bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of low temperature specimen stage and its applications to electron holographic measurement
Authors: Endo J. , Osakabe N. , Matsuda T. , Yano S. , Yamada H. , Kawasaki T. , Tonomura A.
Published: June 2020

Using electron holography, information about a specimen, such as distributions of magnetization and thickness, can be revealed by investigating the shape of reconstructed wavefronts of the laser beam. These distinctive features can be utilized in investigations in the fields of superconductivity and biology. Therefore, we have developed a low temperature specimen stage which is reguired for superconducting specimens and to decrease the radiation damage in biological specimens. Using this cold stage, we have observed magnetic flux quantization in superconductors.
A CCD imaging and analysis system for the VG HB5 STEM
Authors: Mochel M. E. , J. M. Mochel
Published: June 2020

A liquid nitrogen cooled charge coupled device (CCD) camera with DEC 11-73 computer has been fitted to the Vacuum Generators HB5 scanning transmission electron microscope (STEM). Typically, diffraction patterns are produced in the VG HB5 STFM by rocking the beam on the sample or alternatively by rastering the electrons that emerge from the sample and displaying the resultant scanned image on the CRT. The image is often noisy because the beam current is small (≈1010 a) and the range of interesting intensities is large.
Linewidth measurement using the low voltage SEM
Authors: Rosenfield M.G.
Published: June 2020

Minimum feature sizes in experimental integrated circuits are approaching 0.5 μm and below. During the fabrication process it is usually necessary to be able to non-destructively measure the critical dimensions in resist and after the various process steps. This can be accomplished using the low voltage SEM. Submicron linewidth measurement is typically done by manually measuring the SEM micrographs. Since it is desirable to make as many measurements as possible in the shortest period of time, it is important that this technique be automated.

Linewidth measurement using the scanning electron microscope is not well understood. The basic intent is to measure the size of a structure from the secondary electron signal generated by that structure. Thus, it is important to understand how the actual dimension of the line being measured relates to the secondary electron signal. Since different features generate different signals, the same method of relating linewidth to signal cannot be used. For example, the peak to peak method may be used to accurately measure the linewidth of an isolated resist line; but, a threshold technique may be required for an isolated space in resist.
Metal coating thickness and image quality in scanning electron microscopy
Authors: Peters K.-R.
Published: June 2020

In biological scanning electron microscopy (SEM), metals are deposited on specimen surfaces for different applications. In conventional microscopy, 10 - 20 nm thick coatings of gold are used to increase electrical conductivity and secondary electron (SE) emission. The - conventional - SE signal (SE-II) is generated by electrons of the probe scattered in the thick gold layer; useful magnification is limited to ∼25,000 x. At higher magnifications the metal coating is imaged by more than one pixel and contributes significantly to the size of particles. Topographic contrasts are produced at features ∼20 nm. High resolution is obtained through another type of signal which is generated by the electrons of the probe before they scatter within the specimen.
Fractographic study of neutron irradiated pressure vessel steel
Authors: Venables D. , Ebrahimi F. , Hren J. J.
Published: June 2020

The presence of phosphorous as a residual element has been correlated with moderate to severe irradiation embrittlement of nuclear reactor pressure vessel steels (RPV's). The degree of radiation embrittlement is usually characterized by a shift in ductile-to-brittle transition temperature and a decrease in upper shelf energy, as obtained by Charpy V-notch impact testing. The mechanism by which phosphorous enhances the radiation sensitivity is unclear, however several possible mechanisms can be proposed. These include 1) radiation induced segregation to interfaces such as grain boundaries and particle/matrix interfaces 2) modification of possible radiation induced changes in the carbide microstructure; 3) radiation induced precipitation of phosphorous and 4) stabilization of defects produced by irradiation.
Characterization of ceramic fibers in the SEM and TEM
Authors: Sawyer L.C. , Chen R.T.
Published: June 2020

Ceramic fiber composites are currently being developed for potential high temperature structural applications. Fibers for these applications are being produced from polymer precursors, such as Nicalon™ silicon carbide (SiC) fibers, commercially produced from polycarbosilane (Nippon Carbon Co.). Experimental ceramic fibers are also being produced as part of a DARPA funded government program by Dow Corning and Celanese. The physical properties of glass and ceramic fibers are well known to be controlled by microstructural features such as crystallinity and crystallite sizes, the nature of amorphous phases, porosity and internal and surface defects or flaws. Defects are known to act as stress concentrators, and the well known Griffith criteria relates their sizes to the tensile stress.
Beam-radiation effects on wool in the ESEM
Authors: Danilatos G.D.
Published: June 2020

The advent of the environmental SEM (ESEM) has made possible the examination of uncoated and untreated specimen surfaces in the presence of a gaseous or liquid environment. However, the question arises as to what degree the examined surface remains unaffected by the action of the electron beam. It is reasonable to assume that the beam invariably affects all specimens but the type and degree of effect may be totally unimportant for one class of applications and totally unacceptable for another; yet, for a third class, it is imperative to know how our observations are modified by the presence of the beam. The aim of this report is to create an awareness of the need to initiate research work in various fields in order to determine the guiding rules of the limitations (or even advantages) due to irradiation.
Zone-axis ALCHEMI for the rapid assessment of site occupancies in garnets
Authors: Otten M.T. , Buseck P.R.
Published: June 2020

