bluuebunny/crossref_metadata_2025 huggingface dataset:
Plug-in scripts for EFTEM automation
Authors: Evans N. D. , Kundmann M. K.
Published: June 2020

Post-column energy-filtered transmission electron microscopy (EFTEM) is inherently challenging as it requires the researcher to setup, align, and control both the microscope and the energy-filter. The software behind an EFTEM system is therefore critical to efficient, day-to-day application of this technique. This is particularly the case in a multiple-user environment such as at the Shared Research Equipment (SHaRE) User Facility at Oak Ridge National Laboratory. Here, visiting researchers, who may oe unfamiliar with the details of EFTEM, need to accomplish as much as possible in a relatively short period of time.

We describe here our work in extending the base software of a commercially available EFTEM system in order to automate and streamline particular EFTEM tasks. The EFTEM system used is a Philips CM30 fitted with a Gatan Imaging Filter (GIF). The base software supplied with this system consists primarily of two Macintosh programs and a collection of add-ons (plug-ins) which provide instrument control, imaging, and data analysis facilities needed to perform EFTEM.
ALCHEMI of L12-Ordered Ni76Al21Hf3and gamma prime particles
Authors: Bentley J. , Anderson Ian M.
Published: June 2020

Recent improvements to the atom location by channeling-enhanced microanalysis (ALCHEMI) technique now allow reliable measurements of site distributions in ordered alloys. Early zone-axis ALCHEMI of Ll2-ordered Ni3Al-based alloys was plagued by ionization delocalization; for Ni76Al21Hf3at <001> the fraction of Hf on the Al sublattice, pHf ‘Al’, = −127%. Reanalysis by statistical ALCHEMI was no better, giving pHf ‘AT’ = −220 ± 190%, contrary to the claims that delocalization effects should be minimized. The same data set analyzed with the reformulated ALCHEMI method yields pHf ‘Al’, = 80 ± 25%.

Further experiments have been performed at ∼140K in a Philips CM12 operated at 120 kV and equipped with an EDAX 9900 spectrometer and superUTW Si(Li) detector. The diffracting conditions were carefully adjusted with the beam (not specimen) tilt, a probe size of ∼200 nm and beam divergence of ∼3 mrad were used, and the current in the probe was monitored and held constant at ∼1 nA.
Quantitative x-ray microanalysis and thickness determination using ζ factor
Authors: Watanabe M. , Horita Z. , Nemoto M.
Published: June 2020

X-ray absorption in quantitative x-ray microanalysis of thin specimens may be corrected without knowledge of thickness when the extrapolation method or the differential x-ray absorption (DXA) method is used. However, there is an experimental limitation involved in each method. In this study, a method is proposed to overcome such a limitation. The method is developed by introducing the ζ factor and by combining the extrapolation method and DXA method. The method using the ζ factor, which is called the ζ-DXA method in this study, is applied to diffusion-couple experiments in the Ni-Al system.

For a thin specimen where incident electrons are fully transparent, the characteristic x-ray intensity generated from a beam position, I, may be represented as I = (NρW/A)Qωaist.
Platform wars: Macintosh-based digitization systems
Authors: Krivanek O. L. , Hunt J. A.
Published: June 2020

Improvements over the past few years in essentially all modern operating systems coupled with the blurring of the boundaries between mainframes, workstations, and personal computers make the choice of platform often one of personal preference other than of necessity. A decade ago there were very distinct differences between the features offered by IBM PC and clones, the Apple Macintosh, and UNIX workstations. These differences included price, processing performance, OS features, look and feel, graphical capabilities, memory and disk limitations, networking capabilities, ease-of-use, system stability, development tools, etc. Since then many of these distinguishing differences have evaporated making platform selection a more subtle issue, but also much less important. However, there are still a great many users that tend to make such decisions based on their “religious” preference of platform rather than based on objective information.

How does one choose the appropriate platform for image acquisition and processing? The question should first be - “What software packages do what is needed?” From this list the choice of platform can be narrowed.
Software tools for 4D live cell microscopy
Authors: Thomas C. , DeVries P. , Hardin J. , White J.G.
Published: June 2020

The Integrated Microscopy Resource has developed a trio of Macintosh-based software tools which allow the user to collect digital 4D microscopy (3D over time) of live cells. To demonstrate the program, high-resolution Nomarski DIC images were collected from an inverted microscope using a video camera. The output of the camera can be averaged and contrast-enhanced before the resulting signal is digitized using a framegrabber card installed in a Macintosh computer (Figure 1). Collection, subsequent processing, and analysis of the resulting data sets are handled by the following three software tools:

4D Acquisition Software - This IMR-designed software system is based on the popular shareware image processing program “NIH-Image” (author Wayne Rasband, NIH) and controls the actual image acquisition through a series of graphic user interfaces. A framegrabber card, a stage drive motor, and an illumination shutter are manipulated to allow the imaging of either a single focal-plane of the specimen over time, or the acquisition of 3-dimensional volumes at each timepoint. After selecting the top and bottom of the sample, the user can specify the number of focal planes to acquire, the increment for the focus motor between focal planes, the number of timepoints to collect, and a file format in which to save the images. The software will automatically move through the sample from the top to the bottom gathering images of each focal plane as it goes. The result is a stack of images representing the 3D structure of the sample. The software will wait a defined amount of time and the process will be repeated for as many timepoints as the user has designated.
TEM characterization of CMR thin film on [001] SrTiO3 substrate
Authors: Wang Y.Y. , Gupta A. , Dravid V.P.
Published: June 2020

