bluuebunny/crossref_metadata_2025 huggingface dataset:
Photo-Electron Emission Microscopy of Semiconductor Surfaces
Authors: Nemanich RJ , English SL , Hartman JD , Yang W , Ade H , Davis RF
Published: July 2020

The technique of photo-electron emission microscopy (PEEM) is based on imaging of photo excited electrons from a surface. Typically ultra violet (UV) light above the work function of a metal will cause electrons to be emitted from a surface. Since photo excited electrons originate very near to the surface, they essentially reflect the electronic structure of the surface. These electrons may be accelerated and imaged, and the image will reflect the properties of the surface.

While the PEEM technique has been understood in a basic sense for many years, it has been limited by the lack of high intensity UV light sources. The most crucial part of the electron imaging system for PEEM, the objective lens, is essentially the same as for the sister technique of low energy electron microscopy (LEEM), and advances in electron optics capabilities have been exploited both for LEEM and for PEEM.
In Situ Electron Microscopy Studies of Surface Dynamical Processes
Authors: Tromp Ruud M
Published: July 2020

To obtain a full and detailed understanding of the spatiotemporal dynamics of surface processes such as epitaxial growth, strain relaxation, phase transformations and phase transitions, chemisorption and etching, in situ real-time observations have proven to be invaluable. The development of two experimental techniques, i.e. Low Energy Electron Microscopy (LEEM) typically operating at electron energies below 10 eV, and Ultra-High-Vacuum Transmission Electron Microscopy (UHV-TEM) at several 100 keV, has made such in situ studies routinely possible. In many cases, the videodata obtained from such experiments are amenable to detailed, quantitative analysis, yielding statistical, kinetic and thermodynamic information that cannot be obtained in any other way.

I will discuss recent experimental developments, including the design and construction of a new and improved LEEM instrument. Figure 1 shows a schematic diagram of this new machine. There are several features that distinguishes this design from most other LEEMs. One is the use of a 90 degree deflection magnetic prism array,
Tem Observations of Interfacial Defects in Mocvd GaAs on Single Crystal Ge Substrates
Authors: Jones KM , Al-Jassim MM , Olson JM
Published: July 2020

The growth of high quality GaAs layers on single crystal Ge substrates has attracted a great deal of interest in recent years. Currently under development at NREL is the GaAs/GalnP-on-Ge high efficiency multi-junction solar cell. Unlike other heteroepitaxial systems such as GaAs-on-Si, physical properties such as the lattice parameter and thermal expansion coefficient of the Ge and GaAs are much closer. Further, Ge has a bandgap of 0.7 eV that makes it a suitable bottom cell in a multi-junction stack. However, the growth of GaAs on Ge is not a straightforward process, and little is known about the Ge surface and the nucleation of GaAs on Ge in the MOCVD environment. In this study, TEM results show that a number microstructural defects are associated with the Ge/GaAs interface and the initial stages of growth.
Tem Investigation of Co-Si Thin Films on S1-xGex/Si
Authors: Myersa A F , Goeller P T , Steel E B , Boyanov B I , Sayers D E , Nemanich R J
Published: July 2020

Cobalt disilicide (CoSi2) is an attractive contact material for Si1-xGex devices due to its low resistivity, low Schottky barrier, and good thermal and chemical stability. Previous studies have shown that blanket Co films on Si0.80Ge0.20 react at annealing temperatures of 700 °C to form CoSi, CoSi2 and a Ge-rich Si1-xGex alloy; no Co-Ge phases were found. The Co preferentially reacts with the Si, which can lead to Ge segregation and island formation. To gain a better understanding of cobalt silicide formation, an electron microscopy investigation of Co/Si1-xGex/Si was performed.

Si1-xGex films (x ≤ 0.3) 64 nm to 260-nm thick were grown by molecular beam epitaxy at 550 °C on phosphorus-doped Si <001> substrates. Co films ranging in thickness from 5 nm to 25 nm were electron-beam evaporated (evaporator base pressure < 6.67 x 10-8 Pa) at room temperature on the Si1-xGex films.
Ge distribution in Gi80Ge20 Islands Grown in the High Mobility Regime.
Authors: Twesten RD , Floro JA , Chasonf E
Published: July 2020

Strained epitaxial films are often unstable to morphological transformations. The classic example of this phenomenon is the SiGe system. A nonplanar morphology results in non-uniform strains in the system (Figure 1). These strain fields can modify the local chemical potential of a uniform alloy driving Ge enrichment in the regions of low compression.