ALCHEMI (Atom Location by CHannelling-Enhanced Microanalysis) is a TEM technique for determining site occupancies in single crystals. The method uses the channelling of incident electrons along specific crystallographic planes. This channelling results in enhanced x-ray emission from the atoms on those planes, thereby providing the required site-occupancy information. ALCHEMI has been applied with success to spinel, olivine and feldspar. For the garnets, which form a large group of important minerals and synthetic compounds, the channelling effect is weaker, and significant results are more difficult to obtain. It was found, however, that the channelling effect is pronounced for low-index zone-axis orientations, yielding a method for assessing site occupancies that is rapid and easy to perform.
Vitrification of Y zeolite and its effects on HREM images
Authors: Csencsits R.
Published: June 2020

High resolution electron microscopy (HREM) is a valuable technique for studying catalytic zeolite systems because it gives direct information about the structure and defects present in the structure. The difficulty with doing an HREM study on zeolites is that they become amorphous under electron irradiation. This work is a systematic investigation of the damage of Y zeolites in the transmission electron microscope (TEM); the goals of this study are to determine the mechanism for electron damage and to access the effects of damage in Y zeolites on their HREM images using computer simulation.
Computer-simulated images of platinum clusters in Y zeolite
Authors: Csencsits R. , O'Keefe M. A. , Chan I. Y. , Gronsky R.
Published: June 2020

High resolution electron microscopy is a valuable technique for studying catalytic metals and their zeolite supports because it allows direct visualization of their microstructure. The question is: what iis the smallest metal particle that can be detected by electron microscopy in such a system? Our goal is to determine the conditions under which a small (13 atoms) platinum cluster inside a Y zeolite channel can be detected by HREM. We chose the Pt/Y system because modified Y zeolites are widely used in commercial catalyst systems and Pt is a common and important catalytic metal.
Particulate carbon at the cretaceous-tertiary boundary
Authors: Mu Chongjun , Wolbach Wendy S.
Published: June 2020

A worldwide Ir layer occurs at the Cretaceous-Tertiary (K-T) boundary, apparently from the impact of a 10 km meteorite that caused the mass extinctions at that time. The same boundary layer also is enriched in carbon showing the characteristic morphology of soot [grape-like, "aciniform" clusters of 0.03 μm spheroids]. Presumably this carbon came from major fires of forests or fossil carbon that were ignited by the impact.

Extending the earlier SEM study, we have examined several samples by TEM. All samples were isolated by dissolving inorganic minerals with HCI and HF and removing extractable organics and most of the kerogen with organic solvents and NaOH. Fig. 1 shows a sample from the K-T boundary at Woodside Creek, New Zealand. The aciniform carbon is still covered with films of kerogen. Further extractions eventually yield the material in Fig. 2, which is free of kerogen, and shows the delicate, welded structure of soot
Crystallography of self-implanted and laser pulse-annealed silicon
Authors: McClure D.L. , Carpenter R.W. , Barry J.C. , Lowndes D.H.
Published: June 2020

Results of a HREM investigation of the structure of the amorphous silicon (aSi) surface layers and the subsequent polycrystalline microstructure after laser pulse annealing are presented here. Amorphous silicon melts at about 200K less than the crystalline phase, resulting in undercooled liquid silicon as the driving force for crystallization.

Surface layers of aSi ∽400nm thick were prepared by self ion implantation on <100> substrates at 77K. Annealing was done with an excimer laser (KrF, 248nm) of 35nsec pulse duration at 0.4, 0.7, 0.8 J/cm2 energy densities. Melt depth penetration into the aSi layer is proportional to laser pulse energy magnitude. Cross section HREM was done in <110>, perpendicular to the implant direction with a JEOL 4000EX, 200CX, Philips EM400 FEG, and EM400T.
Study of the interfacial reaction zone in a glass-ceramic to metal seal by Analytical Electron Microscopy
Authors: Headley T. J. , Loehman R. E. , Watkins R. D. , Madden M. C.
Published: June 2020

Glass-ceramic to metal seals are commonly used in a variety of electronic components. A complex Li2O-SiO2 glass-ceramic has been developed that forms thermal expansion-matched seals with several Ni-base superalloys allowing its use in high-strength connectors, feed-throughs and headers. Typically, an interfacial reaction zone is created by diffusion of metallic species into the glass during the high-temperature sealing step. The microstructure in this zone is altered from that in the bulk glass-ceramic. This AEM study examined the product phases in the reaction zone of seals between this glass and the Ni-alloy Inconel 718 (1718). Such studies are helpful in identifying the reaction sequence at the interface. The glass was sealed to thin strips of 1718 for 20 min at 1000˚C, followed by a lower temperature cycle that crystallized the glass-ceramic. The alloy was etched away and thin samples of the reaction zone were prepared both in crosssection and by back-thinning (ion milling) from the glass-ceramic side.
Micrograph image processing system
Authors: Mittleman R. K. , Parker N. W.
Published: June 2020

We have developed a computer-driven image processing system for the analysis of STEM micrographs. Modern computer and data storage devices give the researcher the ability to record and store large numbers of images. Our system was designed to allow rapid viewing and to facilitate the measurement process. Making use of an IBM 4381-2 mainframe computer and a high resolution (1024 x 1280 pixels) Metheus Omega 500 display system, we have written a flexible, interactive system to display and process data from our electron microscopes. The system is menu-driven from a mouse and can call subroutines from fortran, pascal or APL.

