidation (activity). This study compares proliferation and activity as endpoints for hepatic peroxisome induction by perfluorodecanoic acid (PFDA). Fluorocarboxylic acids such as PFDA represent a class of compounds possessing commercially important surfactant properties. A single 50 mg/Kg ip. dose of PFDA produces a characteristic “wasting syndrome” in male F-344 rats. Symptoms include hypophagia, weight loss, hepatomegaly, and delayed lethality. Hepatic studies reveal changes similar to those seen with the hypolipidemic agent clofibrate. These include mitochondrial disruption, endoplasmic reticulum and peroxisome proliferation, and increased peroxisomal acyl-CoA oxidase activity.

Male Fisher-344 rats received a single ip. dose of 2, 20, or 50mg/Kg PFDA dissolved in 1:1 propylene glycol/water and were sacrificed 8 days post-dose. All control rats received an equal volume of vehicle ip. Animals were provided food and water ad libitum, except pair-fed controls which received the same restrictive food intake consumed by their weight-paired dosed partners (50mg/Kg PFDA group) to simulate the hypophagia associated with PFDA.
Intracytoplasmic lumina in ependymomas: An ultrastructural study
Authors: Ho Khang L. , Caccamo Dario V. , Garcia Julio H.
Published: June 2020

Intracytoplasmic lumina (ICL) are round to oval, single membrane bound spaces bearing microvilli that may or may not contain secretory material. The morphogenesis of ICL remains controversial and their biological significance and diagnostic implication of this structure have not been fully explored. ICL have been reported in a variety of epithelial tumors outside the central nervous system, but this structure is rarely described in tumors of the central nervous system.

We analyzed ultrastructural features of ICL in six ependymomas (1 from lateral ventricle, 3 from fourth ventricle and 2 from the spinal cord) and three myxopapillary ependymomas of the filum terminale. Two types of ICL were identified: (1) ICL with both microvilli and cilia and (2) ICL with only microvilli (Fig.1). Ciliated ICL were common in ependymomas of the ventricles whereas non-ciliated ICL were frequently seen in myxopapillary variant. Various stages of formation and development were observed in ciliated ICL (Fig. 2,3).
Ependymal morphogenesis in a simple vertebrate spinal cord
Authors: Frey Jill K. , Chen Aileen , David Heathcote R.
Published: June 2020

All cells of the spinal cord originate from the single layer of neuroepithelium that lines the central canal. Since the turn of the century, it has been known that a subclass of these ependymal cells can differentiate into neurons and extend cytoplasmic projections and cilia into the central canal. We have recently used tyrosine hydroxylase immunocytochemistry to identify a catecholaminergic subpopulation of cerebrospinal fluid (CSF) contacting ependymal neurons in the developing spinal cord of the frog Xenopus laevis (Fig. 1). The interneurons are located in the floor plate region of the spinal cord and have axons that extend rostrally toward the hindbrain. During the morphogenesis of the catecholaminergic population of cells, two longitudinal columns gradually appear and then rapidly “converge” at the ventral midline. Transverse sections of embryonic spinal cord (see Fig. 1) showed that the cell bodies decreased in size and underwent changes in shape, number and position within the spinal cord.
Effect of cyclical stretching on cultured rat cerebral vascular smooth muscle cells: A flow cytometric, confocal laser scanning, and TEM Study
Authors: Ghoneim Shams , Waldschmidt Thomas , Yoder Elizabeth , Moore Steven , Baumbach Gary
Published: June 2020

Vascular hypertrophy during chronic hypertension has been linked to a variety of potential determinants including increased intravascular pressure, neurohumoral factors and genetic factors. Of the various determinants, one might assume that arterial pressure per se would play an especially important role. Nonetheless, the role of intravascular pressure has remained unclear. Based on findings in vivo, we recently proposed that increases in pulse pressure, as opposed to mean pressure, may be an important determinant of cerebral vascular hypertrophy (3).