Due to its potential magnetic device application, giant/colossal magnetoresistant (GMR or CMR) material, (bulk material as well as thin film), have been under intense investigation in recent years. In the bulk material, the changes of the resistivity under 4T magnetic field is about 80% at the metal-insulate transition temperature for La0.7Ca0.3MnO3 (LCMO). In 1994, S. Jin et al reported thousandfold change in resistivity in magnetoresistive La-Ca-Mn-0 films on a LaAlO3 substrate. It is suspected that such a large magnetoresistivity change in the thin film is due to the defect structure in the epitaxially grown thin film.

The base structure of LCMO is an MnO6 octahedral perovskite cubic structure. Because of its strong static Jahn-Teller (JT) distortion, the compound is metrically orthorhombic, with a∼b∼√2 ap and c∼2ap where ap is the lattice constant for the base cubic structure. X-ray diffraction study on this compound shows that it belongs to Pnma space group.
On the crystallography of triple points in nonoxide ceramics: epitaxial relationships are a function of statistics and geometry
Authors: MoberlyChan Warren J.
Published: June 2020

Covalent ceramics, such as SiC and Si3N4, can have superior mechanical properties for high temperature structural materials. However, densification difficulties and poor fracture toughness have hindered use. Traditionally, the choice of sintering additives has been a “hit and miss” method to improve properties. The primary purpose of an additive is to provide good density, as residual pore size relates to critical flaw size. Another feature of an additive has recently been to invoke fracture around the matrix grains, thereby enhancing toughness. Most reported nonoxide ceramics include upwards of 10% secondary phase(s) as a continuous phase between many matrix grains and causing poor high temperature strength. Even when this secondary phase is crystallized, thin amorphous interfaces are observed. The ABC-SiC processed for this study incorporates <5% total Al, B, and C as additions. The Al enhances densification, as well as the P-to-a phase transformation at lower temperatures (1600-1900°C); and controlling die microstructural development has provided record toughness.
Site occupancy in gamma alumina
Authors: Bateman C. A. , Ringwelski A.Z. , Broach R.W.
Published: June 2020

Gamma (γ) alumina is referred to as a defect spinel because it has a tetragonally distorted spinel structure (AB2O4) and an insufficient number of cations to fill all cation sites. In the spinel structure, the oxygen lattice is cubic close packed with A- and B-site cations in tetrahedral and octahedral coordination, respectively. The 2l⅓ Al atoms per unit cell of γ alumina can distribute themselves across 16 octahedral and 8 tetrahedral sites.

The literature differs on where the 2⅔ cation vacancies per unit cell are located. Wilson and McConnell proposed that the vacancies in γ alumina, as first formed by calcining boehmite, are predominantly on the tetrahedral lattice but, with further heat treatment, move to occupy random positions on both octahedral and tetrahedral lattices. One study using NMR showed that the vacancies lay exclusively on the tetrahedral lattice, independent of the calcination temperature. A more-recent study using Rietveld refinement of powder neutron diffraction data suggested that both octahedral and tetrahedral lattices were partially occupied.
Pentagonal and heptagonal carbon-rings in growth of nanosize graphitic carbon spheres
Authors: Kang Z.C. , Wang Z.L.
Published: June 2020

Fullerene C60 and nano-tubes are a group of unique structures of carbon. These structures are producei using a carbon electrode arc-discharge technique, but it has not been successful in producing carbon spheres Recently, a new mixed-valent oxide-catalytic carbonization (MVOCC) process has been invented that can b used to synthesize monodispersed nano-size graphitic carbon spheres at low cost and with large quantities [3] The carbon spheres were produced at 1100° C by decomposition of natural gas (methane) under the catalytic assistance of transitional/rare earth metal oxides with mixed valences. The product is pure and separated fron the catalyst, thus, no purification is needed. The MVOCC process does not produce any environmenta hazardous chemicals, and the catalyst is reusable. The carbon spheres are expected to have extraordinary mechanical, physical and chemical properties and potential applications in the areas such as high-strengfi composite materials, environmental filtering, catalysis, lubrication and surface coating.
On the influence of specimen thickness in TEM images of super-conducting vortices
Authors: Bonevich J. , Capacci D. , Pozzi G. , Harada K. , Kasai H. , Matsuda T. , Tonomura A.
Published: June 2020

The successful observation of superconducting flux lines (fluxons) in thin specimens both in conventional and high Tc superconductors by means of Lorentz and electron holography methods has presented several problems concerning the interpretation of the experimental results. The first approach has been to model the fluxon as a bundle of flux tubes perpendicular to the specimen surface (for which the electron optical phase shift has been found in analytical form) with a magnetic flux distribution given by the London model, which corresponds to a flux line having an infinitely small normal core. In addition to being described by an analytical expression, this model has the advantage that a single parameter, the London penetration depth, completely characterizes the superconducting fluxon. The obtained results have shown that the most relevant features of the experimental data are well interpreted by this model. However, Clem has proposed another more realistic model for the fluxon core that removes the unphysical limitation of the infinitely small normal core and has the advantage of being described by an analytical expression depending on two parameters (the coherence length and the London depth).
Direct measurement of chromatin diffusion and constraint in living cells using a gfp-lac repressor fusion protein
Authors: Marshall W.F. , Straight A.F. , Murray A. , Fung J.C. , Marko J. , Agard D.A. , Sedat J.W.
Published: June 2020