APL is an interpreted, interactive language that is particularly well suited to real time image processing. Algebraic manipulations can be performed with equal ease on arrays of any rank and the user can affect many complex operations on dataarrays, usually with a single line of code (e.g., gray scale expansion, zoom, inner and outer product, etc.).
Electron microscope of the 21st century
Authors: Crewe A.V.
Published: June 2020

I have been asked to talk about the future of the electron microscope by the Chairman and cannot refuse because he was one of my students. On the other hand, I do not feel comfortable with the position because there is a very poor history in science on the matter of making predictions; they are always wrong.

After giving it some thought, it would seem that the best thing that I can do is to discuss some of the problems that I can see with the instruments and suggest that the solutions to these problems will slide into the next century. That puts me on safe ground and perhaps may allow me to suggest some solutions myself.

To start with, one can say that the business shows every sign of being, or becoming, a mature industry. The instruments become more and more expensive every year. They become generally larger, and there is more attention given to appearances. This is very similar to the automobile industry and is at gross variance with the computer industry where the functionality increases and the cost decreases every year. In the present climate of financial support (or the lack of it), this means that fewer instruments are sold every year, and this increases the price of those that are sold.
Altered renal proximal tubule cell ion homeostasis during diabetes
Authors: Freudenrich Craig C. , Hockett Daniel , Kirk Benjamin , Gilliland Kurt , Scherer Brian , Winter Kerri , Ingram Peter , LeFurgey Ann
Published: June 2020

A major characteristic of human and experimental diabetes mellitus is enhanced urinary excretion of electrolytes (e.g. Na, K, Mg, Ca). The increased urinary electrolyte excretion can lead to low serum electrolyte concentrations (e.g. hypomagnesemia, hyponatremia, hypocalcemia, hypokalemia), although this issue remains controversial. Ultimately, elevated urinary electrolyte excretion can result in mineral deficiencies which have important consequences such as retinopathies, convulsions, osteoporosis, arrhythmias, and sudden death ischemia. The increased urinary electrolyte excretion could be due to osmotic diuresis and/or altered renal tubule cell ion homeostasis; however, few studies have addressed whether diabetes alters specific tubule cell ion homeostasis. In this study, we used electron probe x-ray microanalysis (EPXMA) to obtain quantitative microchemical images of proximal tubule cells from diabetic rats so that we could define how subcellular ion homeostasis is altered during diabetes.

Adult male Sprague-Dawley rats were housed in individual metabolic cages and urine output was measured daily; peri-orbital plasma samples were collected every 3 days. Urine and plasma ion concentrations (Na, K, Ca, Mg) were assayed by atomic absorption spectroscopy, blood glucose was measured with a commercial glucometer, and urine glucose and ketones were detected with commercial strips.
Characterization of a human endothelial cell line from a hepatic angiosarcoma
Authors: Hoffpauir John M. , Hoover Marie L. , Větvička Václav , Tamburro Carlo H.
Published: June 2020

Angiosarcoma of the liver is extremely rare, but there is an increased incidence in Louisville, Kentucky due to worker exposure to gaseous vinyl chloride. The tumor is endothelial in origin as it possess factor VIII, a coagulation factor and known endothelial cell marker. The malignant cells have been described as pleomorphic malignant endothelial cells, malignant spherical and spindle cells or enlarged spindle-shaped endothelial cells. The lack of a cell line derived from a hepatic angiosarcoma has significantly limited studies of this rare lethal malignancy. This study describes the characterization of a human endothelial cell line from a liver biopsy of a hepatic angiosarcoma patient who had been previously exposed to gaseous vinyl chloride. This cell line denoted, HAEND, does not require growth factors such as endothelial cell growth factor for cultivation. Also, this line can be cultivated on untreated plastic surfaces and does not require gelatin or fibronectin coated surfaces.
Role of the exosporial membrane in attachment and germination of C. sporogenes
Authors: Panessa-Warren B.J. , Tortora G.T. , Warren J.B.
Published: June 2020

Some bacteria are capable of forming highly resistant spores when environmental conditions are not adequate for growth. Depending on the genus and species of the bacterium, these endospores are resistant in varying degrees to heat, cold, pressure, enzymatic degradation, ionizing radiation, chemical sterilants,physical trauma and organic solvents. The genus Clostridium, responsible for botulism poisoning, tetanus, gas gangrene and diarrhea in man, produces endospores which are highly resistant. Although some sporocides can kill Clostridial spores, the spores require extended contact with a sporocidal agent to achieve spore death. In most clinical situations, this extended period of treatment is not possible nor practical. This investigation examines Clostridium sporogenes endospores by light, transmission and scanning electron microscopy under various dormant and growth conditions, cataloging each stage in the germination and outgrowth process, and analyzing the role played by the exosporial membrane in the attachment and germination of the spore.
Microtubule motors associated with kinetochores in mitotic cells
Authors: Wordeman Linda
Published: June 2020

