bluuebunny/crossref_metadata_2025 huggingface dataset:
The Microstructure of Oxidized Consolidated Nanocrystalline NiAl
Authors: Chen T , Hampikian JM
Published: July 2020

The intermetallic, stoichiometric compound NiAl is a candidate for aggressive environments such as those encountered in aircraft engines due to its excellent oxidation resistance and high melting point. However, NiAl is limited in application by its insufficient ambient temperature ductility. This may potentially be overcome via synthesis of NiAl with a nanocrystalline microstructure. Since microstructure can also affect oxidation resistance [1], the focus of this investigation is to examine the oxidation characteristics of nanocrystalline NiAl.

Nanophased NiAl specimens were prepared by mechanical alloying followed by shock wave consolidation, which yielded consolidated material with an average grain size of 11 ±8 nm [2]. Thermogravimetric analysis (TGA) of nanosized NiAl was used to characterize the material's oxidation resistance, and performed for three different times (0.5 h, 5 h, 48 h) in compressed air at 1373 K. It was found that the thickness of the scale increased with increasing oxidation time, in accordance with parabolic oxidation kinetics.
Structure Characterization of Colossal Magnetoresistive Oxides
Authors: Berta Y , Studebaker DB , Todd M , Baum TH , Wang ZL
Published: July 2020

Magnetoresistance describes a phenomenon in which the electrical resistance of the material depends strongly on an externally applied magnetic field. Colossal magnetoresistance (CMR) was observed in a new class of oxides, (La,A)MnO3 (A = Ca, Sr, or Ba), and the Magnetoresistance ratio of ΔR/R(H)> -100,000% has been reported in epitaxially grown La0.67Ca0.33MnO3 films. The new types of intrinsic magnetoresistive oxides offer exciting possibilities for improved magnetic sensors, magnetoresistive read heads, and magnetoresistive random access memory (MRAM).

The CMR magnetic oxides have a perovskite-type crystal structure with ferromagnetic ordering in the a-b planes and antiferromagnetic ordering along the c-axis (Figure 1). The ferromagnetically ordered Mn-O layers of the a-b planes are isolated by a non-magnetic La(A)-O monolayer. This spin-coupling structure is intrinsic. La1-xAxMn03 compounds having the extreme values x = 0, 1 are neither ferromagnetic nor good electrical conductors; they are semiconductors. Only compounds with intermediate values of x are ferromagnetic,
Microstructure of PbTi03/SrTi03 Superlattice Grown by MBE
Authors: W. Tian  , J. C. Jiang  , X. Q. Pan  , C.D. Theis  , D.G. Schlom
Published: July 2020

Ferroelectric superlattices have been actively and intensively studied in recent years for their great scientific and technological interest. Superlattice containing Pb-based ferroelectric layers are important among ferroelectric superlattice systems, however, it is difficult to grow such superlattice due to the high volatility of Pb. Recently, great progress has been made in fabricating superlattice structure of PbZrO3/PbTiO3 by multi-ion-beam sputtering’ and molecular beam epitaxy (MBE). In this paper, we report the microstructural investigations of PbTiO3/SrTiO3 superlattice films, which were epitaxially grown on the SrTi03 substrate by MBE, using transmission electron microscopy (TEM).

[(PbTiO3)l0/(SrTiO3)l0]15 superlattice films were stacked on (100) SrTiO3 substrate alternately by MBE. Before growing the superlattice structure, a baffle layer including the 1000Å La-doped SrTi03 and the subsequent 500 Å PbTiO3 thin films was grown on the substrate. Above the PbTi03/SrTi03 superlattices, another PbTi03 thin film (1000 Å) was grown. Cross-section TEM specimens were prepared by standard methods.
Domain Structure of Epitaxial SrRuO3 Thin Films on (001) LaA1O3
Authors: Jiang J C , Pan XQ , Gan Q , Eom C B
Published: July 2020

Epitaxial thin film of SrRuO3 is very useful in device applications, due to its important electrical and magnetic properties. For example, (Pb,Zr)TiO3 ferroelectric capacitors with SrRuO3 thin film electrodes exhibit superior fatigue and leakage characteristics. Epitaxial SrRuO3 thin films grown on different substrates, such as on (001) SrTiO3 and (001) LaA1O3, have different magnetic properties, owing to the different microstructures in the film. Microstructures in epitaxial SrRuO3 thin films grown on (001) SrTiO3 have been studied in our previously work. In this paper, microstructure of epitaxial SrRu03 thin films grown on (001) LaA103 is reported.