The polymer dynamics of interphase chromatin is at present a poorly understood aspect of nuclear organization, but one with profound consequences on events within the nucleus. The rates of many important processes, such as meiotic homolog pairing, site-specific recombination, and chromatin condensation, all involve the motion of chromatin within the nucleus. How fast can such rearrangements occur? Because interphase chromatin is likely to behave as a tangle of random-walk polymers, there is likely to be a substantial hindrance to diffusion. Therefore, the rate at which a given site on a chromosome can diffuse within the nucleus may limit the rate at which events requiring chromatin motion can occur. Furthermore, the polymer physics of interphase chromatin is an interesting line of theoretical research in its own right, and knowledge of diffusion rates is an important experimental parameter to consider when evaluating physical models. Finally, a fundamental aspect of nuclear architecture is the extent to which chromatin is anchored to a nuclear skeleton. Such matrix attachments would result in constrained diffusion, which can be detected by analysis of chromatin motion.
Effects of pacing and isoproterenol on mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during the contractile cycle
Authors: Ohata Hisayuki , Herman Brian , Lemasters John J.
Published: June 2020

Using laser scanning confocal microscopy, we have developed methods to measure mitochondrial free Ca2+ in cultured adult rabbit cardiac myocytes during excitation-contraction. Here, we examined the effect of isoproterenol and the rate of electrical pacing on the magnitude and duration of mitochondrial Ca transients. The mitochondria and cytosol of myocytes were loaded with the Ca2+-indicating fluorophore, Fluo-3-AM, at 4°C. Subsequently, the mitochondria were labeled with tetramethylrhodamine methylester (TMRM). In line scans, red TMRM fluorescence and green Fluo-3 fluorescence were simultaneously imaged by laser scanning confocal microscopy. Large transients of Fluo-3 fluorescence after electrical stimulation were observed both in cytosol and mitochondria (Figures 1). Isoproterenol (1 μM) increased the magnitude of Ca2+ transients caused by field stimulation and their subsequent rate of decay (Figure 2). This effect of isoproterenol was more marked in the cytosol than in mitochondria. In particular, the effect of isoproterenol on Ca2+ transients was least in perinuclear mitochondria.
Brain Homogenate Standards for Analysis of Aluminum in Alzheimer’s Disease
Authors: Swyt C. R. , Smith Q.R. , Deng Q.S.
Published: June 2020

Aluminum doped brain homogenates have been proposed as a common set of standards to help resolve the disparity among the reported measured concentrations in plaques and tangled neurons in postmortem Alzheimer’s Disease brain specimens. Among the many analytical techniques utilized electron probe microanalysis (EPMA) alone provides both the capability to identify, by backscattered electron imaging, cells in unstained sections and to achieve, using wavelength dispersive x-ray spectrometry (WDS), a sensitivity for aluminum approaching the higher reported normal aged brain concentrations. We have prepared a set of freeze-dried 30 μm cryosections of aluminum doped homogenized rat brain. We report here the preliminary results of analysis by EPMA and briefly discuss some of the practical considerations in using such homogenates as a standard set for quantitation by WDS.
Evaluation of platelet dense granules for determining storage pool deficiencies by HVEM image and quantitative analysis
Authors: Gunning W.T. , Turner J.N. , Buttle K. , Calomeni E.P. , Lachant N.A. , Smith M.R.
Published: June 2020

There are a variety of conditions which have been associated with prolonged bleeding times. If other etiologies including von Willebrand's disease have been ruled out, a platelet function disorder must be considered. The best, if not only, technique to make this diagnosis is the electron microscopic evaluation of whole air dried platelets. Bull first described the presence of dense granules in whole platelets in 1968 and the technique has been utilized extensively The electron dense or delta granules are easily distinguished from the larger more numerous alpha granules which are electron lucent. The significance of the dense granules is that they are known to be “storage pools” of serotonin, calcium, adenosine di- and triphosphate, and pyrophosphate. Prolonged bleeding times may be directly related to an insufficiency of these substances. The diagnosis of a storage pool deficiency is made when either the storage content of the dense granules is abnormal or their number is diminished. We observe normal platelets to have 4-6 dense granules, which agrees with the literature.
Seminal Phagocytes Detection at Light-Microscopy Level using Three Different Techniques
Authors: Benítez L. A. , Díaz O. G. , Eguía R. M. M. , Gallegos M. G. A.
Published: June 2020

The phagocytes are normally present in the conective tissue at different organs, and their number increased in immunological or inflammatory processes. In these cases, they are present too in corporal fluids.

The presence of phagocytes in semen was referred by several authors, but that phenomenon has not been suficiently analysed. At smear Papanicolaou stained analysis, the seminal phagocytes can be inadverted thus, their relationship to the male infertility or another reproductive tract pathology can not be established.