Chromosomes in dividing tissue culture cells exhibit three types of movement along mitotic spindle microtubules: l)Fast minus-end directed movement (prometaphase), 2)Plus-end directed movement, and 3) Slow minus-end directed movement (anaphase) . In all cases these movements are mediated by the kinetochore region of the centromere of mitotic chromosomes. This region consists of three domains based on both immunocytochemistry and electron microscopy. The outermost or kinetochore domain is composed of the distal fibrous corona and trilamminar plate. The central and pairing domains are located in the chromatin beneath the kinetochore. Both plus- and minus-end directed microtubule motors have been localized to the kinetochore region of mitotic CHO chromosomes. I have used double-label immunocytochemistry to map the location of these motors within the centromere region at the level of the light microscope. Furthermore, I have cloned and expressed a number of novel kinesin-related motors, two of which (Clone 26 and Clone 14) are localized to kinetochores and kinetochore microtubules, respectively.
Plant anatomy, histochemistry, and gene expression as visualized by tissue printing
Authors: Reid Philip D. , Bickar David
Published: June 2020

Tissue printing on nitrocellulose membranes followed by localization with appropriate probes has been used to study the distribution of protein and mRNA as well as the timing of gene expression in a variety of plant tissues. Included in these studies are cell wall proteins in soybean and cellulase activity in relation to abscission in kidney bean. Our recent studies have focused on the distribution of enzymes involved in crassulacean acid metabolism in the leaves of various species of Peperomia, the localization of ubiquitinated protein in various plant tissues, and the development of a stain for total protein transferred to nitrocellulose by tissue printing.

Tissue prints made on dry nitrocellulose membranes (0.45μm pore size) and viewed with side illumination at low (10X) magnification show the characteristic morphology and anatomical organization of the printed tissue (Figs. 1 and 2). Treatment of the membranes with 12 mM HC1 followed by a two minute incubation in an acidic 0.05% solution of copper phthalocyanine 3,4',4'',4'''-tetrasulfonic acid tetrasodium salt (CPTS) (Figs. 3 and 4) shows the distribution of total protein transferred to the membrane.
Spray-freezing apparatus for cryofixation of unicellular algae
Authors: Fields Stephen D. , Strout Gregory W. , Russell Scott D.
Published: June 2020

Spray freezing unicellular organisms directly from the culture medium is an attractive alternative to other methods of rapid freezing because the possibility of pre-freezing artifacts is minimized. However, the utility of spray-freezing has been limited due to difficulties in obtaining satisfactory freezing in large specimens and in collecting and processing specimens after freezing. Consequently, the design of many spray-freezing devices is somewhat complicated, of limited application and often difficult to use. A simple, inexpensive spray-freezing apparatus of our own design was developed with the purpose of rapid freezing unicellular algal suspensions for freeze substitution. A naked, freshwater dinoflagellate was used to determine the effectiveness of the freezing device and the associated freeze substitution protocol.
Two-photon excitation fluorescence microscopy in living systems
Authors: Piston David W.
Published: June 2020

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.
Imaging in 5 dimensions: Quantitative measurement of membrane potential from single mitochondria in living cells
Authors: Loew Leslie M. , Tuft Richard A. , Carrington Walter , Fay Fredric S.
Published: June 2020

The energy released during the oxidation reactions in the mitochondrial respiratory chain is stored as an electrochemical potential consisting of a transmembrane electrical potential (Vmit) of about −180 mV and a proton gradient of about 1 pH unit; this drives the synthesis of the ATP required to fuel the cell’s energy-dependent processes. This key cellular system has been the subject of thousands of studies. However, data on how it is regulated by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have been precluded by their small size, their high motility, and the absence of appropriate methodologies.

An approach toward a more quantitative assessment of Vmit within intact cells was based on the development of the potentiometric fluorescent dyes TMRE and TMRM, which rapidly equilibrate across membranes in accord with the Nernst equation.
Three-dimensional reconstruction of the golgi complex and trans-golgi network: A HVEM tomographic study
Authors: Ladinsky Mark S. , Kremer James R. , Furcinitti Paul S. , Richard McIntosh J. , Howell Kathryn E.
Published: June 2020

The Golgi complex is the central organelle in the secretory pathway of eukaryotic cells. It is composed of multiple cisternae that contain various enzymes involved in posttranslational modification of newly synthesized proteins. The cisternae are connected to a reticular lattice, the trans-Golgi Network (TGN), which is responsible for sorting and targeting proteins and lipids to other cellular locations. Transport between the Golgi cisternae is thought to occur largely via carrier vesicles, but recent studies suggest that some transport may be via tubules which link the cisternae and TGN. Although the Golgi complex has been well characterized biochemically, only a few studies have addressed its 3-D fine structure.