The goal of the present study was to further explore the possible link between pulse pressure and hypertrophy using a combination of tissue culture, fluorescent-activated cell sorting (FACS), transmission electron microscopy, and confocal laser fluorescent microscopy techniques. Cultured smooth muscle cells derived from rat cerebral microvessels were used for these experiments. Flex culture plates with rigid (control) or flexible (elastomer), collagen-coated bottoms were seeded with smooth muscle cells. After 48 hours to allow for attachment, the culture plates were placed in a Flexercell Strain Unit (model FX-2000) (1, 2), and exposed to continuous cyclical stretching at 20 k Pascals unit pressure and 60 cycles per minute for 5 days.
Properties and dose dependence of embedding media for cryo-automated electron tomography
Authors: Braunfeld M.B. , Koster A.J. , Sedat J.W. , Agard D.A.
Published: June 2020

Electron tomography is well suited to the study of complicated, non symmetric biological structures. In our laboratory, we use intermediate voltage electron microscopic tomography to follow complex paths of chromatin fibers within intact sections of Hela telophase chromosomes. In order to accurately reconstruct these features at resolutions beyond 50Å, precise imaging conditions and data collection schemes have been developed and employed.

To obtain useful high resolution information, the specimen needs to be well preserved. Data collection must also be accurate and self-consistent. However, a serious limitation has been radiation damage to the specimen during scanning, and data collection. Because of the high doses required for tomography, the standard approach has been to accept the inevitability of serious radiation damage, and to heavily pre-irradiate the sample in an attempt to provide stability and consistency during data collection.

The use of fully-automated data collection methods allows a substantial decrease in beam dose, suggesting that the entire approach should be reevaluated.
Biomineralization in loricate green algae
Authors: Dunlap John R. , Walne Patricia L.
Published: June 2020

Members of the green algal family Phacotaceae are characterized by the presence of an extracellular wall-like structure termed a lorica. A complex assemblage of mucopolysaccharides and metals, primarily calcium and iron, the lorica is considered to be a highly conserved structure. Hence, lorica morphology has served as a basic criterion for classification at both the generic and species levels. Although lorica development is thought to be genetically controlled in the phacotacean algae the extent of cellular control over development and mineralization is largely unknown. This study was initiated to gain a better understanding of lorica microarchitecture and mineralization patterns in this unusual group of organisms.

Three members of the Phacotaceae are reported on here: Pteromonas, Phacotus and Dysmorphococcus. Samples for the present study were fixed in glutaraldehyde; some were post-fixed in osmium tetroxide and all were dehydrated in alcohol and embedded in Spurr's low-viscosity resin. Thin and thick sections were examined and analyzed on an Hitachi H-800 TEM/STEM operating at 200kV.
Design and performance of instrumentation for automated single-tilt-axis electron tomography
Authors: Koster A.J. , Chen H. , Clyborne W. , Sedat J.W. , Agard D.A.
Published: June 2020

One of the driving questions in our group is into understanding how chromosomes are constructed from fibers of DNA wrapped around histones in their native form. To permit high resolution analysis of these highly complex fibers, we use intermediate voltage electron tomography. To obtain 50Å resolutions, we incorporate new approaches to overcome the resolution limiting factors determined by specimen fixation and staining techniques, data collection and 3D reconstruction algorithms.

With our present instrumentation we can automatically collect a series of projection data of large, radiation sensitive objects with only a minimum of manual operation, with high accuracy and consistency. The images are recorded directly in digital format to overcome the time consuming task of digitizing negatives. Furthermore, the system offers automated eucentricity setting, automatic tracking of image shifts, and automatic focusing during data collection. Highly reliable data collection is ensured by closely monitoring the variation in image shift, defocus, average image intensity, and exposure time throughout the tilt series.
Scanning electron microscopy studies on the morphology and release of vesicular stomatitis virus
Authors: Moninger Thomas , Rodriguez Jose
Published: June 2020

In spite of convincing evidence to the contrary, the overwhelming majority of virologists still refer to vesicular stomatitis virus (VSV) as “bullet-shaped”. The term bacilliform, which refers to a cylinder with 2 rounded ends, is used infrequently and conditionally to describe VSV.

We have used scanning electron microscopy (SEM) to study the morphology of the Indiana strain of VSV attached to, being, or having been released from 3 types of cultured cells, namely, a human melanoma line known as MEWO, human foreskin cells, and primary cultures of chick embryo cells. We have used crude virus suspensions and virus concentrated ten-fold by highspeed centrifugation and resuspension, as well as fresh virus preparations and virus suspensions that have been kept frozen at -70°C and thawed before use.