Recent work has shown that annealed films of SiGe on Si{001} exhibit both vertical and lateral segregation during the transition from a metastable planar film to an array of ﹛100﹜ directed island. Vertical segregation is evidenced by an enrichment of Ge in the last few monolayers of the film while lateral segregation is seen as an enhancement of this surface layer in the region near the island peaks. This segregation is due to surface diffusion as the bulk diffusion lengths are too short. The case of annealed, metastable films can be compared to growing films.
Kinetics of the C-Axis Aligned YBa2Cu3O7 Thick Film By a BaF2 Process
Authors: Wu Lijun , Zhu Yimei , Solovyov VF , Wiesmann H , Suenaga M
Published: July 2020

Over the last few years, considerable efforts have been made in developing fabrication processes for thick superconducting YBa2Cu3O7-δ film on a metallic substrate for electric power applications. Since these applications require the composite tape to be longer than 4-500m, any methods chosen need to be compatible with such length requirement. One possible approach is the so-called BaF2 process which involves deposition of a unreacted mixture of a BaF2-Y-Cu-O layer on a substrate by an electron beam evaporation method, followed by heat-treatment of the layer in a mixed-gas atmosphere of a low partial pressure O2, saturated H2O, and N2. Although it was shown previously that c-axis aligned films up to ∼ I m long can be produced under these conditions it is uncertain how thick the films can be made and this is of crucial importance as most applications require film thickness to be greater than ∼5μm to carry a large, dissipationless current.
Microstructure of YBCO/Co-PBCO/YBCO edge Josephson junctions
Authors: Huang Y , Moeckly BH , Merkle KL
Published: July 2020

In recent years, many efforts have been made to fabricate high-Tc superconductor/normal-metal/ superconductor (SNS) Josephson junctions, but no breakthrough has been made. The major difficulties are the poor reproducibility of the junction parameters due to the structural complexity and defects in the barrier layer. Some junctions, such as the one with Co-doped YBCO barrier, have relatively small spreads in properties. However, the value of their normal state resistance Rn is low, which limits their use for many applications. One approach to solve this “low-Rn” problem is to use materials of high resistivity for the barrier layer. PBCO is an insulator near the metal-insulator transition. Doping with Co drives it further into the insulating regime. The lattice constants of PBCO are close to that of YBCO, even closer when doped with Co. Therefore, Co-PBCO may form a high-resistivity barrier which matches with YBCO very well and hence is a potential candidate barrier material.
Graphitic Disks or Polygons? Faceting of Graphite Disks
Authors: Krishnan A , Dujardin E , Ebbesen TW , Treacy MMJ
Published: July 2020

The recent discovery of graphite cones [1,2] has raised some interesting questions about the nucleation and growth of curved graphitic structures. Here we report the structural peculiarities of one member of the ensemble, i.e. the flat graphite disks. Unlike graphite flakes seen in carbon soot which are irregularly shaped, the disks in this sample often show regular faceting which might give us additional insight into the growth mechanisms.

The samples were examined in a Hitachi H9000 NAR TEM. Samples were sonicated in high purity methanol and dispersed on a 300 mesh Cu grid coated with a 20 nm thick amorphous carbon layer. Typical disk diameters range from 0.5 to 3.0 μm, with thickness ranging from 10 nm to 50 nm.

A close examination reveals that the edges of the disks are faceted and typically have 12 sides. In most cases, the edges show a long facet alternating with a shorter facet.
A Study of Amorphous Chalcogenides by Electron Microscopy and Analysis
Authors: Fitzgerald A G , Mietzsch K
Published: July 2020

Amorphous chalcogenides films coated with certain metals are known to possess a remarkable sensitivity to radiation. Based on these effects they have found several important applications in the production of microcircuits, e.g. as resists in photo- and electron beam lithography. A variety of chalcogenides in combination with a range of metals has been extensively investigated with regard to potential applications in the fabrication of semiconductor devices.

The objectives of the present project are to extend knowledge of the behaviour of amorphous chalcogenides in contact with metals. This includes studying the metal diffusion process, understanding the film-electron beam interaction process and evaluating the potential of the metal lines formed by the electron beam in fabrication of photomasks for the production of higher densities of silicon microcircuits.
Structural Stability of Nanocrystalline NiAl
Authors: Chen T , Hampikian JM , Thadhani NN , Wang ZL
Published: July 2020

NiAl is an important high temperature structural material, with a high melting point (1640°C), low density and excellent high temperature oxidation resistance. The room temperature ductility of NiAl may potentially be improved with the use of nanocrystalline grain size. However, a key question concerning the application of nanostructured NiAl is about its structural stability at high temperature. The current study is thus focused on the investigation of the structural stability of nanocrystalline NiAl using in-situ transmission electron microscopy (TEM) and differential thermal analysis (DTA).