In this study we implemented three techniques to detect the presence of phagocytes in semen of 28 infertile patients: Myeloperoxidase for polymorphonuclears (leukocyte), Neutral red for macrophages and the semithin sections analysis at light microscopical level of the seminal pellet obtained by centrifugation and included in epoxyresin.
Adherence of temperature and iron-stressed enterohemorrhagic escherichia coli growing at 9.5°C to HEp-2 cells
Authors: Schwach Tina S. , Zottola E. A.
Published: June 2020

Conditions in the intial growth environment are known to effect growth and expression of numerous bacterial virulence factors. Thus, we were interested in studying whether growth and adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal-like epithelia was affected by temperature and the amount of available iron in the initial growth environment. Adherence of E. coli 026:H1l, strain 400, to HEp-2 cells was quantified using the assay of Sherman and Soni and monitored by both SEM and TEM. Bacteria were grown at 9.5°C with and without iron-restriction in 0.3% Trypticase Soy Broth plus 1% Nobel Agar (TSBA). Ethylenediaminedihydroxyacetic acid was added (0.75 mM and 2.0 mM) to induce both low (EL) and high (EH) levels of iron-restriction and 100 μM FeCl was added to induce iron-replete conditions.
Bacterioids in the Rectal Organ of the Lantern Dug Pyrops Candelaria Linn (Homoptera: Fulgoridae)
Authors: Cheung W.W.K. , Wang J.B.
Published: June 2020

The lantern bug harbours three symbionts, namely a, x and i in its body. These microorganisms are supposed to be transmitted transovarially to the future progeny. The x-symbionts are found in a special pair of organs called the x-organ which bulges to form a rectal organ in adult females. The purpose of this study is to investigate into the fine structure of the x-symbionts. This will serve as a basis for understanding the interactions of this microorganism with its host.

The rectum of the lantern bug Pyrops candelaria Linn, was dissected out in buffered insect saline and fixed in 2.5% glutaradehyde in 0.1M sodium cacodylate buffer (pH 7.2) for 1 hr. The rectal organ was subsequently post-fixed in 1% osmium tetroxide (pH 7.2) and dehydrated in alcohol/acetone series. These were blocked in Spurr resin and cut with a Reichert Ultratome. Sections were stained with uranyl acetate and lead citrate and examined with a JEOL JEM-1200EX electron microscope. Thick sections (1 μn) were stained with 1% toluidine blue and examined under a Nikon Optiphot light microscope.
Attachment, entry, and destruction of cultured human B-lymphocytes by the lyme-disease spirochete borrelia burgdorferi
Authors: Fischer Elizabeth R. , Dorward David W.
Published: June 2020

The most prevalent tick-borne disease in the United States, Lyme disease, is caused by the spirocheteBorrelia burgdorferi. Studies have suggested that Lyme disease spirochetes, like other bacteria, may enter host cells thereby evading the immune system.

In order to investigate possible interactions between Lyme disease spirochetes and immune system cells, we co-incubatedB. burgdorferistrain Sh-2-82 with cultured human B- and T-cells (TIB-215 and H9, ATCC, respectively) and primary lymphocytes at an MOI of 100:1.

Nomarski interference microscopy revealed that as early as 10 minutes after coincubation, as previously described with other cell lines, low passage spirochetes (<8 in vitro passages) attached tip first to the B-cells and then invaded the B-cells (Fig. 1,4). Scanning electron microscopy revealed that by 24 hours post-infection, 90+% of B-cells had spirochetes attached to their surfaces (Fig.2). Transmission electron microscopy suggested that intracellular spirochetes may exist both in vacuoles and free in the cytoplasm of primary lymphocytes (Fig. 3a-b).
Evaluation of alternative fixatives/protocols for the ultra-structural preservation of fast and slow growing mycobacteria
Authors: Hayes S. F , Small P. L. C.
Published: June 2020

Various fixation formulas and protocols were examined to determine a routinely optimal fixation of Mycobacterial species in and out of tissues. These were evaluated by TEM and compared to results obtained using freeze substitution methods upon other Mycobacteria such as Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, M. kansasii and M. thermoresistible ATCC 19527

Samples consisted of two slow growing mycobacterial species, both human pathogens; Mycobacterium tuberculosis, grown in M7H9 broth, and Mycobacterium marinum (1218 R & S variants), grown on M7H10 agar, with the latter also grown in tissue culture, and in guinea pig skin. Fixatives included: (1) glutaraldehyde/parafoimaldahyde/tannic acid in a phosphate/sucrose buffer, (2) paraformaldehyde/polylysine/periodate/glutaraldehyde (PLPG) in phosphate buffer followed by tannic acid and reduced osmium respectively, (3) PLP followed by tannic acid only without osmium, and (4) fixative 84-40 containing carbodiimide, glutaraldehyde and ruthenium red. Protocols varied in the length of time for fixation, types of buffers, solvents and in embedding schedules for Spurr's low viscosity resin.
FESEM imaging of the xenopus oocyte nuclear envelope
Authors: Ris H.
Published: June 2020