Virus suspended in medium containing 10% fetal bovine serum was used for transmission electron microscopy (TEM). The suspension were negative stained with 1% phosphotungstic acid containing 0.1% bacitracin and viewed in an Hitachi H-7000 TEM. For scanning electron microscopy, virus was adsorbed for 1.5 hours at 37°C unto confluent layers of cells grown in 35 mm plastic petri dishes.
Fractal geometry and its uses for characterizing microstructure
Authors: Russ John C.
Published: June 2020

Observers of nature at scales from microscopic to global have long recognized that few structures are actually described by Euclidean geometry. Mountains are not cones, clouds are not ellipsoids, and surfaces are not planes. Classical geometry allows dimensions of 0 (point), 1 (line), 2 (surface), and 3 (volume). The advent of a new geometry that allows for fractional dimensions between these integer topological values has stirred much interest because it seems to provide a tool for describing many natural objects. As is the case for many new tools, this fractal geometry is subject to some overuse and abuse.

A classic illustration of fractal dimension concerns the length of a boundary line, such as the coast of Britain. Measuring maps with different scales, or striding along the coastline with various measuring rods, produces a result that depends on the resolution. More than this is required for the coastline to be fractal, however: It must also be self-similar.
Quantitative measure of mixture homogeneity of an alumina silicon carbide composite
Authors: Kelly James F. , Fuller Edwin R.
Published: June 2020

Homogeneity, defined in terms of spatial distribution uniformity of one phase or component within another, can be determined from the variation of the standard deviation of the measured volume fraction of one component with sampling area. We have applied this concept to the evaluation of dispersions of Sic whiskers in an alumina matrix by using energy dispersive x-ray spectroscopy (EDS) . The quantitative analysis measures the area to area variation in SiC fraction. The homogeneity distribution was obtained by calculating the variance in this fraction as a function of magnification (sampling area) in a scanning electron microscope. These data are compared to calculations of expected values based on a Poisson distribution model for the dispersion of SiC.

Compositions were prepared from two different SiC size distributions. The blending procedures and volume fractions (0.30) were the same for each composition. Six polished specimens were analyzed from each of two 100mm diameter hot-pressed alumina - SiC billets.
Cryopreservation of biological specimens
Authors: Joseph Costello M.
Published: June 2020

Rapid cooling is the first, critical step in the preservation of biological specimens for examination using a wide variety of electron microscopic techniques. The choice of which cooling method to use depends on many factors, such as the restrictions of specimen geometry, the type of structural information desired and the microscopic techniques to be employed. This summary will highlight some recent technical innovations and novel applications of cryopreservation of biological specimens to study ultrastructure, chemical composition, and dynamic processes.

For cryofixation to give realistic preservation of structural details, the specimen must be rapidly brought into intimate contact with the coolant and the contact must be maintained throughout the critical stages of the cooling process. During the last decade, four cooling methods have become prominent because they produce excellent preservation without cryoprotectants, are commercially available and are relatively simple to use:plunge cooling, jet cooling, slam cooling, and high-pressure cooling.

Plunge cooling, the most commonly used method for cryofixation, is simply the immersion of a specimen into a liquid coolant usually held near its melting point.
Atomic-force microscopy: Exotic invention or practical tool?
Authors: Chernoff Donald A.
Published: June 2020

The Scanning Tunneling and Atomic Force Microscopes are well-known due to their extraordinary capability of imaging atoms using a simple mechanism. However, atomic resolution is usually not needed to solve most problems in development and manufacturing. So, many scientists and engineers (mistakenly) regard these new microscopes as more “exotic” than practical.

Both the AFM and the STM make 3-dimensional images of solid surfaces, but the AFM has much broader applications. The reason for this is that the AFM uses a universal sensing mechanism (repulsive and attractive mechanical forces), whereas the STM uses an electrical signal, which requires that the surface be at least somewhat conductive.
Using the extraordinary height sensitivity and wide scan capability of the AFM, we easily answer simple (but important) questions about surfaces and surface features, including:

-Is a contaminant present?

-Is a feature a pit or a peak?

-How tall is it?

-What is the grain size of a coating?