SrRuO3 thin films on (001) LaA1O3 were deposited by 90° off-axis sputtering. For cross-section TEM studies the SrRuO3/LaA1O3 heterostructural samples were cut along the [100] direction of LaA103. The cut slides were glued face-to-face by joining the SrRu03 surfaces. Plan-view and cross-section TEM specimens were prepared by mechanical grinding, polishing and dimpling, followed by Ar-ion milling.
Electron Microscopy and Microanalysis of Metal Phases in Meteorites
Authors: Williams D B , Goldstein J I
Published: July 2020

Meteorites are remnants of the primordial material from which the solar system condensed. Most meteorites originated in the asteroid belt between Mars and Jupiter and fell to earth when their orbits were disturbed by collisions. Metal phases are present in all types of meteorites and are alloys of Fe and Ni containing S and P. The study of metal meteorites has yielded valuable information about the early thermal history of the solar system, since their heat treatment has been preserved in the microstructure and microchemistry of the meteorites and can be discerned by electron microscopy and microanalysis. A full understanding of the structure and chemistry of meteorites requires detailed knowledge of the Fe-Ni, Fe-Ni-S and Fe-Ni-P phase diagrams and determination of these diagrams has been carried out over more than three decades of electron-beam analysis by the authors.
Electron Microscopy in the Real Semiconductor Processing World
Authors: Anderson Ron
Published: July 2020

Introduction: The “real” semiconductor processing world consists of the universe of industrial and government semiconductor laboratories and manufacturing facilities plus a small number of university departments with heavy funding from industry—or a consortium of the above. We are bombarded with reports of how the drive toward faster, denser, lower power consuming and more reliable semiconductor products will accelerate with time. The smallest features of today's devices have already shrunk from view in visible light microscopes! Electron microscopy studies of semiconductor device materials, interconnects, insulators, and device packaging materials play a vital role in these extraordinarily “high tech” and financially important arenas.

Shifting semiconductor analytical paradigms: For the most part, the analytical paradigm has shifted: from visible light microscopy to SEM analysis, at low magnification and high accelerating voltage… to much more sophisticated SEM analysis, at high resolution and/or low accelerating voltages… to the many variations of TEM available today… and to new types of scanning probe microscopy.
Transmission Electron Microscopy Studies of Tin Oxide Thin Films Grown on the Sapphire Substrate
Authors: Fu L , Pan X
Published: July 2020

Tin dioxide (SnO2) with rutile type structure exhibits unique electronic and optical properties. In applications of this material as gas sensors, a film-type of SnO2 provides high ratio of surface area to volume and lead to high sensitivity and fast responses. It has been found that substrate material, deposition conditions, and annealing procedure may directly control the microstructure of thin films, hence control gas-sensing properties. In this paper, we present transmission electron microscopy (TEM) studies of the microstructure and crystal defects of tin oxide thin films on sapphire substrate with subsequent annealing at high temperatures.

Tin oxide thin films were deposited on the surface of sapphire by e-beam evaporation of high purity SnO2 (99.999%) at 350°C followed by annealing in air at 600°C - 700°C. Microstructures of the films were characterized by X-ray diffraction (XRD) and TEM techniques.
Microstructural Characterization of GaN on (0001) Sapphire
Authors: Mao Zhigang , Johnson Matthew T , Barry Carter C
Published: July 2020

III-V nitrides grown on (0001) sapphire substrates often contain a very high density of defects; this high density is usually attributed to the poor lattice and thermal match between the nitride and the sapphire [1]. There is thus a need to understand the defect structures in these materials. In the present study, a microstructural characterization of MBE-grown GaN films on (0001) sapphire using various TEM techniques was performed to evaluate defect structures in this material. This paper will outline some of the different types of defects found in these films and suggest some possible mechanisms by which they were formed.
Focused Ion Beam (FIB) Milling Damage Formed During Tem Sample Preparation of Silicon
Authors: Susnitzky David W , Johnson Kevin D
Published: July 2020

The ongoing reduction of scale of semiconductor device structures places increasing demands on the sample preparation methods used for transmission electron microscopy (TEM). Much of the semiconductor industry's failure analysis and new process development effort requires specific transistor, metal or dielectric structures to be analyzed using TEM techniques. Focused ion beam (FIB) milling has emerged as a valuable technique for site-specific TEM sample preparation. FIB milling, typically with 25-50kV Ga+ ions, enables thin TEM samples to be prepared with submicron precision. However, Ga+ ion milling significantly modifies the surfaces of TEM samples by implantation and amorphization. Previous work using 90° milling angles has shown that Ga+ ion milling of Si produces a surface damage layer that is 280Å thick. This damage is problematical since the current generation of semiconductor devices requires TEM samples in the 500-1000Å thickness range.
Fluctuation Microscopy: A New Class of Microscopy Techniques for Probing Medium Range Order in Amorphous Materials
Authors: Gibson J M , Treacy M M J , Voyles P M
Published: July 2020

Amorphous materials are devoid of periodic long range order, but at the nearest-neighbor level they possess a high degree of short-range order. In amorphous tetrahedral semiconductors, such as Si and Ge, this short-range order arises because each atom attempts to satisfy four bonds arranged as a regular tetrahedron. It is the rotations about each bond, from the second-nearest-neighbor outwards, that result in the loss of long-range order. It is apparent from modeling of amorphous materials, that there is considerable flexibility as to how rapidly the medium-range-order diminishes. The continuous random network (CRN) is a hypothetical tetrahedral extended structure wherein the atoms possess full four-connected coordination, but have minimal medium-range order. However, real amorphous materials infrequently exhibit true CRN-like topologies. Traditionally, diffraction has been used to study short- and medium-range order in amorphous materials. Assuming kinematical scattering, and that every atom has a similar environment, a radial distribution function (RDF) can be extracted which is sensitive only to the averaged atom pair-correlations out to ∼1 nm.
Extended Electron Loss Fine Structure Analysis of Silicon-K Edges Using an Imaging Filter
Authors: Former J A , Buck E C , Strachan D M , Hess N J
Published: July 2020