Nanocrystalline B2-NiAl was prepared by ball milling (24 hrs) from elemental Ni and Al powders. Subsequent consolidation into bulk form was performed using dynamic consolidation employing a 3-capsule plate-impact fixture at approximately 400 m/s [1-3]. Powder nanocrystalline NiAl was dispersed on a holey carbon film for TEM observation. TEM specimens of shock compacted bulk NiAl nanocrystals were prepared by cutting, polishing, dimpling and ion milling.
The Experimental Accuracy of Lattice Spacing Determination on Small Metal Particles in Commercial Catalysts
Authors: Tsen S-C Y , Crozier P A , Liu J
Published: July 2020

A description of the microstructure of heterogeneous catalysts is important in understanding the mechanisms for catalysis and may result in improvements in catalyst performance. Many commercial catalysts consist of small metal particles and/or metal oxide particles dispersed on a porous support. For bimetallic catalysts, the metals may be in the form of alloys, metal mixtures or completely phase separated. HREM has been demonstrated to be a powerful technique for determining the lattice spacing from small particles[l]. In many studies of catalysts it is necessary to prepare TEM samples by microtomy so that the particle location relative to the porous support is preserved. However, such samples show a reduction in particle visibility and signal-to-noise ratio of lattice fringes. In spite of these difficulties, we have been able to routinely use HREM to rapidly determine the relative distributions of metal and metal oxides in many systems because of the large difference in spacing between strong reflections from the metal and metal oxide (typically 20 %).
Microstructural Analysis of Disordered and Ordered Mesoporous Silica Films
Authors: Drewien C A , Lu Y , Brinker C J , Ganguli R , Anderson M T
Published: July 2020

Processing can be controlled to produce a family of mesoporous silica films with either disordered, lamellar, hexagonal, or cubic pore distributions[l]. These films, formed by surfactant-templated synthesis and exhibiting a unimodal pore size, promise potential use as inorganic membranes, catalysts, and optically-based sensors[l,2]. The mesoporous films can be formed from initially homogeneous silica sols by a continuous, surfactant-templated process, which relies upon solvent evaporation during the sol-gel dip-coating process. Films of 100-500 nm thick are formed within seconds in a continuous coating operation. The microstructure of the films is dependent upon the cationic surfactant concentration CTAB (CH3(CH2)15N+(CH3)3Br-) and the dip-coating rate. Several films, processed under differing conditions, were investigated by TEM to characterize pore size, structure, and orientation.

Figures 1 a & b show the plan view and cross-sectional microstructure of a 2-d hexagonal mesoporous silica film deposited on silicon; the sample was calcined at 400 °C for 3 hours in air. The images were obtained on a Philips CM30 TEM, operated at 300 kV.
Interfacial Solute-Atom Segregation: An Atomic-scale Ahenomenon with Implications for the Oxidation Kinetics of Pt-Al Alloys
Authors: Dickey EC , Alexander KB , Pint BA
Published: July 2020

Interfacial solute-atom segregation is known to have profound effects on macroscopic physical properties (e.g. electronic transport or fracture toughness) in interface-influenced or controlled materials. It has recently been shown that solute-atom segregation also can play an influential role in the dynamics of phase transformations, in particular the oxidation kinetics of alumina-forming alloys such as PtAl. By coupling analytical electron microscopy (AEM) with conventional macroscopic oxidation studies, we have demonstrated that oxidation kinetics are affected by atomic-scale segregation of dopant species to the grain boundaries and metal-oxide boundaries in the system. Furthermore, these studies illustrate the utility of AEM techniques in elucidating the atomic-scale aspects of macroscopic physical phenomena such as phase transformations.

Oxidation studies were carried out on PtAl alloys with and without small additions of Zr to the alloy. While the pure PtAl alloys exhibited oxide scale spallation and very fast oxidation rates, the Zrcontaining alloys maintained a well-adhered oxide scale and significantly lower oxidation rates.
The Measurement of Light Element Segregation Using EDS and EELS
Authors: Busby JT , Kenik EA , Was GS
Published: July 2020