-How rough is the surface?
Scanned-probe microscope roughness spectroscopy
Authors: Fraundorf P. , Armbruster B.
Published: June 2020

Air based scanning tunneling and scanning force microscopes are capable of producing images very easily, but oft-times that is all that they produce. Since microscopic regions of specimens of course differ in detail from one to another, often judgements about how two regions differ fundamentally are left to qualitative intuition. Given a quarter of a million numbers, in the case of a typical large SPM image, we should be able to quantitatively compare the statistical properties evidenced therein from one specimen to another. Such quantitative insight may also yield information on artifacts (like timedomain noise and feedback-loop effects) in an image. To accomplish this most intuitively, we recommend simply decomposing the rms height variations in the specimen into two and one dimensional (azimuthally averaged) logarithmic (per decade frequency) roughness spectra. The basic properties of such a decomposition of topographic images are discussed in a paper submitted separately to this conference. We here discuss scanned probe microscope results.
Lateral displacement microscopy of holograms using scanned tip image pairs
Authors: Shen C. , Fraundorf P.
Published: June 2020

Tip displacements associated with lateral forces between specimen and tip can be quite large (e.g. microns in size) during repulsive force microscopy (SFM or AFM) and air-based scanning tunneling microscopy (STM) of soft samples. Both x and y components of the lateral tip displacement due specifically to drag between the specimen and tip, as separated from tfiose components of the tip's displacement due solely to the geometry of mechanical equilibrium between the tip and a tilted surface, may be inferred from STM or SFM image pairs of the same region taken with the tip traveling in different directions (e.g. with the tip fast scan going left versus up). We refer to this process of inferring displacement from image pairs as displacement mapping. By comparison, absolute measurements of lateral force like those obtained by imaging cantilever twists in Digital Instrument's commercial lateral force microscope: (a) don't remove equilibrium (purely geometric) components of the lateral displacement from the often more interesting friction components, (b) measure the lateral force in only one of the two transverse directions, and (c) require special hardware, not available on conventional STM's for example, to measure transverse deflection.
Software for the reconstruction of off-axis electron holograms
Authors: Joy David C , Bunn Rodney D
Published: June 2020

Holography is a two step imaging process. After a hologram has been recorded it must then be reconstructed in order to obtain the stored image information. Gabor originally envisaged the use of optical reconstruction methods, but these are difficult to set up and limited in their flexibility. The alternative is to use digital techniques to reconstruct and manipulate the hologram and its reconstructions. This paper describes a program designed for the reconstruction of off-axis electron holograms.

An off-axis electron hologram is an interferogram between a reference electron wave and the signal wave which has passed through the specimen. The hologram consists of a pattern of fringes modulated in position and amplitude as a result of phase shifts in, and scattering of, the signal wave. To recover phase and amplitude images from the hologram it is necessary to perform three operational steps. Firstly, the digitized hologram is Fourier transformed producing an array of complex numbers, one for each pixel in the original hologram.
Determination of the defocus value of micrographs of ice-embedded specimen without carbon support film
Authors: Hong Zhou Z.
Published: June 2020

It is well recognized that the contrast transfer function (CTF) of an electron microscope modulates the image contrast The effects of this CTF are to reverse the sign of the phases and to alter the amplitudes at different spatial frequencies. These changes are dependent on the defocus of the objective lens in a given microscope setting. Therefore, it is necessary to determine the defocus experimentally in order to correct the phase reversal and the amplitudes due to the CTF for attaining a high resolution reconstruction. The most straightforward way of determining the defocus value is to determine the positions of the Thon rings in the CTF by optical or computed transforms. In a crystalline specimen, the defocus value of an image can be refined against the electron diffraction amplitude. For specimen of which the x-ray structure is known, one can also use the x-ray structure factor to determine the CTF parameters.
Atomic resolution characterisation of interface structures by Electron Energy Loss Spectroscopy
Authors: Browning N. D. , McGibbon M. M. , Chisholm M. F. , Pennycook S. J.
Published: June 2020

The recent development of the Z-contrast imaging technique for the VG HB501 UX dedicated STEM, has added a high-resolution imaging facility to a microscope used mainly for microanalysis. This imaging technique not only provides a high-resolution reference image, but as it can be performed simultaneously with electron energy loss spectroscopy (EELS), can be used to position the electron probe at the atomic scale. The spatial resolution of both the image and the energy loss spectrum can be identical, and in principle limited only by the 2.2 Å probe size of the microscope. There now exists, therefore, the possibility to perform chemical analysis of materials on the scale of single atomic columns or planes.