Specific staining, high voltage electron microscopy (HVEM), and computer axial tomography were used to visualize the structure of the Golgi cisternae and the TGN in 3-D. A technique developed by R. E. Pagano was used to facilitate the visualization of the TGN and trans-most cisternae: Normal rat kidney (NRK) cells are treated with a fluorescent analog of ceramide (BODIPY-cer) which is internalized and metabolized in the Golgi complex (Fig. 1a).
The organization of the microtubule associated protein tau in Alzheimer paired helical filaments
Authors: Ruben George C. , Iqbal Khalid , Grundke-Iqbal Inge , Johnson John E.
Published: June 2020

Neurofibrillary tangles (NFT) of paired helical filaments (PHF) are the most characteristic brain lesions of Alzheimer disease and their abundance determines the diagnosis. This histopathological hallmark occurs with greater frequency in patients with clinical dementia of the Alzheimer type than in normal individuals of the same age. Therefore, it is important that we establish and understand the arrangement of subunits in PHF. This knowledge, we believe, could be key to understanding the pathogenesis of neurofibrillary degeneration.

Intracellular Alzheimer NFT fixed in OsO4or KMnO4in the absence of glutaraldehyde treatment were first observed by transmission electron microscopy (TEM) of thin sections and reported by Kidd. The tangles he observed contained helical pairs of ~10 nm filaments separated center to center by 15 nm with a double helical period of ~160 nm that he named paired helical filaments. Subsequent thin sectioning work with a similar fixation treatment confirmed that the PHF were helical with crossover width, T=12.8±2 nm, and a wide region, W=24±3.1 nm, which occured every L=65−80 nm.
Correlative atomic-force microscopy and high-resolution scanning electron microscopy of proteins attached to platelet surfaces
Authors: Simmons S.R. , Eppell S.J. , Marchant R.E. , Albrecht R.M.
Published: June 2020

The atomic force microscope (AFM) has provided images at submolecular or atomic scale resolution of biological macromolecules attached to surfaces such as mica, graphite, or synthetic phospholipid membranes. Because the AFM can be operated with the sample in air, vacuum, or immersed in a liquid such as a biological buffer, it has the potential for high resolution imaging of the structure and organization of macromolecules on surfaces of cells in the hydrated or even living state. Realization of this potential would allow observation of molecular processes at the cell surface without the necessity for preparation of the sample for electron microscopy. To date, however, the AFM has yielded images of cell surfaces only at relatively low magnifications, and has not provided the atomic resolution achieved on hard, crystalline surfaces.

Previously we have utilized correlative video-enhanced light microscopy, high voltage transmission electron microscopy, and low voltage, high resolution scanning electron microscopy (HRSEM)
Development of high-performance objective lens polepiece for ultrahigh resolution Analytical Electron Microscope
Authors: Fukushima K. , Kaneyama T. , Hosokawa F. , Tsuno H. , Honda T. , Kersker M.
Published: June 2020

Recently, in the materials science field, the ultrahigh resolution analytical electron microscope (UHRAEM) has become a very important instrument to study extremely fine areas of the specimen. The requirements related to the performance of the UHRAEM are becoming gradually severer. Some basic characteristic features required of an objective lens are as follows, and the practical performance of the UHRAEM should be judged by totally evaluating them.

1) Ultrahigh resolution to resolve ultrafine structure by atomic-level observation.

2) Nanometer probe analysis to analyse the constituent elements in nm-areas of the specimen.

3) Better performance of x-ray detection for EDS analysis, that is, higher take-off angle and larger detection solid angle.

4) Higher specimen tilting angle to adjust the specimen orientation.

To attain these requirements simultaneously, the objective lens polepiece must have smaller spherical and chromatic aberration coefficients and must keep enough open space around the specimen holder in it.
Discrimination of the assembly states of cytoskeletal proteins in cultured cells using confocal microscopy
Authors: Carpenter David A. , Khan Sohaib A. , Ip Wallace
Published: June 2020

The cytoskeleton is a three-dimensional network of cytoplasmic filaments that mediates many processes involving motility, and the specification and maintenance of cell form. In recent years, it has become evident that all three major components of the cytoskeleton- microfilaments, microtubules, and intermediate filaments (IF)-are dynamic structures that undergo reversible assembly-disassembly as required by the physiologic needs of the cell. While theassembledform of the cytoskeleton-the filamentous network-is readily visible by conventional immunofluorescence microscopy, it is often difficult to visualize the nonfilamentous form of a cytoskeletal protein because the subunits or oligomeric assemblies are small and because the images tend to be diffused due to interference from fluorescently labelled subunits above and below the plane of focus. Confocal microscopy offers a convenient solution to this problem at the light microscope level, because optical sections of fluorescently immunolabelled cytoskeletal networks do not suffer from such out-of-focus interference.
Demonstration of GAD in purkinje-cell terminals with silver enhanced gold immunocytochemistry
Authors: Gilerovitch Helen G. , Bishop Georgia A. , King James S. , Burry Richard W.
Published: June 2020

The neurotransmitter gamma-amino butyric acid (GABA) is synthesized in presynaptic terminals by glutamic acid decarboxylase (GAD). Antibodies to GAD have been used to localize presynaptic terminals in the cerebellar cortex that use GABA as a neurotransmitter. In the cerebellum, the Purkinje cell makes synaptic contacts on dendrites and somata of neurons in the cerebellar nuclei. We were interested in developing a technique that combined light microscopic analysis of GAD distribution in Purkinje cell terminals with high quality electron microscopic localization of GAD.