Radiation-induced segregation (RIS) is the spatial redistribution of elements at defect sinks such as grain boundaries and free surfaces during irradiation. This phenomenon has been studied in a wide variety of alloys and has been linked to irradiation-assisted stress corrosion cracking (IASCC) of nuclear reactor core components. However, several recent studies have shown that Cr and Mo can be enriched to significant levels at grain boundaries prior to irradiation as a result of heat treatment. Segregation of this type may delay the onset of radiation-induced Cr depletion at the grain boundary, thus reducing IASCC susceptibility. Unfortunately, existing models of segregation phenomena do not correctly describe the physical processes and therefore are grossly inaccurate in predicting pre-existing segregation and subsequent redistribution during irradiation. Disagreement between existing models and measurement has been linked to potential interactions between the major alloying elements and lighter impurity elements such as S, P, and B.
Interface Characterization of Metallized CVD Diamond
Authors: Menon E S K , Saunders M , Dutta I
Published: July 2020

Progress towards the large scale integration of active devices within electronics packages has imposed stringent heat dissipation requirements necessitating the development of innovative materials solutions. One possibility being considered is the use of chemically vapor deposited diamond (CVDD) thin films as heat sinks. However, there are technological challenges which must be overcome before these materials become commercially viable. For example, the CVDD substrate must be metallized to provide interconnections between the various devices in the package. Conventional metallizations, such as Au, Cu or Al, display poor adhesion to the diamond causing problems associated with the reliability and stability of metallized diamond packages. A novel solution has been proposed involving the consecutive deposition of a thin layer of Cr and an electrically insulating layer of alumina on the diamond substrate such that the thermal conductivity of the treated substrate is not degraded significantly.
Hrem Characterization of Magnetic Tunnel Junctions and Discontinuous Multilayers
Authors: Smith David J , Ge Fuding , Piatt CL , Sankar S , Berkowitz AE
Published: July 2020

There is much current interest in the magnetotransport properties of systems consisting of two (or more) magnetic metal layers separated by thin insulating layers. Traditional magnetic tunnel junctions (MTJs) are comprised of simple ferromagnet-insulator-ferromagnet trilayer structures and the conductance depends on the relative alignments of the magnetizations in the two ferromagnets. In the case of discontinuous metal/insulator multilayers, negative magnetoresistance (MR) results from spindependent tunneling. In both types of systems, the tunneling phenomena are strongly influenced by the microstructure of the films, particularly the metal/insulator interfaces and the nature and uniformity of the thin oxide barrier layers. In this study, we have used cross-sectional HREM to characterize a variety of magnetic tunnel junctions and discontinuous multilayers.

The MTJs were prepared by rf and dc magnetron sputtering onto thermally oxidized (100) silicon wafers at room temperature. The magnetic layers consisted of thin films of Co, Fe and/or CoFe alloys with thicknesses ∼ 30-50nm, and the barriers included MgO, HfO2, CoO, SiO2 as well as Al2O3 (thicknesses in the range 2-10nm).
Quantitative Chemical Speciation of Multi-Phase Polymers Using Zone Plate x-ray Microscopy
Authors: Hitchcock AP , Urquhart SG , Ade H , Rightor EG , Lidy W
Published: July 2020

Phase segregation is important in determining the properties of many complex polymers, including polyurethanes. Achieving a better understanding of the links between formulation, chemical nature of segregated phases, and physical properties, has the potential to aid development of improved polymers. However, the sub-micron size of segregated features precludes detailed chemical analysis by most existing methods. Zone-plate based, scanning transmission X-ray microscopes (STXM) at NSLS and ALS provide quantitative chemical analysis (speciation) of segregated polymer phases at ∼50 nm spatial resolution. Image sequences acquire much more data with less radiation damage, than spot spectra. After alignment, they provide high quality near edge spectra, and thus quantitative analysis, at full spatial resolution.

Fig. 1 shows an image and spectra acquired with the NSLS STXM of a macro-phase segregated TDI polyurethane. Spectral decomposition using model polymer spectra is used to measure the local urea, urethane and polyether content.
The Morphology of Carboxylated Composite Latex and Latex Film
Authors: Shaffer O L , Sandor M W , El-Aasser M S
Published: July 2020

Carboxylated latex has become very important in the formation of polymer films. In order to study the film and its properties it is important to know the morphology of the latex that is forming the film. The latex for this study has been examined by transmission electron microscopy(TEM) using positive preferential stains such as ruthenium tetroxide (RuO4) and cesium hydroxide(CsOH); and uranyl acetate(UAc) as a negative stain.