The recent availability of Gatan's imaging filter (GIF) has improved detail in electron energy loss spectroscopy (EELS) owing to the greatly reduced channel-to-channel gain variation. In particular, extended electron loss fine structure (EXELFS) can be rapidly obtained with relatively low beam exposures to the sample. Previous studies using EXELFS (of which there are relatively few) have been limited to rather beam-insensitive samples and have required extensive effort to correct detector gain variation. [1,2] We will discuss here some of our ongoing work on use of EXELFS as a complement to other analytical microscopy techniques to study changes in materials caused by radiation damage (ion and alpha) and transmutation. Using an imaging filter as a spectrometer, we have obtained EXELFS from a rather beam-sensitive alkali aluminosilicate, pollucite (CsAlSi2O6).
Porous Spherical Particles of ZnS Nanocrystallites
Authors: Arnold M , Wang ZL , Scholz SM , Vacassy R , Stadelmann P
Published: July 2020

ZnS particles consisting of nanocrystallites have potential applications as light emitters for displays. The possibility of encapsulating chemicals into porous particles and the temperature-controlled release of them could also be of interest in chemical engineering. The objective of this study is to determine the size distribution, shape and morphology of ZnS particles synthesized under different conditions, since they strongly influence the optical and electric properties.

The ZnS powders were prepared by precipitation from homogeneous aqueous solutions, following a procedure described elsewhere1-3 The initial solution consists of a zincsalt precursor dissolved in ultrapure water under acidic pH conditions. Adding Thioacetamide (TAA: CH3C(S)NH2) to this acidic solution at elevated temperature leads to the precipitation of ZnS nanocrystals. Four different types of zincsalt precursors were used to study their influence on the precipitation process and the final powder morphology: acetate (CH3COO), chloride (Cl-), trifluoromethanesulfonate (tFMS: CF3SO3  -) and dimethyldithiocarbamate (dMdTC: S4N2C6H12  -). The dried powder were subjected to 350°C and 700°C heat treatments, respectively.
Transmission Electron Microscopy Studies of Pd Encapsulation by Ceria-Zirconia Oxides
Authors: Jiang J C , Pan X Q , Graham G W , McCabe R W , Schwank J
Published: July 2020

High-temperature catalysts containing Pd supported on high-surface area ceria-zirconia are optimum materials for fuel economy when automotive engines operate under high speed or load conditions. A prerequisite for developing such thermally stable catalysts is to gain a good understanding of the thermal deactivation modes contributing to the aging and degradation of catalysts in harsh thermal environments. It was discovered by X-ray diffraction that upon hightemperature aging, Pd may sinter into large (about 10 nm diameter) particles and become encapsulated in the ceria-zirconia. To confirm this conclusion, a prototype high-temperature catalyst containing Pd supported on high-surface area ceria-zirconia, aged at temperature above 1100 °C is studied by transmission electron microscopy (TEM).

The ceria-zirconia supported Pd (0.25 wt%) catalyst was aged at 1105 °C and calcined at 700 °C for 2 h. For TEM sample preparations, first of all, a large-size aggregate with diameter about 1 mm was selected and sandwiched between by two pieces of silicon crystal.
Ordered Assembling of Size and Shape Selected Nanocrystals
Authors: Wang ZL
Published: July 2020

Nanoparticles and the physical and chemical functional specificity and selectivity they possess, suggest them as ideal building blocks for two- and three-dimensional cluster self-assembled superlattice structures, in which the particles behave as well-defined molecular matter and they are arranged with long-range translation and even orientation order [1]. Self-assembled arrays involve self-organization into monolayers, thin films, and superlattices of size-selected nanoclusters encapsulated in protective compact organic coating. The macroscopic properties of the nanocrystal superlattice (NCS) are determined not only by the properties of each individual particle but by the coupling/interaction between nanocrystals interconnected and isolated by a monolayer of thin organic molecules.

Periodic packing of nanocrystals is different from the 3-D packing of atoms. First, to an excellent approximation atoms are spherical, while nanoparticles can be faceted polyhedra, thus, the 3-D packing of particles can be critically affected by their shapes and sizes.
Self-Assembled and Micro-Patterned Mesoscopic Thin Films
Authors: Yao N , Trau M , Nakagawa N , A. Aksay I
Published: July 2020

Recently we have reported that the silica mesoscopic thin films can be formed at water/solid interface at room temperature. These self-assembled thin films are comprised of hexagonally packed nanotubules ((∼ 5 nm in diameter), with a percolating one-dimensional channel network that extends from one side of the film to the other. The solid substrate can affect strongly the overall alignment of assembly of surfactant micelle array. These films hold much promise for applications such as their use as orientated nanowires, sensor/actuator arrays and optoelectronic devices. Here, we report further electron microscopy studies of free standing thin film formed at air/water interface and patterned silica mesoscopic thin film formed with the guidance of micro-molding and electric field.