The approach taken in this study was to combine high glutaraldehyde/metabisulfite fixation technique with the preembedding N-propyl gallate (NPG) silver enhanced gold procedure. Mice were perfused with buffered 3% glutaraldehyde and 0.4% sodium metabisulfite. Vibratome sections (60 μm) were permeabilized with 0.3% Triton, and incubated with a monoclonal anti-GAD antibody. The sections were incubated in goat-anti-mouse 1 nm Nanogold, and the gold was enhanced with the NPG silver enhancement solution for 10 minutes.
Immunoelectron microscopic localization of actin in developing rat leg muscle
Authors: Fassel Theresa A. , Greaser Marion L.
Published: June 2020

Studies were conducted on the arrangement of α-actin in muscle cells in Sprague-Dawley rats at 1, 3, 5, 7, 30 and over 100 days of age. The α-actin isoform was distinguished from the nonmuscle β- and τ- isoforms by using the α-specific monoclonal antibody HUC1-1. The antibody was used in a two stage post-embedding procedure with colloidal gold conjugated to goat anti-mouse IgG.

Muscle samples were placed in a relaxing solution (100 mM KCl, 10mM Imidazole, 10mM MgCl2, 2mM EGTA, 4mM ATP, 1mM NiCl2, 1mM PMSF, 100 ¼M leupeptin, 50% glycerol) for 1 hr on ice or overnight at -20°C. Samples were transferred to a skinning solution (100mM KCl, 10mM KH2PO4, 5mM MgCl2, ImM EGTA, 1% glucose, 0.2% Triton X-100, 0.02% NaN3) for 1 hr on ice and then into 3.7% formaldehyde in rigor buffer (RB - 75mM KCl, 5mM K2HPO4, 2mM MgCl2, 2mM EGTA, 2mM NaN3) for 1 hr for fixation.
Ultrastructural localization of antigenic sites in pancreatic tissue using in situ (metal-mirror) cryofixation
Authors: Lightfoot Fred G.
Published: June 2020

There have been previously reported methods using in situ cryofixation via metal mirror freezing to study the ultrastructural characteristics of lung tissue while in its native state (inflated), as well as the elemental composition in subcellular compartments of the proximal tubule in whole (unexcised) tissue. Many studies have been done to determine the most appropriate way to conserve both antigenicity and structural integrity of various tissues. In most of these protocols there are steps which are likely to reduce or destroy the tissue antigenicity. In this report, an application using in situ cryofixation of pancreatic tissue for optimal ultrastructure and antigenicity will be described.

Male rats were anesthetized with sodium pentobarbital (50mg/kg). The abdomen was opened and the pancreas was carefully elevated, using forceps, being careful to maintain integrity of the mesentery of the duodenum and pancreas along with their blood supply. The tissue was rapidly frozen using the PS1000 cryofixation unit (Delaware Diamond Knives, Wilmington, DE), a pneumatically operated metal mirror instrument precooled to nitrogen temperature.
Ultrastructural application of the patsc-GMS staining in various kidney diseases
Authors: Namimatsu Shigeki , Nakamura Shin-ichi , Nakagami Yoshizo , Oguro Tatsuo , Asano Goro
Published: June 2020

We have previously reported a new staining method, PATSC-GMS, for transmission electron microscopy. In the present study, we evaluated the stainability and usefulness of the PATSC-GMS method in tissues from renal diseases.

Fifty human kidney biopsies comprising normal (n=2), membranous glomerulonephritis (n=10), Alport's syndrome (n=l), IgA nephropathy (n=4), and other cases (n=33) were studied. Samples were pre-fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, and embedded in Epok-812 after dehydration. Ultrathin sections were made and placed on nickel grid mesh. The sections of the tissues were Pre-incubated in ammonia water(25%) for 10minutes, oxidized in 1% periodic acid for 20 minutes, reacted in 0.1% thiosemicarbazid (TSC) for lminute, post-incubated in ammonia water(25%) for 5minutes and stained with gelatin methenamine silver(GMS) solution: (50ml of 3% methenamine, 5ml of 2% gelatin, 5ml of 5% silver nitrate, 40ml of distilled water, and 5-6ml of 5% sodium tetraborate) was performed for 50C 50minutes in the oven.
Localization of atrial natriuretic peptide in normal human atrial tissue by immunoelectron microscopy
Authors: Wei Chi-Ming , Hukee Margaret , McGregor Christopher G.A. , Burnett John C.
Published: June 2020

Atrial natriuretic peptide (ANP) is a 28-amino acid peptide which is secreted from atrial myocytes and possesses natriuretic, vasoactive and renin inhibiting actions. In chronic congestive heart failure (CHF), plasma concentrations of ANP are significantly increased in the circulation secondary to enhanced atrial and ventricular synthesis. In a variety of non-human species, ANP has been detected by light and electron microscopy. However, the localization of ANP in normal human cardiac tissue remains unclear. Therefore, the current studies were designed to determine the localization of ANP in normal human atrial tissue.