In order to achieve atomic resolution energy loss spectroscopy, the range over which a fast electron can cause a particular excitation event, must be less than the interatomic spacing. This range is described classically by the impact parameter, b, which ranges from ~10 Å for the low loss region of the spectrum to <1Å for the core losses.
Acquisition of EELS spectra for high-resolution spectroscopy
Authors: Qian Maoxu , Sarikaya Mehmet , Stern Edward A.
Published: June 2020

It is difficult, in general, to perform quantitative EELS to determine, for example, relative or absolute compositions of elements with relatively high atomic numbers (using, e.g., K edge energies from 500 eV to 2000 eV), to study ELNES (energy loss near edge structure) signal using the white lines to determine oxidation states, and to analyze EXELFS (extended energy loss fine structure) to study short range ordering. In all these cases, it is essential to have high signal-to-noise (S/N) ratio (low systematical error) with high overall counts, and sufficient energy resolution (∽ 1 eV), requirements which are, in general, difficult to attain. The reason is mainly due to three important inherent limitations in spectrum acquisition with EELS in the TEM. These are (i) large intrinsic background in EELS spectra, (ii) channel-to-channel gain variation (CCGV) in the parallel detection system, and (iii) difficulties in obtaining statistically high total counts (∽106) per channel (CH). Except the high background in the EELS spectrum, the last two limitations may be circumvented, and the S/N ratio may be attained by the improvement in the on-line acquisition procedures. This short report addresses such procedures.
Hole count and secondary excitation in several analytical electron microscopes
Authors: Kenik E.A. , Allard L.F. , Bentley J.
Published: June 2020

Hole count and secondary excitation are two concerns the analyst must address for energy dispersive X-ray spectrometry (EDXS) in the analytical electron microscope (AEM). Identification and minimization of the uncollimated radiation responsible for the hole count should be as important to the AEM manufacturer as it is to the microscopist, especially as "good" hole count performance can be a sales advantage. Secondary excitation (which we previously referred to as secondary fluorescence) arises from excitation of the specimen by electrons or high energy x-rays generated by the interaction of the primary electron beam with the specimen. A test specimen (modified chromium thin film/molybdenum washer) described previously, has been used to evaluate levels of both hole count and secondary excitation in a number of AEMs.

Hole count spectra from the test specimen have been measured for several Philips AEMs and have been compared to those produced by direct excitation of a thick region of the molybdenum support by the high energy electrons at reduced beam current.
Real-space difference spectroscopy at interfaces
Authors: Bruley J. , Müllejans H.
Published: June 2020

Parallel acquisition of spatially resolved electron energy-loss spectra (SREELS) enables the characterization of interfaces on a nanometer scale using the fairly weak but chemically specific coreabsorption edges. A major hurdle for the routine qualitative and quantitative analysis is the very low signal-to-background ratio (and consequently low signal-to-noise ratio, SNR) of the absorption edge of a dilute element. In situations where the background is only slowly varying with energy, the weak edge may be exposed from the intense background either by empirically fitting a power-law curve prior to the edge and extrapolating, or by acquiring the spectrum in the 1st or 2nd energy difference modes (In the limit of small energy shifts such difference spectra are equivalent to derivatives with respect to energy loss). However, these techniques are unsuitable when: the background is strongly energy dependant, such as when the weak edge overlaps severely with the ELNES or EXELFS of a preceding edge; the region of interest is radiation sensitive; or there is specimen drift.
Characterization of ultrafine copper oxide aerosols for animal exposure studies
Authors: Godleski John J. , Krishna Murthy G.G. , Skornick William , Hatch Victoria , Stearns Rebecca , Katler Marshall
Published: June 2020

People are exposed to complex mixtures of air pollutants in the urban environment. The inhalation of acid-coated metal oxide fine particles is a well-recognized health effects problem, but the mechanisms of toxicity remain undefined. Ultrafine particles (<100 nm in diameter) are difficult to detect by conventional air sampling methodology. In order to characterize such particles, electron microscopy techniques are needed. In this abstract, we outline techniques to produce, characterize, and expose animals to ultrafine (1-30 nm) copper oxide aerosols.

The ultrafine copper oxide aerosols were generated using a high temperature tube furnace with molybdenum windings (0.93 m long), capable of operating at 1923° K. The temperature at the center of the furnace was maintained at 1709° K during the experiments. Copper metal shot of 99.99+ purity (Aldrich Chemical Co. Inc.) and 2-8 mm in size was used as the copper source. An alumina boat (30 x 8 x 3 mm) containing 2 g copper was placed at 0.187 m from the center of the furnace where the measured temperature was 1683° K. The copper vapors from the boat were carried by a pure argon stream and were oxidized by added oxygen.