We employ tetraethoxy silane (TEOS), dissolved in acidic solution, as a silicate source and cetyltrimethyl ammonium chloride (CTAC) as the templating surfactant. Typical molar ratios are 1 TEOS : 1.2 CTAC : 9.2 HC1 : 1000 H2O.
Effects of Cation Promoters in Selective Catalyzation of N-Butane
Authors: Gai Pratibha L , Kourtakis Kostantinos
Published: July 2020

The role of cation promoters on the microstructure and stability of complex vanadyl pyrophosphate ((VO)2P2O7 or VPO) catalysts in the selective oxidation of n-butane to maleic anhydride (MA) is of major technological importance. Although several studies have been cited in the literature on the specific activity and MA selectivity of promoted VPO, no direct evidence for the formation of solid solutions with VPO has been reported. A better fundamental understanding of the location and the complex role of promoters in the catalyst-adsorbate interactions is therefore required. We have synthesized novel promoted catalyst powders with several cations including iron and antimony, as well as with ceria. We have probed their nanostructures in n-butane directly (in-situ) at the atomic level using environmental-HREM (EHREM) and their composition by high spatial resolution depth profiling using EDX (and STEM probe size) on the nanometer (nm) scale.
Selected Reflection Imaging of Nanostructured Materials
Authors: Liu Yi , Sellmyer David J
Published: July 2020

Nanostructured materials are finding increasing applications. In characterizing the nanostructured materials, we have developed a technique using a conventional TEM to characterize the nanostructure. The technique is named selected reflection imaging and could be used for measuring the grain size, measuring the volume fraction of a second phase in a dual phase material, measuring the texture and identifying the crystal structure in multiphase materials.

The technique is evolved from dark field imaging which is known to generate strong contrast. In conventional materials with a grain size larger than 1 μm, selected area diffraction pattern is from a single crystal. Dark field image could be formed by allowing one of the diffracted beam to go through the objective aperture. In nanostructured materials, however, the diffraction pattern becomes a ring pattern. Ordinary dark field image could be formed by allowing one of the spot in the ring to go through the aperture. However, only a limited number of grains are differentiated from the rest.
Core Level Shifts and Grain Boundary Cohesion
Authors: Muller D A
Published: July 2020

The role of core level shifts at metallic interfaces has often been ignored in electron energy loss spectroscopy (EELS) even though very small changes in bond length can lead to large core level shifts. However, the popular interpretation of core level shifts as measures of charge transfer is highly problematic. For instance, in binary alloys systems, the core level shifts can be the same sign for both atomic constituents[l]. The simple interpretation would require that both atomic species had lost or gained charge. Further, the signs of the core level shifts can be opposite to those expected from electronegativity arguments[2]. A core level shift (CLS) is still possible, even when no charge transfer occurs. As illustrated in Fig. 1, if the valence band width is increased, the position of the center of the valence band with respect to the Fermi energy will change (as the number of electrons remains unchanged).
Grain Boundary Dislocations and Stacking Defects in the 9R Phase at an Incoherent Twin Boundary in Copper
Authors: Medlin DL , Campbell GH , Barry Carter C
Published: July 2020

Experiment and modeling show that there is a general mode of grain boundary dissociation, common in low stacking fault energy FCC metals, that can be well understood in terms of the emission of arrays of stacking faults from the grain boundary plane. Most extensively studied of such dissociated interfaces are the Σ=3 incoherent twin boundaries. Numerous observations now exist of grain boundary dissociation at such interfaces showing a layer that is well described as a narrow, several nanometer wide slab of 9R stacked material. The 9R stacking sequence is equivalent to a close-packed stacking of FCC ﹛111﹜ planes with an intrinsic stacking fault inserted every three planes (i.e., a stacking sequence of ABC/BCA/CBA …). The width of the 9R layer is sensitive to the local stress state. Figure 1 shows results of an atomistic calculation simulating the effect of an applied shear strain parallel to the boundary.
Investigating Ca Segregated to a Grain Boundaries in MgO Using Multiple Scattering Analysis of Electron Energy Loss Spectra
Authors: Buban J P , Zaborac J , Moltaji H , Duscher G , Browning N D
Published: July 2020

Although grain boundaries typically account for only a small fraction of a material, they can have far reaching effects on the overall bulk scale properties. These effects are usually simply linked to the boundary having a different atomic arrangement to the bulk. A necessary first step in understanding the structure-property relationships is therefore a detailed determination of the boundary structure.