The polybutyl acrylate(PBA)/ polymethylmethacrylate(PMMA) composite latex particles consist of a soft core phase and a hard second phase with varying amounts of acrylic acid in the core, the shell and in both core and shell. The latexes were examined before and after cleaning. The cleaning was necessary in order to remove the surfactant and any small particles that might have formed during the emulsion polymerization. Prior to cleaning, the small particles adhered to the surfaces of the particles making it difficult to determine the final morphology of the latex particles.
UV Absorption Microspectrophotometry and Histochemistry of Flax and Kenaf
Authors: Akin DE
Published: July 2020

Flax (Linum usitatissimum L.) and kenaf (Hibiscus cannabinus L.) are the sources of fibers used for textiles and other industrial applications. Both flax and kenaf produce fibers in the bast region (Fig. 1, 2) which must be separated from other tissues by retting. Although both flax and kenaf are bast fibers, their properties are vastly different. UV absorption microspectrophotometry and histochemistry elucidate their chemistry and structure related to enzymatic retting.

Aromatics such as lignins are produced by plants for protection and strength, but their presence inhibits microbial degradation, which is necessary in retting. Histochemical tests indicated variations in the site and type of aromatics within these two plants (1,2). In flax, acid phloroglucinol but not chlorine-sulfite gave positive reactions occasionally in fiber cell walls in the bast. The other cell walls in the bast did not contain aromatics by these tests, although aromatics occurred in the cuticle.
Microwave Processing of Cell Monolayers in Situ for Postembedding Immunocytochemistry with Retention of Ultrastructure and Antigenicity
Authors: Madden Victoria J
Published: July 2020

Increasingly, cells isolated from blood and body fluids and cells grown in culture are becoming the experimental models of choice for biological research. The demand for demonstrating biochemical processes morphologically is also becoming commonplace in the electron microscopy laboratory. Successful fixation, in situ embedment, and ultrathinsectioning of cell monolayers can be difficult to achieve for routine transmission electron microscopy. For postembedding immunocytochemistry, processing becomes more complex due to fixation constraints and the use of acrylic resins. The object of this paper is to present a reliable, rapid method for processing monolayers that preserves both the ultrastructure of the cells and antigenicity.

The equipment used for this procedure was a Pelco Model 3440 MAX laboratory microwave oven equipped with a temperature probe and a maximum power output of 800 watts. Using a neon bulb array, the oven cavity is calibrated to determine the microwave energy distribution.
Blind Deconvolution of Low and High Signal-to-noise 3-D Images of Fluorescent Subcellular Structures
Authors: Rajwa B , Czyz J , Dobrucki J
Published: July 2020

This report summarizes our experiences gained with deconvolving 3-d images of various types of biological samples - animal and plant cells; live and fixed. We compared raw, unmodified images with images processed with standard filtering methods and images improved by using deconvolution. The blind decovolution software package from AutoQuant, Inc. was used. All images were acquired using a BioRad MRC 1024 confocal microscope (attached to a Nikon Diaphot microscope), using fluorescence or reflected light. For thin (up to 25 μm) flat specimens a 60x PlanApo NA1.4 lens was used while for thick (25-200 μm) water containing samples a 40x Fluor NA1.15 water immersion lens was used.

In an attempt to test the usefulness and validity of deconvolution for various types of biological samples we adopted the following approaches:

1. collect images of structures that are known to be both fluorescent and reflective - compare the results of deconvolution of fluorescence images with reflected light images that are not deconvolved.
Patterning of Surface Chemistry and Topography for Biological Applications
Authors: Craighead H G , Davis R C , Foquet M , Isaacson M , James C , Turner S , Kam L , Turner J N , Shain W , Banker G
Published: July 2020

The technologies of nanofabrication as applied to inorganic materials and substrates are advanced and continue to develop. These sophisticated processes enable the formation of complex electronic, optical and mechanical devices with feature sizes down to tens of nanometers. Adaptation of these types of processes to surface chemical patterning and topographical patterning provides a new set of experimental tools for investigating biological systems and realizing sensors and devices that require the interaction of biological systems and fluids with inorganic materials and surfaces.

In this talk we discuss methods of pattering self assembled monolayers, proteins and antibodies on silicon and glass surfaces by lithography and microcontact printing. This is of utility in a variety of experiments in cell-surface interactions and in sensor devices. In certain types of devices the manipulation of fluids and sieving of molecules is a critical function such as in DNA sequencing6 by electrophoretic separation.
The Confocal Microscope: How to Choose it and Use it
Authors: Drazba JA
Published: July 2020

The decision to buy a confocal microscope presents the huge task of wading through designs and options from a number of excellent companies. While the principle of confocal imaging - rejection of out-of-focus light - is consistently achieved, each manufacturer does so with a unique design. There is strong competition among manufacturers to have the microscope with the best possible resolution and light transfer efficiency so that maximum specimen detail is detected with the least light damage. There are a number of excellent instruments currently available. In deciding among them consideration should be given to the following: type and number of users, types of imaging required, and your budget. If the microscope is going to be used by a single lab for a single purpose i.e. gfp expression in embryos, you should consider one of the less elaborate “personal confocals” with one laser, one or two detectors and a price tag under $150,000.
The Importance of Optimizing Operational Procedures and Calibrations in Materials Characterization Using an Analytical Electron Microscope
Authors: Echer CJ
Published: July 2020