The transport of proteins and nucleic acids into and out of the nucleus is controlled by a unique membrane transport system, the nuclear pore complex (NPC), a large macromolecular assembly inserted into the pores of the nuclear envelope (NE). The large nucleus of the Xenopus oocyte has been a favorite object for both functional and structural studies. TEM of thin sections and negatively stained membranes had revealed the eightfold symmetry of the NPC, but the complex anatomy of the cytoplasmic and intranuclear surface of the NPC was first revealed by high resolution, field emission SEM. On the cytoplasmic side, the NPC is limited by a flat ring, about 20 run wide and 120 nm in diameter. It carries 8 twisted filaments which, in the presence of divalent cations are condensed into 20x40 nm cylinders. On the nuclear side there is a similar ring with 8 long , thin filaments attached to its outer rim.
Imaging of surface-adherent human-blood platelets by atomic-force microscopy (AFM)
Authors: Murthy N. , White J. G. , Rao G. H. R.
Published: June 2020

Studies in our laboratory over the past three decades have explored platelet ultrastructural morphology by light microscopy as well as transmission, scanning and low-voltage, high-resolution scanning electron microscopy. Unlike these optical microscopes, scanning probe microscopes measure real space images of cells on flat surfaces. In brief, the AFM works by scanning a very sharp conducting tip (SiN) over the surface of a conducting biological membrane. Cantilivers used for probing were short tipped with thin legs (0.22 nm spring constant). Scanning was done with the J size scanner (150 x 150 u scan area). The difficulty in achieving high resolution images of biological samples relates to the damage caused by the probing tip in spite of its minute force on delicate biological cell surface membranes. In the present study we have evaluated surface topography of fixed resting and activated platelets using AFM. Resting platelets were treated with cytochalasin B to prevent actin filament assembly shape change prior to being placed on polylysine-coated grid surfaces.
New insights into the Ras onco-protein and its interactions with the Raf-1-1 kinase
Authors: Campbell Sharon , Mott Helen , Zhong Sheng , Drugan Jonelle , Carpenter John
Published: June 2020

The Ras proteins are members of a large superfamily of nucleotide-binding proteins that cycle between active GTP- and inactive GDP-bound states. They are positively regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and negatively regulated by GTPase activating proteins (GAPs) that stimulate formation of the inactive GDP-complexed protein. Structural mutations that activate Ras oncogenic potential either impair GAP-stimulated GTPase activity or promote enhanced intrinsic nucleotide exchange. The net result of either biochemical effect is to favor elevated levels of Ras-GTP in vivo.

We are investigating selected Ras variants containing substitutions at conserved sites thought to be important for guanine nucleotide binding, GTP hydrolysis, GTP/GDP interconversion and protein recognition, to better understand the role of these conserved amino acids in Ras-mediated signal transduction. Recent studies conducted at the U. of North Carolina will be presented, where we have characterized a novel activating mutation in Ras using multi-dimensional NMR spectroscopy and established a region involved in direct interaction with guanine nucleotide exchange factors.
Visualization of individual astrocytes in three-dimensional cerebellar tissues using green fluorescent protein
Authors: Andersen M. D. , Szarowski D. H. , Turner J. N. , Shain W.
Published: June 2020

The central nervous system is a complex structure traditionally studied through selective stains and two-dimensional images of fixed tissues. A long term goal of this laboratory is the study of dynamic cellular interactions in three dimensional tissues such as organotypic cultures. Due to the large volume of tissue, traditional microscopy methods contain excessive out-of-focus data making it difficult to observe clearly specific cellular interactions. The visualization of morphological changes in specific cells in living, three-dimensional tissues is commonly done with vital dyes. An alternate approach is labeling individual astrocytes with Green Fluorescent Protein (GFP) and injecting them into organotypic cultures.

GFP is a naturally fluorescent protein originally isolated from the jellyfish Aequorea victorea. To label individual astrocytes, LRM55 astroglial cells were transfected with GFP using calcium phosphate. The percentage of stable transfectants was low (< 1%) resulting in expression of varying amounts of GFP, as seen in Figs 5 and 6.
TEM comparison of osmium vs osmium with potassium ferricyanide secondary fixatives and the impact of secondary fixative temperature on tissue preservation/contrast quality
Authors: Horn J. W. , Dovey-Hartman B. J. , Meador V. P.
Published: June 2020

Osmium tetroxide (OsO4) is a universally used secondary fixative for routine transmission electron microscopic evaluation of biological specimens. Use of OsO4 results in good ultrastructural preservation and electron density but several factors, such as concentration, length of exposure, and temperature, impact overall results. Potassium ferricyanide, an additive used primarily in combination with OsO4, has mainly been used to enhance the contrast of lipids, glycogen, cell membranes, and membranous organelles. The purpose of this project was to compare the secondary fixative solutions, OsO4 vs. OsO4 with potassium ferricyanide, and secondary fixative temperature for determining which combination gives optimal ultrastructural fixation and enhanced organelle staining/contrast.