One means of obtaining detailed information on the structure of grain boundaries is through correlated Z-contrast imaging and electron energy loss spectroscopy (EELS). The Z-contrast image generates a map of the grain boundary which can be used to position the probe in defined locations for spectroscopy. In the case of oxides, a structural model of the metal atom positions can be determined directly from the image. Furthermore, using a simple bond-valence sum minimization routine, the oxygen atoms can be placed so that the structure contains atoms that have valences consistent with their expected formal valence state.
Profiling Pvp/Ps Homopolymer Interfaces Using Core-Loss Electron Energy-Loss Spectroscopy
Authors: Siangchaew K , Prayoonthong P , Libera M
Published: July 2020

Despite the physical and technological significance of polymer interfaces (1), they have not been extensively studied using techniques based on electron scattering. Spontak et al. (2) used digital analysis of bright-field images to study Os-stained polystyrene-polydiene block copolymer interfaces, but work in this area has been lacking largely because of difficulties with staining or uncertainties in the distribution of stain at the nanometer length scales relevant to interfaces. Studies of polymer interfaces are now largely done by neutron scattering (3) This research explores the application of spatially-resolved electron energyloss spectroscopy to measure compositional widths of polymer-polymer interfaces. These first studies focus on blends of poly(2-vinyl pyridine) (PVP) dispersed in poly(styrene) (PS) to establish the various electron-optical, electron-scattering, and specimen-dependent limitations (4) to resolving such an interfacial width. The uncompatibilized PS/PVP interface should be relatively sharp.

Blends of PS (M.W. 190,000) and PVP ( M.W. 200,000) were made using a 5wt% solution of polymer in THF with a PS/PVP ratio of 7/3. The solution was cast into methanol. The resulting solid precipitate was removed and annealed under vacuum at 120°C for 36 hours.
Quantitative Thickness Mapping of Dewetting Polymer Bilayers
Authors: Winesett D A , Ade H , Smith A P , Rafailovich M , Sokolov S , Slep D
Published: July 2020

The study of wetting and dewetting in thin polymer films has many implications for technological applications of polymer films, including dielectric films to control conductivity and colloidal paint systems. We have started to use a novel approach, Near Edge X-ray Absorption Fine Structure (NEXAFS) microscopy, to characterize dewetting in thin polymer films. We have investigated a polystyrene/brominated polystyrene (PS/PBrS) bilayer model system with the Scanning Transmission X-ray Microscope (STXM) at the National Synchrotron Light Source. Our method offers quantitative thickness mapping of all constituent components in these thin polymer films.

NEXAFS imaging of polymers has advantages over other forms of microscopy primarily because of its low damage, good chemical sensitivity, and the possibility for quantitative analysis. Figure 1 shows four micrographs of one PS/PBrS bilayer annealed for one week at 170 C.
Microscopical Procedures for Investigating Natural Textile Fibers
Authors: Boylston E K
Published: July 2020

Microscopical procedures for the evaluation of cotton textiles are important to the textile industry in evaluating mixed fiber blends, dyes and chemical finishes on fabrics. A new procedure for embedding cellulosic textiles has been developed for FT-IR microscopy whereby fibers are embedded in polystyrene. This polymer does not absorb in the same regions of the infrared spectrum as cellulose or traditional acrylate and epoxy resins that contain chemical groups in common with cellulose. Additionally, use of cross-sections mounted on a KBr disk (Fig. 1) has the advantage of better resolution (Fig. 2B) than grinding and pressing fibers in a KBr disk (Fig. 2A).

A new process for the evaluation of yarns has been developed. Approximately 2000 fibers before spinning, 50 yarn segments after spinning, or yarns removed from fabric after processing, can be encased in a tube, embedded in methacrylate plastic, quickly UV polymerized, and sectioned.
Uncoated Lvsem and Imaging Tof-Sims of Unfixed, Plunge Frozen, Freeze Dried, Fungal and Plant Material
Authors: Basgall E J , Winograd N
Published: July 2020

A Cryosorption Freeze Drying (CFD) system was evaluated for its effectiveness in preparing delicate biological materials for both low voltage-field emission scanning electron microscopy (LVFESEM) and imaging liquid metal (Ga) ion beam, static time-of-flight, secondary ion mass spectrometry (TOF-SIMS). The primary goals of these studies were to investigate the retention of both structural and chemical integrity using fresh cryoprepared biological material which had not been exposed to any chemical fixation and which would not be coated by any conductive material in order to obtain information from the native surfaces. Duplicate chemically fixed samples were processed for comparison. LV-FESEM (2-2.5kV) was used to assess the quality of the structural preservation of the freezing and freeze drying (FD) protocols. Imaging static TOF-SIMS was used to investigate the surface chemical compositions of the biological samples.
Comparison of Focused Ion Beam and Conventional Techniques on Tem Specimen Preparation of Metal-Ceramic Interfaces
Authors: Ramirez de Arellano López A , Chiou W-A , Faber K T
Published: July 2020

The results of TEM analyses of materials are critically dependent on the quality of the sample prepared. Although numerous techniques have been developed in the last two decades, differential thinning of inhomogeneous materials remains a serious problem. Recently, focused ion beam (FIB) technique has been introduced for cross-sectional sample preparation for TEM and SEM.

A novel system for depositing a fine-grain (∼ 200 nm) ceramic coating on a metal surface via a patent pending Small-Particle Plasma Spray (SPPS) technique has been developed at the Basic Industry Research Laboratory of Northwestern University. To understand the properties of the coated surface, the ceramic/metal interface and the microstructure of the ceramic coating must be investigated. This paper presents a comparison of the microstructure of an A12O3 coating on a mild steel substrate prepared using conventional and FEB techniques.
Designing an Electron Microscopy Facility: Step by Step Procedure
Authors: Murphy Judy A
Published: July 2020

Designing a microscopy facility can be completed in a step by step approach, whether it be a completely new facility or an existing one moving into expanded quarters. The procedure is independent of size. There are several major factors that must be taken into consideration. Check lists can be compiled by the microscopist. One should easily be able to start at any point along the way. The time and effort initially put into the design will pay for itself many times over in convenience and efficiency of the resulting laboratory.