When managing multi-disciplinary materials characterization facilities to help solve scientific materials problems, one needs to confidently operate the instrumentation knowing that the calibrations and operational procedures are as accurate as possible and optimized by periodic evaluation. An overview of the calibrations and some operational pitfalls will be reviewed for the Analytical Electron Microscope. I intend to show examples of various results produced over the years characterizing materials, including some recent data. A short chronological history of my career pertaining to image documentation will show examples from the old days to the present. I pride myself on having a top-notch laboratory so when scientists need relatively fast answers, one knows which direction to proceed and the equipment is capable of achieving the results. When scientific users come to an AEM laboratory, each individual brings a potpourri of materials problems to try and solve.
Corrosion Casting in the Reproduction of the Micro-Surface Topography of Fibrillar Collagen
Authors: Sims PA , Albrecht RM
Published: July 2020

The interaction of cells with biomaterials is influenced by a number of factors including the chemical composition, surface charge, hydrophobicity, porosity and texture of the biomaterial. Changes in texture alone have been shown to result in reduced platelet adhesion on ventricular assist devices, reduced scar tissue formation around implanted prostheses, and enhanced biocompatability of vascular stents. In vitro growth of certain cells requires extracellular matrix components to provide the right physical and chemical stimuli for adherence, growth and differentiation. What role the texture of these materials plays, and the mechanisms responsible for the effects of substrate texture on the behavior of cells are not well understood. Previously we have used microcorrosion casting technology to replicate lymph node and other vasculature. More recently, we have used corrosion casting to replicate of the subendothelial matrix exposed when cells are damaged due to the passage of air emboli through a vessel.
Microvascularization of the uterine tube in the rabbit (Oryctolagus cuniculus)
Authors: R. P. Chopard , I. Watanabe , C. I. Conegero
Published: July 2020

Several authors have been studying the microvascularization pattern of the ovary in different animals. To determine the microvascularization pattern using the corrosive-resin casting method, casts of the vascular network were prepared and examined by scanning electron microscopy. However, the same procedure has rarely been used to study the microvascularization pattern of the uterine tube itself. The microvascularization of the uterine tube has always been associated microvascularization studies of the ovary. This study analyses the microvascularization of the uterine tube, pointing out differences between the region of the tube itself and it's infundibulum. In this study we used uterine tubes from adult female rabbits. Anesthesia was carried out with Ketalar (5mg/kg body weight); after that saline solution and 2,5% glutaraldehyde were injected through the left ventricle. This procedure was followed by mercox 2B injection. After polymerization the samples were collected and immersed in warm water (60°C). Then samples underwent tissue digestion.
Formation of Proteasome-PA700 Complexes Directly Correlates with Activation of Peptidase Activity
Authors: Adams G , Crotchett B , Slaughter C , DeMartino G , Gogol E
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 β rings while the outer α 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.
Green Fluorescent Protein as a Non-Invasive Probe of Viscosity and pH in Cell Cytoplasm and Organelles
Authors: Verkman AS
Published: July 2020

Green fluorescent protein (GFP) was evaluated as a non-invasive probe to quantify the rheological properties and pH of intracellular aqueous compartments. Spectroscopic studies were done on several bacterially-expressed and purified GFP mutants. GFP-S65T was brightly fluorescent in solution (ƛex 492 nm, ƛem 509 nm) with lifetime 2.9 ns and rotational correlation time ( tc ) ∼20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 ± 2 ms (5 μm diameter spot) giving a diffusion coefficient of 8.7 x 10-7 cm2/s. The t1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein, GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), independent of spot diameter, and not affected by O2 or quenchers.
Efficient, Retro Viral-Mediated Expression Of GFP Fusion Proteins as Inhibitors of Intracellular Signal Transduction Pathways: A Case Study
Authors: Murphy TJ , Wang Xiaofei
Published: July 2020

A variety of extracellular signals, including hormones, neurotransmitters, growth factors and cytokines, are capable of activating vascular smooth muscle cells (VSMC). These can modulate the VSMC phenotype, which underlies the progression of vascular diseases. We study gene expression control responses in VSMC that are regulated by different classes of cell surface receptors. Taken as simpler model systems for the pleiotropic sensitivity of VSMC to extracellular stimuli, we focus on understanding the elements of intracellular signal transduction pathways involved in controlling gene expression. In particular, we are interested in identifying signaling elements that can be activated by multiple classes of cell surface receptors.