Fresh rat liver samples were diced to ∼1 mm3 blocks, placed into porous processing capsules/baskets, preserved in buffered 2% formaldehyde/2.5% glutaraldehyde solution, and rinsed with 0.12 M cacodylate buffer (pH 7.2). Tissue processing capsules were separated (3 capsules/secondary fixative.solution) and secondarily fixed (table) for 90 minutes. Tissues were buffer rinsed, dehydrated with ascending concentrations of ethanol solutions, infiltrated, and embedded in epoxy resin.
Ultrastructural study of human fetal appendix: A “novel” cytoplasmic organelle
Authors: Siddiqui K. M.
Published: June 2020

Vermiform appendix (VA) present in humans and other vertebrates may have a similar function to gut-associated lymphoepithelial tissue (GALT). The function of GALT in the gastrointestinal tract (GIT) is well documented. The functional significance of VA, as a “lymphoid” tissue in developing human fetus has not been established. However, VA is the narrowest part of GIT that protrudes from the base of cecum at the ileocecal juncture. Several diseases such as chronic appendicitis, mucocele, argentaffin carcinoma, and developmental diverticula causing inflammation are associated with VA (Delikaris, et al., 1983). An agenesis of VA and complications associated with it in a child born to a mother who had taken thalidomide during her pregnancy has been documented (Bremner & Mooney, 1978). These pathologies and also occurance of appendicitis with cystic fibrosis (Bockman, 1983) suggest a functional significance, and warrants a close observation of VA in human fetuses. On the other hand, studies detailing the subcellular architecture and characterization of various cell types of it have been limited,
Using the secondary electrons (SE) of SEM with NIST‘s MONSEL-II program to obtain improved linewidth measurements and slope angles of line edges on a MMIC GaAs device
Authors: Sartore Richard G.
Published: June 2020

In the evaluation of GaAs devices from the MMIC (Monolithic Microwave Integrated Circuits) program for Army applications, there was a requirement to obtain accurate linewidth measurements on the nominal 0.5 micrometer gate lengths used to fabricate these devices. Preliminary measurements indicated a significant variation (typically 10 % to 30% but could be more) in the critical dimensional measurements of the gate length, gate to source distance and gate to drain distance. Passivation introduced a margin of error, which was removed by plasma etching. Additionally, the high aspect ratio (4-5) of the thick gold (Au) conductors also introduced measurement difficulties. The final measurements were performed after the thick gold conductor was removed and only the barrier metal remained, which was approximately 250 nanometer thick platinum on GaAs substrate. The thickness was measured using the penetration voltage method. Linescan of the secondary electron signal as it scans across the gate is shown in Figure 1.
Shallow drain extension by angled ion implantation
Authors: Alvis Roger , Liming Scott , Griffin Peter
Published: June 2020

In a technology driven enterprise, such as the semiconductor industry, pure research and development often overshadows manufacturing concerns. Typically, “discovery” is the result of miniaturization efforts with integration into existing process flows a secondary concern. However, it is often the case that fabrication of devices is complicated by next generations of technologies. Since revenue and profits are ultimately determined by the speed and ease with which a wafer may be processed, the streamlining of process flows assumes a certain level of importance. In this work, we describe the construction and microstructural characterization of a simple spacerless metal-oxide semiconductor (MOS) transistor with a self-aligned shallow drain extension.

Transistor structures were fabricated at Stanford University's Center for Integrated Systems usinga single masking step to pattern the gate mask for the self-aligned structures. A 200Å gate oxide wasgrown and a 3000Å polysilicon blanket film was subsequently deposited on the wafer. Thepolysilicon was patterned into an array of 2.0μm lines and 3.0μm spaces.
In situ TEM observations of melting in nanosized eutectic Pb-Cd inclusions embedded in Al
Authors: Hagège S. , Dahmen U. , Johnson E. , Johansen A. , Tuboltsev V.S.
Published: June 2020

Small particles of a low-melting phase embedded in a solid matrix with a higher melting point offer the possibility of studying the mechanisms of melting and solidification directly by in-situ observation in a transmission electron microscope. Previous studies of Pb, Cd and other low-melting inclusions embedded in an Al matrix have shown well-defined orientation relationships, strongly faceted shapes, and an unusual size-dependent superheating before melting.[e.g. 1,2].

In the present study we have examined the shapes and thermal behavior of eutectic Pb-Cd inclusions in Al. Pb and Cd form a simple eutectic system with each other, but both elements are insoluble in solid Al. Ternary alloys of Al (Pb,Cd) were prepared from high purity elements by melt spinning or by sequential ion implantation of the two alloying additions to achieve a total alloying addition of up to lat%. TEM observations were made using a heating stage in a 200kV electron microscope equipped with a video system for recording dynamic behavior.
Effect of electron irradiation on the 3C-4H transformation in a Co-Fe alloy
Authors: Allen Charles W. , Mori Hirotaro
Published: June 2020

In general, irradiation of materials by high energy electrons, protons or neutrons has been shown to stabilize the existing microstructure against subsequent martensitic transformation. Alloys which have been studied include the binary intermediate phases NiTi and Ni63Al37 and ternary phases between Cu37,5Al62.5 and CU63.6Al36.4 as well as the intermetallic Cu69.01Al17.92 Zn12.17. In these materials, the martensitic transformations are known to be sensitive to small changes in chemical composition and to the degree of long range order. In addition, one should expect the kinetics of martensitic transformations to be sensitive to other forms of structural damage introduced during irradiation also. This spectrum of irradiation-induced phenomena renders the interpretation of results in studies of such concentrated binary and ternary phases rather complicated, therefore.