Prior to drawing the actual design of the microscopy facility but critical to the design specifics, it is necessary to compile the following information: considerations for determining the size of the facility; general EM lab requirements which affect site determination; activity functional flow diagram; EM room requirements; a list of all the equipment to be placed in the EM facility and exact specifications for each; design features not associated with specific equipment; activities requiring space that are not specific to the equipment; general building requirements not specifically related to microscopy operations; and ergonomic and psychological factors.
Some Simple Quantitative Applications of Vascular Corrosion Casting
Authors: Hossler Dr Fred E
Published: July 2020

The primary use of vascular corrosion casting has been and likely will continue to be for describing the 3-dimensional anatomy of the microvasculature of organs and tissues. In these studies the assumption is made that the corrosion cast faithfully replicates the anatomy, dimensions, and distribution of the vasculature of these tissues and organs. If this assumption is accurate, and it appears to be nearly so in tissues where measurements have been made, it follows that the casts should lend themselves to quantitative measurements. I summarize here some simple quantitative measurements we have made from corrosion casts. For these studies, cannulas were inserted into arteries leading to the organ of interest in anesthetized animals, and the blood was removed by flushing with Ringer solution or buffered saline, at physiological temperatures and pressures. Resin consisting of Mercox (or Mercox diluted with methyl methacrylate monomer) and catalyst was infused via the same cannula until the onset of polymerization.
Cryo-Electron Microscopy and Image Reconstruction of PBCV-1, an ALGAL Virus with T=169, Icosahedral Lattice Symmetry
Authors: Yan X , Olson N H , Van Etten J L , Baker T S
Published: July 2020

Paramecium bursaria Chlorella virus l(PBCV-l), the type species of the Pycodnaviridae family, is a large, plaque-forming virus that replicates in certain unicellular, exsymbiotic, Chlorella-like green algae. The virion (∼lxl09 daltons) has a 330,740 base pair dsDNA genome encapsulated within an icosahedral shell of ∼190nm diameter. Purified virions contain more than 50 different proteins, which account for 65% of the total virion mass and range in size from 10 to more than 200kDa. Vp54, the major capsid protein (40% of total virion protein) is a myristilated glycoprotein but it is not phosphorylated. Three other proteins are located on the virus surface. Virions contain 5-10% lipid, located inside the glycoprotein shell, which is required for virus infectivity. Little is known about the detailed ultrastructure of PBCV-1 virions, though some information has been obtained with sectioned, negatively-stained, and metal-shadowed specimens. We have used cryo-electron microscopy and three-dimensional (3D) image reconstruction methods to examine the native morphology of PBCV-1.
On The Unique Structural Organization of the Saccharomyces Cerevisiae Pyruvate Dehydrogenase Complex
Authors: Stoops James K , Zhou Z Hong , Schroeter John P , Kolodziej Steven J , Cheng R Holland , Baker Timothy S , McCarthy Diane L B , Yazdi Mohammed A , Maeng Cheol-Young , Reed Lester J
Published: July 2020

Dihydrohpoamide acetyl transferase (E2), a catalytic and structural component of a multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate, forms the central core to which the other components are bound. We have utilized protein engineering and 3-D electron microscopy to study the structural organization of the largest multienzyme complex known (Mr ∼ 107). The structures of the truncated 60-mer core (tE2) and complexes of the tE2 associated with a binding protein (BP), and the BP associated with its dihydrohpoamide dehydrogenase (BP'E3) and the intact E2 associated with BP and the pyruvate dehydrogenase (E1) were determined (Figs. 1 and 2). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges.

Previous studies have given rise to the generally accepted belief that BP and BP'E3 components are bound on the outside of the E2 scaffold and that E1 is similarly bound to the core in variable positions by flexible tethers.
The Ribosome - Three-Dimensional Structure and Ligand-Binding Studies
Authors: Frank J , Penczek P , Malhotra A , Gabashvili I , Grassucci R , Heagle A , Srivastava S , Burkhardt N , Jünemann R , Nierhaus KH , Agrawal RK
Published: July 2020

To date, cryo-electron microscopy has become the most successful technique for exploring the structure of the ribosome and for studying binding positions of its various ligands, with the resolution slowly extending toward 10 Å. Obstacles in the attempts to improve resolution are the limited stability and coherence of the electron microscope, the statistics of data collection, and the conformational heterogeneity of the specimen. The last factor in this list proved to be the reason why it has been difficult to go past 18-20 Å with many specimens despite the use of state-of-the-art electron microscopes and inclusion of tens of thousand of projections.