Cardiac tissue of three normal human right atria were obtained from the donor heart undergoing primary cardiac transplantation. Atrial tissue was fixed for 24 hours at 4°C in 4% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer. These specimens were rinsed in phosphate buffer, dehydrated in a series of ethyl alcohols, embedded in Quetol 651, and polymerized at 42°C for 48 hours.
Cytochemical localization of free-radical-derived oxidants in human coronary atherectomy plaques
Authors: Ann Ellis E. , Grant Maria B. , Wargovich Thomas J.
Published: June 2020

Atherosclerosis is a multifactorial disease which leads to occlusion of vessels and potentially fatal intravascular thrombosis. Biochemical and immunocytochemical studies of oxidized low density lipoprotein (LDL) and treatment with antioxidants have demonstrated a role for oxidation in antherogenesis in animal models. The enzyme NADH-oxidase produces superoxide radicals (O2·-) and the free radical derived oxidant, hydrogen peroxide (H2O2). We used the cerium NADH-oxidase cytochemical localization technique to identify sites of free radical derived oxidation in coronary artery atherosclerotic lesions removed from patients undergoing coronary atherectomy.

Atherectomy plaques were fixed in cold, sodium cacodylate buffered (pH 7.4) 5% acrolein within 5 min after removal from patients. Specimens were washed in buffer and processed as previously described for cytochemical localization of NADH-oxidase. Cross sections of the plaques were examined and photographed, without post staining, at 75 kV in the transmission electron microscope.

NADH-oxidase activity, shown by cerium perhydroxide precipitate, was confined to the extracellular matrix and foam cells of the fatty streak.
Serial thin sectioning for pre- and post-embedding immunohistochemistry: Achieving consistent and reliable results for a 3-D reconstruction
Authors: Milroy Antonia M.
Published: June 2020

In recent years many new techniques and instruments for 3-Dimensional visualization of electron microscopic images have become available. Higher accelerating voltage through thicker sections, photographed at a tilt for stereo viewing, or the use of confocal microscopy, help to analyze biological material without the necessity of serial sectioning. However, when determining the presence of neurotransmitter receptors or biochemical substances present within the nervous system, the need for good serial sectioning (Fig. 1+2) remains. The advent of computer assisted reconstruction and the possibility of feeding information from the specimen viewing chamber directly into a computer via a camera mounted on the electron microscope column, facilitates serial analysis. Detailed information observed at the subcellular level is more precise and extensive and the complexities of interactions within the nervous system can be further elucidated.

We emphasize that serial ultra thin sectioning can be performed routinely and consistently in multiple user electron microscopy laboratories. Initial tissue fixation and embedding must be of high quality.
Ultrastructural study of basement membrane re-formation after intimal denudation
Authors: Yokoyama Munehiro , Ishiwata Toshiyuki , Asano Goro
Published: June 2020

The basement membrane (BM) underneath the endothelial cell(EC) layer plays important roles in the maintenance of cell polarity and the regulation of cell migration, adhesion, proliferation and differentiation. The model of denudation of the aortic intima with a balloon catheter is useful in research into interactions among cell components and extra-cellular matrix in the regenerative process of ECs in atherogenesis. In this study, re-formation of BM was examined during re-endothelialization after intimal denudation, using transmission electron microscopy.

Male Wistar rats, weighing about 400 g were used and endothelial denudation of the thoracic aorta was performed with a vascular balloon catheter (1.5 Fr., DOW CORNING, CORP). The catheter was inserted into the thoracic aorta via the left common carotid artery. After inflation of the balloon with saline solution, the catheter was passed through the length of the aorta about 2.5 cm three times. The animals were sacrificed at 15 min, 3, 7, 14, 21 and 28 days after endothelial denudation and the thoracic aortas were removed.
Adherence of enterotoxigenicKlebsiella pneumoniaeto hela cells and expression of type 3 fimbriae
Authors: Miliotis M.D. , Tall B.D. , Gray R.T. , Hall R.H.
Published: June 2020

In 1986, an outbreak of diarrheal disease in Texas was attributed to contaminated iced tea in whichKlebsiella pneumoniaewas the predominant flora. Although the ten strains tested were of different serotypes, they all produced a heat labile enterotoxin. Some of these enterotoxigenic strains (ETKP) also exhibited mannose-resistant hemagglutinins, which were later found to be type 3 MR/K fimbriae. In this study we extended our observations and investigated fimbrial expression, as well as HeLa cell adherence behavior, using negative staining, immunogold labeling, and scanning electron microscopy (SEM).