We present here the preliminary results of a study of the effect of electron irradiation on the character of
the martensitic transformation in a relatively dilute terminal solid solution of Co+5.75 wt pet Fe, similar in
composition to that employed for studies of the transformation mechanism itself.
An Application of a Method for Correlating Mechanical Property Changes with Microstructural Changes in Ion-Irradiated Specimens
Authors: Rice P. M. , Stoller R. E.
Published: June 2020

A method for correlating mechanical property changes and microstructural changes as a function of depth in ion irradiated specimens has been applied to a series of model pressure vessel steels. The technique employs nanoindentation with very small indent depths, on cross sectional specimens, to measure changes in hardness with sub-micron spatial resolution. Conventional TEM imaging of the cross sectional specimens allows the investigation of the defect microstructure as a function of depth below the original irradiated surface. For ion irradiations (implantations) the dose varies as a function of depth in the specimen and can be calculated with reasonable accuracy using TRIM calculations. Thus changes in both hardness and defect microstructure can be measured and correlated, over a range of doses, from a single specimen.

The technique has been applied to a set of model ferritic pressure vessel alloys to study the effect of various solutes on embrittlement. 2.5 MeV He ion irradiation was used to produce qualitatively similar microstructural features to those created in a nuclear reactor environment allowing the study to be carried out without the added complications associated with radioactive specimens. Figure la is an SEM image showing a set of nano-indents that starts on the left, beyond the end of range of the ions which is easily seen as the dark band. The indents eventually cross the thin oxide that marks the original irradiated surface (the white line) and enter the Fe electroplating seen here as the fine grained material at the bottom of the images. Figure lb is a higher magnification SEM image of the same indents showing the three-sided pyramid shaped indents reflective of the Berkovich diamond used in nanoindenting. Each of these residual indents is the result of a multipule indent process. The first indent is 50nm deep to acquire the sub-micron resolution hardness data, and the second is twice as deep (thus twice as wide) to clearly mark the position of the shallower indent before moving to the next position. Figure 2a is a bright-field TEM micrograph showing the defect microstructure of the entire irradiated region of an Fe + low N alloy, taken with g = {330} such that the dislocation loops appear dark. Figure 2b is a plot of the actual change in hardness, AH, as a function of depth below the original irradiated surface at the same scale, and for the same alloy shown in figure 2a.
Correlative TOF-SIMS and fluorescence microscopy analyses of surfaces used to control mammalian cell function
Authors: Healy K.E. , Thomas C.H. , Rezania A. , McKeown P.J. , McFarland C. D. , Steele J.G.
Published: June 2020

A common theme in engineering surfaces for biomedical materials and devices is the control of cell behavior at the material-tissue interface. Multiple analytical techniques are required to fully characterize a material surface both prior to and after exposure to the biological environment. In addition, a full cadre of microscopy techniques are essential for understanding cell behavior to these surface engineered materials. At the heart of understanding the mechanisms that control cell function on solid materials is the adsorption of serum proteins, which ultimately dictates how a cell responds to a material. A great deal of complexity is introduced into the system by adsorbed proteins, since there are over 200 proteins in human blood, and that post adsorption changes in conformation could lead to altered function. Until recently it has been extremely difficult to correlate cell behavior with the initial surface chemistry of a material and the type of protein adsorbed to the surface.
Nuclear coiled bodies and the nucleolus
Authors: Brasch K. , Williams J. , Gallo D. , Lee T. , Ochs R. L.
Published: June 2020

Though first described in 1903 by Ramon-y-Cajal as silver-staining “accessory bodies” to nucleoli, nuclear bodies were subsequently rediscovered by electron microscopy about 30 years ago. Nuclear bodies are ubiquitous, but seem most abundant in hyperactive and malignant cells. The best studied type of nuclear body is the coiled body (CB), so termed due to characteristic morphology and content of a unique protein, p80-coilin (Fig.1). While no specific functions have as yet been assigned to CBs, they contain spliceosome snRNAs and proteins, and also the nucleolar protein fibrillarin. In addition, there is mounting evidence that CBs arise from or are generated near the nucleolus and then migrate into the nucleoplasm. This suggests that as yet undefined links may exist, between nucleolar pre-rRNA processing events and the spliceosome-associated Sm proteins in CBs.

We are examining CB and nucleolar changes in three diverse model systems: (1) estrogen stimulated chick liver, (2) normal and neoplastic cells, and (3) polyploid mouse liver.
Dynamics of calcium control in local compartments
Authors: Guthrie P. B.
Published: June 2020

Neurons undergo a number of morphological changes during the establishment, maintenance, and restructuring of neuronal circuits. During these morphological transformations, different portions of a single neuron may simultaneously exhibit markedly different behaviors. Calcium has been strongly implicated as a regulator of morphological plasticity. We have investigated the extent of autonomous calcium regulation within plastic structures during various stages of development and remodelling. We have found that these plastic structures have highly compartmentalized calcium regulation.