Previous studies had supported both a role of protein kinase A (PKA) activity and transcriptional induction of an unknown factor in control of ATI angiotensin receptor gene expression in VSMC (1). The mRNA from this gene is down-regulated by a variety of extracellular stimuli, and we hypothesized that stimulation of PKA by several classes of receptors might explain the apparently pleiotropic nature of ATI-receptor down regulation.
Optical Mesurements of Presynaptic Function: What Keeps Vesicle Traffic Going
Authors: Ryan Timothy A
Published: July 2020

The nervous system has evolved to make use of a variety of mechanisms that allow information to flow and be processed among a large collection of individual cells. The communication between individual brain cells occurs largely at chemical synapses. In these compartments, chemical messengers are packaged into small vesicles that fuse with the cell membrane upon stimulation, releasing neurotransmitter.. The average total number of synaptic vesicles in a typical central nervous system synapse is only a few hundred and as a result an efficient local recycling mechanism operates in order to replenish this pool during periods of even modest neuronal activity. Without this membrane recycling, synapses quickly become depleted of vesicles, and soon fail to communicate information between cells.

We make use of optical techniques to follow the trafficking of synaptic vesicles at synapses formed between hippocampal neurons grown in culture. Recycling synaptic vesicles can be readily labeled using the fluorescent amphipathic membrane dye FM 1-43.
Characterization of Endocytic Pathways by Quantitative Fluorescence Microscopy
Authors: Maxfield Frederick R , Ghosh Richik N , Mallet William G , Thwe Soe Thwe , Leopold Philip L , Cohen-Gould Leona , Mayor Satyajit , Mukherjee Sushmita , Zha Xiaohui , McGraw Timothy E
Published: July 2020

We have used light and electron microscopy to analyze endocytic trafficking pathways. In one set of studies, we have used fluorescently labeled antibodies to trace an endocytic pathway from the cell surface to the trans- Golgi network (TGN). Cells were transfected with a construct consisting of the transmembrane and cytoplasmic domains of TGN38 and the extracellular domain of Tac. TGN38 is predominantly in the TGN, but a small fraction is found on the cell surface. We used FITC-labeled anti-Tac monoclonal IgG to analyze the pathway from the surface to the TGN. We compared the distribution of internalized Tac-TGN38 to internalized transferrin. We found that most Tac-TGN38 enters the same early endosomes as transferrin. Furthermore, most Tac-TGN38 returns to the cell surface from the endocytic recycling compartment (ERC) at the same rate as transferrin. However, on each pass through the cell approximately 18% of Tac-TGN is retained, and this Tac-TGN38 is delivered to the TGN.
Fluorescent and Electron Microscopic Studies Suggest That Confronting Cisternae Arise from Nuclear Envelope in Mitotic Cells
Authors: Palisano J R , Thacker J L , Piromalli C S , Morrison A M , Tate J E
Published: July 2020

The origin and role of confronting cisternae (CC) have been clouded in mystery since their discovery in 1955. Early investigations suggested that CC arose from either nuclear envelope (NE) or by stacking of endoplasmic reticulum (ER) cisternae. Electron microscopic studies of mitotic HeLa cells suggest that CC are fragments of NE (Fig. 1) that arise as portions of the NE fold back on one another. Because lamin B is a NE specific protein, a monoclonal antibody to lamin B was used to probe the origin of CC. The monoclonal antibody to lamin B was then detected by utilizing a secondary antibody conjugated to the fluorochrome rhodamine which fluoresces red when exposed to UV light. Fluorescent microscopy of interphase HeLa cells demonstrates that only the NE fluoresces red when the cells are probed with a monoclonal antibody to lamin B (Fig. 2). There is no label localized in the cytoplasm which supports the evidence that lamin B is only localized to the NE in interphase cells.
Electron Microscopy of Bound Polysomes on in Vitro Rough Endoplasmic Reticulum Prepared by Cryohomogenization
Authors: Kent Christensen A
Published: July 2020