ETKP strains were grown on colonization factor antigen agar overnight at 37° C for expression of fimbriae. The presence of fimbriae and immunogold labeling of the cells was determined as described by Wolfeet al.Antiserum was prepared against purified fimbriae in rabbits and dilutions of primary and secondary antibodies (goat anti-rabbit IgG conjugated with 10 nm gold particles) in phosphate buffered saline, pH 7.2, containing 1 % bovine serum albumin and 0.5% Tween 20 were used to label cells.
Bacterial adherence and biofilm behavior ofVibrio vulnificus
Authors: Tall B. D. , Gray R. T. , Shah D. B.
Published: June 2020

Vibrio vulnificus, an opportunistic human pathogen, is found as member of the normal microflora of shellfish and other seafoods, many of which are eaten raw. Though usually not harmful,V. vulnificusis responsible for causing fulminating septicemia in immunocompromised individuals. In previous light microscopic studies, we showed data suggesting that isogenic unencapsulated phase variants were more adherent to HeLa cells than were counterpart encapsulated phase variants. In this study, we extended our observations by comparing phase variant capsular morphology stained with Alcian blue (AB) and Ruthenium red (RR), and investigated the dynamics of biofilm formation by these organisms to glass coverslips (CS) using quantitative plate counts and scanning electron microscopy (SEM).

To characterize the morphology of capsules expressed by these organisms, we stained cells grown on trypticase soy agar containing 1 % NaCl (TSA/NaCl) with AB and then prepared them for electron microscopy (EM) according to the method described by Hendleyet al.
SEM studies of membrane endocytosis in toad urinary bladders following withdrawal of mezerein
Authors: Mia A.J. , Davidson A.D. , Oakford L.X. , Yorio T.
Published: June 2020

Vasopressin (ADH), enhances fluid reabsorption through a complex process of water channel insertion into the apical membrane and retrieval during hormonal removal. These water channel membranes are thought to internalize by endocytosis resulting in down-regulation of the apical membrane permeability. ADH V1-receptors, like ADH V2-receptors, increased water flow, when mezerein (MZ), an activator of protein Kinase C, was added to the mucosal surface. This is accompanied by the activation of several protein Kinase C isozymes, as determined by protein A-immunogold labeling. However, little is known about the surface ultrastructural changes accompanying membrane retrieval in amphibian bladder tissues following removal of hormone. We reported an apparent membrane internalization process using scanning electron microscopy (SEM) following withdrawal of ADH. The current report is concerned with time-response surface studies of the apical membrane internalization process following withdrawal of MZ, and the role of protein Kinase C in this process.
Thickness measurement in quantitative Electron Microscopy
Authors: Heng Yew M.
Published: June 2020

Quantitative electron microscopy generally implies the measurement of elemental content and/or structures in a given specimen or the 3D reconstruction of a structure. When a feature in a specimenis being quantified in a transmission electron microscope (TEM), the specimen thickness has to bedetermined for at least two reasons. Firstly, thickness is a required parameter in the calculation, for example, of volume and secondly, it is required for the calibration of instrumental conditions ortheoretical parameters such that correction for thickness effects due to conditions such as radiation damage, x-ray absorption or multiple scattering of electrons can be performed. However, it is a common knowledge that specimen thickness is difficult to be determined accurately. The difficulty is mainly due to the nature of TEM specimens. There is no universal method which can be used.Therefore, in order to select a few appropriate methods, it is necessary that a wide range of methods be reviewed and critically assessed.

Numerous methods are available for the measurement of the thickness of a specimen to be analysed in a TEM (Table 1).
Quantitative image analysis and morphometry of electron micrographs using a comptuer-assisted user-defined interface
Authors: Evans Barbara A. , Howard Tamara A. , Mahoney Thomas M. , Becich Michael J.
Published: June 2020

Quantitative Image Analysis and Morphometries (QIAM) is a tedious and time-consuming process. The large number of micrographs examined leads to special data management problems. Analysis of multiple subcellular compartments in cells is complicated by the inability to resolve adjacent cellular compartments. In addition, capturing data via grey scale or thresholding techniques on electron micrographs is very difficult at the organelle level. Visual feedback from an image that is also captured by an integrated digitizing pad (to identify and edit the cellular compartments) is critical. Statistical evaluation of the large amounts of information generated further complicates data management and analysis. Computer assisted QIAM with a user defined interface in a Windows environment has helped make data collection and analysis a much simpler and efficient process. We used BioScan’s Optimas software (Edmonds, WA) linked to a spreadsheet to analyze multiple intracellular compartments and other features of a variety of epithelial cells in vivo and in vitro.
Basic literacy in electron-excited x-ray microanalysis
Authors: Newbury Dale E.
Published: June 2020

Electron beam x-ray microanalysis with energy dispersive x-ray spectrometry (EDS), as performed in electron probe microanalyzers (EPMA)/scanning electron microscopes (SEM) for thick specimens and analytical electron microscopes (AEM) for thin sections, is a powerful technique with wide applicability in the physical and biological sciences and technology communities. The operation of an EDS x-ray microanalysis system has been automated to the point that many users now consider EDS to be a routine tool where the results reported by the automation system are always correct Unfortunately, there are numerous pitfalls awaiting the unwary analyst. All EDS users require a basic level of literacy in x-ray microanalysis to properly interpret spectra and develop a sensible analysis strategy for their problems. This “basic literacy” includes knowledge of the factors controlling the efficiency of production of characteristic and continuum x-rays, the characteristic energies and structure of x-ray families that provide the basis for qualitative analysis, the operational characteristics of energy dispersive x-ray spectrometers, including artifacts, and the systematic procedures for qualitative and quantitative analysis.