A breakthrough has been achieved with a protein synthesis initiation-like complex in which mRNA and fMet-tRNA is bound to the E. coli ribosome. The high occupancy and extraordinary conformational homogeneity of this specimen has enabled us to reach a resolution of 15 Å.
Molecular Architecture of Ribonucleoprotein Vaults
Authors: Stewart P L , Rome L H , Kong L B
Published: July 2020

Vaults are ribonucleoprotein complexes highly conserved between species as diverse as Dictyostelium, rat, and human. Thousands of vault particles exist per cell in various tissues including liver, lung, kidney and spleen. These complexes have been found in the cytoplasm as well as associated with the nuclear membrane. Vaults have a large barrel-like middle section with a protruding cap on each end. These structures have an average mass of 12.9 ± 1 MDa, as measured by STEM, and are composed of 3 proteins, p210, pi92 and pi04, as well as an RNA strand of approximately 90 bases. The major vault protein, pl04, constitutes the majority of the particle mass, existing in 96 copies per structure. The major vault protein was recently recognized as the lung-resistance related protein (LRP), a known multidrug resistance gene that is over-expressed in certain cancer cells, including ovarian and myeloid leukemia cells.

A three-dimensional structure of the vault complex was generated using the QVIEW and IMAGIC-5 software packages (Fig. 1).
Macromolecular Assemblies Designed for Controlled Proteolysis
Authors: Walz J , Koster AJ , Tamura T , Baumeister W
Published: July 2020

Since cellular structures are rebuilt continually, protein degradation is essential for the maintenance of homeostasis. Misfolded proteins ensuing from genetic defects or environmental stress, are prone to aggregation; chaperones and proteases cooperate in minimizing such unproductive interactions. Last, but not least, protein degradation serves to terminate the lifespan of many regulatory proteins at distinct times and thus features as a key regulatory element itself. Proteins destined for degradation must be recognized and selected within the crowded environment of the cell. The stratagem of self-compartmentalization is key to controlling cellular proteolysis (1).

In recent years, a number of multisubunit proteolytic complexes have been described which possess large internal cavities or nano-compartments. This allows them to confine the proteolytic action to their interior; access to these inner compartments is usually restricted to the unfolded proteins. This, in turn, makes it necessary for these proteases to interact - either in a transient or in a permanent manner
Formation of Proteasome-PA700 Complexes Directly Correlates with Activation of Peptidase Activity
Authors: Gogol E , Adams G , Crotchett B , Slaughter C , DeMartino G
Published: July 2020

The eukaryotic 20S proteasome (700 kDa) serves as the core complex for the breakdown of most cytosolic proteins by the ubiquitin-dependent pathway. This cylinder-shaped proteolytic assembly (17 nm long, 11 nm diameter) consists of four stacked heptameric rings that help to form a central cavity where degradation occurs. The catalytic sites are located on the cavity surface of the two middle (3 rings while the outer a rings serve as docking sites for various regulatory complexes.

PA700 (700 kDa, 20 protein subunits) is the major regulatory complex of the proteasome, forming a “cap” that attaches to either or both proteasome ends. The resulting superassembly structurally resembles the larger, more active 26S proteasome and exhibits both a higher intrinsic peptidase activity as well as a wider range of substrate specificity, including ubiquitin-tagged proteins. A second regulatory complex, the modulator (300 kDa), further stimulates the effects of PA700 at subsaturating concentrations of the activator with no detectable effect on the proteasome in the absence of PA700.
Proteolytic Control of Bacteriophage HK97 Capsid Maturation.
Authors: Duda Robert L , Conway James F , Cheng Naiqian , Steven Alasdair C , Hendrix Roger W
Published: July 2020

HK97 is a tailed temperate bacteriophage of E. coli that builds an icosahedral capsid using steps that include regulated assembly, proteolysis, radical conformational changes and the formation of novel covalent bonds (Fig. 1). This pathway is being exploited as a model system to explore how the formation of multiprotein complexes can be regulated by each of these mechanisms. We have identified and purified at least four intermediates (Prohead I, Prohead II, Head I and Head II) and examined them by cryo-electron microscopy and three dimensional reconstruction procedures (Fig. 2). Comparison of particle reconstructions at resolution of about 25 - 30 A have lead to major insights into the causes and purposes of the regulated changes that we have also characterized biochemically and genetically.

Prohead I consists of 420 copies of the 42 kDa gp5 capsid protein arranged as 72 blister-shaped morphological capsomers in a thick walled hollow T=7 icosahedral particle with a diameter of -470 Å.
Dual-Labeled Probes for Fluorescence and Electron Microscopy
Authors: Powell Richard D , Halsey Carol M R , Gutierrez Edmund , Hainfeld James F , Furuya Frederic R
Published: July 2020

Fluorescein and the 1.4 nm Nanogold cluster have been separately attached to polyclonal antibody Fab’ fragments to generate combined fluorescent and gold secondary antibody probes (1, 2), and in comparisons with Nanogold, colloidal gold and fluorescein-labeled secondary antibodies, neither label was found to be significantly compromised by the presence of the other. The proximity to a gold label with which a fluorophore retains sufficient fluorescence emission intensity to be useful is limited by non-radiative fluorescence resonance energy transfer (FRET) (3). This is a function of the degree of overlap of the fluorophore emission spectrum with the metal particle absorbtion spectrum, and of the separation of the metal particle and the fluorophore (4); for an effective dual-labeled probe, separation should be greater than the Forster distance (3), at which 50 % of excited-state decay occurs by FRET.
Correlative Instrumental Neutron Activation Analysis, LM, And TEM to Track in Vivo Distribution of Colloidal Gold Spheres in BALB/C Mice
Authors: Hillyer JF , Albrecht RM
Published: July 2020