During neurite pathfinding, growth cones encounter many different guidance cues. We have found that the autonomous calcium regulation capabilities of specific compartments can also be influenced by intercellular interactions. In the developing grasshopper limb, the pioneer neurons extend processes form transient electrical connections appropriate guidepost cells (Fig 1). These electrical connections allow the guidepost cells to locally reduce calcium concentrations within specific regions of the pioneer neurite, creating local regions of autonomous calcium regulation.

We have used calcium ionophores to demonstrate the existence of local sub-cortical calcium compartments, and at least one mechanism of their maintenance.
Organization of intracellular calcium regulatory proteins in the neuronal endoplasmic reticulum
Authors: Martone Maryann E. , Edelman Victoria M. , Alba Saul A. , Deerinck Thomas J. , Ellisman Mark H.
Published: June 2020

The smooth endoplasmic reticulum (SER) has been established as an intracellular calcium storage site in neurons. Although the SER appears to form a continuous membrane system within neurons, immunolocalization studies suggest that calcium regulatory proteins are not evenly distributed within the SER but are selectively concentrated or excluded from certain domains. The subcompartmenalization of the SER has been clearly demonstrated in Purkinje neurons where two proteins involved in the release of calcium from intracellular stores, the IP3 and ryanodine receptor, were differentially localized within dendrites. Both proteins were found associated with the SER in cell bodies and dendrites of chick Purkinje neurons but only labeling for the IP3 receptor was found within dendritic spines. A similar differential localization was described in Purkinje cell dendrites for the SER Ca++ATPase and calsequestrin, a lumenal calcium binding protein. The Ca++ATPase was found throughout dendrites and dendritic spines while calsequestrin was restricted to membranous profiles within the dendritic shaft.
In vitro differentiation in a human cortical neuronal cell line (HCN-1A): Ultrastructure and immunocytochemistry
Authors: Moses R. L. , Haycock J. W.
Published: June 2020

The study of neuronal differentiation would be facilitated by the availability of a homogeneous neuronal cell line capable of differentiation. Ronnett, et al. have cloned a cell line (HCN-1A) from the cerebral cortex of a megalencephalic patient. These cells undergo process formation after treatment with nerve growth factor and cAMP and, after differentiation, express numerous neuronal markers. Subsequent to treatment, the normally epithelioid HCN-1A cells became highly branched, often within the first two hours (FIG. 1A), creating a meshwork of closely associated processes with several orders of branching (FIG 1B). Not all cells remained in the differentiated state, resulting in phenotypically mixed cultures (FIG. 1A). Light microscopy of treated cells suggested that process formation occurred via cytoplasmic retraction rather than neurite outgrowth. In situ TEM analysis revealed that the cell bodies (FIG. 2A) of treated and untreated cells both possessed a number of neuronal characteristics including large euchromatic nuclei with prominent nucleoli, numerous profiles of rough endoplasmic reticulum (FIG. 2B), and prominent Golgi lamellae (FIG. 2C). Processes (FIG. 2D) were not ultrastructurally identifiable as axonal versus dendritic. Longitudinally arranged microtubules and intermediate filaments were both present in the majority of processes.
Otoconia perfect/imperfect crystals
Authors: Fermin César D. , Martin Dale
Published: June 2020

Otoconia of higher vertebrates are interesting biological crystals that display the diffraction patterns of perfect crystals (e.g., calcite for birds and mammal) when intact, but fail to produce a regular crystallographic pattern when fixed. Image processing of the fixed crystal matrix, which resembles the organic templates of teeth and bone, failed to clarify a paradox of biomineralization described by Mann. Recently, we suggested that inner ear otoconia crystals contain growth plates that run in different directions, and that the arrangement of the plates may contribute to the turning angles seen at the hexagonal faces of the crystals.

Using image processing algorithms described earlier, and Fourier Transform function (2FFT) of BioScan Optimas®, we evaluated the patterns in the packing of the otoconia fibrils of newly hatched chicks (Gallus domesticus) inner ears. Animals were fixed in situ by perfusion of 1% phosphotungstic acid (PTA) at room temperature through the left ventricle, after intraperitoneal Nembutal (35mg/Kg) deep anesthesia. Negatives were made with a Hitachi H-7100 TEM at 50K-400K magnifications. The negatives were then placed on a light box, where images were filtered and transferred to a 35 mm camera as described.
Geochemical applications of synchrotron x-ray microspectroscopy
Authors: Rivers M. L. , Sutton S. R. , Bajt S. , Delaney J. S.
Published: June 2020

The synchrotron x-ray fluorescence microprobe at bending magnet beamline X-26A at the National Synchrotron Light Source has been used for a number of years for geochemical trace element microanalysis using collimated white radiation. More recently an incident beam monochromator and 8:1 focusing mirror have been added. These optics permit the formation of a small (30-100 micron) intense beam of monochromatic radiation suitable for use in x-ray absorption spectroscopy, including both near edge (XANES) and extended fine structure (EXAFS) techniques. This system has been used to study a number of problems in the earth and environmental sciences, including: oxidation state of uranium in contaminated waste sites; oxidation state of Cr in olivine inclusions in diamonds from the Earth's mantle; oxidation state of Mn in wheat roots infected with the take-all disease. We report here some recent results obtained on studies of the oxidation state of Fe in cosmochemical systems.

Oxygen fugacity is one of the most important parameters in determining the cosmochemical and geochemical evolution of a system.