Colloidal gold is routinely used conjugated to ligands or antibodies as a label for the detection of cell structures by transmission (TEM) and scanning (SEM) electron microscopy. Silver enhancement, a procedure that amplifies the size of gold spheres, is used for light microscopic (LM) observation of colloidal gold. These techniques are valuable in localization studies but it is difficult to quantitate the level of labeling in larger volumes of tissue solely by microscopic means.

Instrumental Neutron Activation Analysis (INAA) can be used to provide a quantitative index of gold accumulation at the organ and tissue levels. Given that gold is not naturally found in the body in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be determined at the organ and tissue levels in whole animal systems.
Properties and Applications of EGFP, Enhanced Color Variants of GFP, and Unstable Derivatives of GFP
Authors: Kitts PA , Li X , Piston DW , Chervenak R , Kain SR
Published: July 2020

The green fluorescent protein (GFP) has great potential as a tool for biologists because it can be used as an in vivo real time reporter of protein localization and gene expression in a variety of experimental systems. Wild type GFP, however, has several undesirable properties including low brightness, a significant lag in the development of fluorescence, complex photoisomerization, inefficient protein folding at 37°C, and poor expression in several species. To improve upon these qualities, we have combined an ultra-bright variant of GFP, GFPmutl, with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yields an enhanced variant (EGFP) (Table 1) that greatly increases the sensitivity of this reporter.

The natural green emission of GFP can conveniently be monitored by optics designed to detect fluorescein. There are, however, many potential applications for GFP that require additional emission colors.
GFP Fusions for Fluorescence Detection of Ca2+ and Ca2+-Calmodulin in Living Cells
Authors: Persechini Anthony
Published: July 2020

We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore.

We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).
Molecular pathology - Implications for Diagnosis and Pathogenesis of Infectious Diseases.
Authors: Zaki SR
Published: July 2020

Molecular pathology represents a distinctive new discipline in medicine with many potential applications that have been heretofore untapped. This discipline is introducing important changes in the concepts of disease causation and in the classification of diseases. Transfer of molecular genetic technology and knowledge to the pathology laboratory is giving the pathologist new and powerful tools to diagnose, classify, and monitor disease. Methodologies such as in situ hybridization (ISH), immunohistochemistry (IHC), and the polymerase chain reaction (PCR) will continue to allow more systematic and accurate classification of human diseases. IHC and ISH allow the identification and localization of cells containing specific antigens or nucleic acid sequences. The main advantage of these methods is their ability to localize targets and define their morphologic features in various tissues, allowing correlations with histopathologic changes.
Ultrastructural Characterization of Virus-Like Particles Produced by a Recombinant Baculovirus Expression Vector
Authors: Wasserman A J , Wang H , Clark G , Megill J R , Durham SK
Published: July 2020

Analysis of Hepatitis C virus (HCV) assembly and packaging, and the study of virus-cell interactions, have been impeded by the lack of a robust cell culture system and a viable small animal model. Recently, an alternative approach utilizing recombinant gene technology has been implemented. We describe a system in which specific HCV genes are cloned into a baculovirus to form a recombinant baculovirus expression vector that produces HCV proteins that self-assemble into enveloped virus-like particles (VLP). Four HCV structural genes were cloned into the baculovirus expression vector under the control of the polyhedrin promoter, and were then recombined with baculovirus DNA. A baculovirus with no HCV insert served as a negative control (wild-type). Ovary cells (Spodoptera frugiperda) were infected with either the recombinant or wild type baculovirus. Five-days post-infection, a group of cells were harvested, fixed in McDowell-Trumps solution, and routinely prepared for transmission electron microscopy (TEM).
Centrosome Proliferation in the Human Androgen-Responsive LNCaP and the Androgen-Independent DU145 Prostate Cancer Cell Lines
Authors: Schatten Heide , Ripple Maureen , Balczon Ron , Taylor Meghan , Crosser Michael
Published: July 2020

Cancer is a disease characterized by uncontrolled cell divisions in which the molecular controls for cytoskeletal regulation are bypassed. Cell division is governed by centrosomes, microtubule-organizing cell organelles which are crucial for the organization of the mitotic apparatus during mitosis and cell division. Because centrosome abnormalities are observed in the most common human cancers, we used immunofluorescence and transmission electron microscopy to determine centrosome organization in the human androgenresponsive prostate cancer cell line LNCaP and the androgen-independent prostate cancer cell line DU145. During interphase, centrosomes are located in close vicinity to the outer nuclear membrane, duplicate during S-phase, and become separated to the mitotic poles during the transition from interphase to mitosis. Centrosome regulation is based on a number of different factors which are only partly understood. Hormones play a role during developmental regulation of prostates which might trigger the activation of centrosome proteins and consequent cell divisions in order to ensure tissue growth.