We have previously described a cryohomogenization method for making in vitropreparations of rough endoplasmic reticulum (RER) and other organelles. The goal of that approach was to minimize the extensive vesicular fragmentation of the endoplasmic reticulum that occurs during homogenization for conventional cell fractionation. The microsome fraction of cell fractionation consists primarily of small vesicles derived from rough and smooth endoplasmic reticulum, and has been of great value in biochemical studies of protein synthesis and secretion. However, the small size of the microsome vesicles has made them less useful for in vitro studies of bound polysomes by electron microscopy. As a further development of our cryohomogenization approach, we here describe a method for removing larger particles and debris from the cryohomogenate by filtration, and the application of in vitro RER and other organelles to EM grid membranes for negative staining and viewing by electron microscopy.
Nature of ADH-Induced Endosomes In Toad Urinary Bladder Granular Epithelial Cells
Authors: Mia AJ , Oakford LX , Dibas A , Yorio T
Published: July 2020

Serosal ADH stimulation enhances water flow under an imposed osmotic gradient through insertion of water channels (aggrephores) into the mucosal plasma membrane of toad urinary bladder sacs. Following cessation of ADH actions, water channels are retrieved as endosomes that can be visualized by mucosal inclusion of horseradish peroxidase (HRP) into round vesicles, long tubules and multivesicular bodies within the cytosol (1,2,3). Endosomes also occur adjacent to golgi bodies or lysosomes (1,2,3). However, true nature of endosomes including their formation at the mucosal surface and their shuttling in granular cells is still unclear (4,5). Current studies were undertaken to understand the role of endosomes in water channel cycling in this renal membrane model.

Urinary bladder sacs removed surgically from doubly-pithed toads, were suspended at ends of glass tubes. Control (no hormone) and experimental bladder sacs were exposed to ADH for 10 min in the absence of osmotic gradient.
Ultrastructural Changes in Hepatocellular Rough Endoplasmic Reticulum in Rats Given a Pyrimidine Derivative
Authors: Maleeff Beverly E , Newsholme Stephen J , Hart Timothy K
Published: July 2020

Hepatomegaly, characterized by increased liver weight due to hyperplasia or hypertrophy, can be induced in many species by various drugs or environmental toxins. In rats, hepatomegaly is more usually associated with increases of smooth endoplasmic reticulum in hepatocytes and the associated induction of cytochrome P450 enzymes or with hepatocellular peroxisomal proliferation. In this study, a substituted pyrimidine derivative given to rats resulted in hepatomegaly with an unusual selective increase in hepatocellular rough endoplasmic reticulum (RER).

Female Sprague-Dawley rats were given repeated oral doses of a substituted pyrimidine derivative for 28 consecutive days. Livers were collected at necropsy, weighed, fixed in 10% neutral buffered formalin and processed to paraffin sections stained with hematoxylin and eosin (H&E) for light microscopic evaluation. Portions of formalin-fixed liver were transferred to buffered 2.5% glutaraldehyde for ultrastructural evaluation. Specimens were processed into epoxy resin and thin sections were examined with a JEOL 1200EX transmission electron microscope.
Morphometry of Liver Lesions in an Ultrastructural Pathology Study
Authors: Singh A , Ireland WP , Chu I
Published: July 2020

Some pathologic alterations in animal tissue sections, because of low resolution, are not identified using light microscopy. Availability of higher resolution in electron microscopy (EM) confers a decisive role on EM for diagnostic pathology that is well accepted. Much has already been contributed by EM in understanding of liver diseases. However, EM has been further refined by adding a quantitative facet to the results. Morphometric technique allows transfer of facts acquired from 2-dimensional samples, e.g., electron micrographs, to a 3-dimensional perspective. In addition, the technique is valuable in determining the significance of the alterations especially in instances where the changes numerically are either scanty or indistinguishable from a normal appearance of the cells or tissues (Fig. 1).

In our work on the effects of several different PCB congeners, one of the compounds, namely, congener number 153 was studied to obtain morphometric data on the lesions in the liver of rats given the congener in the diets for 13 weeks.
Muscle Biopsy: Use of Cryosections, Histochemistry, and Electron Microscopy for Diagnosis
Authors: Hastings Cindy L , Jones Yvonne M , Mrak Robert E
Published: July 2020

Modern diagnosis of biopsied muscle requires a multidisciplinary approach, including routine histology and immunohistochemistry, cryosectioning for histochemistry, and (in selected cases) electron microscopy or biochemical evaluation. Routine paraffin embedding offers excellent histological detail. Cryosections provide fresh tissue for histochemical reactions that delineate fiber types and reveal special architectural alterations. Electron microscopy is applied in selected instances to provide diagnostic detail not otherwise apparent. Biochemical analysis of frozen tissue is used to define certain inborn errors of metabolism. This integrated approach allows diagnostic precision far beyond the limited information available from routine paraffin sections. We present examples of diagnoses made possible using these special techniques.

Chronic, slowly progressive neuropathies show grouping of fiber types and target fiber formation, apparent in special histochemical preparations. Routine histological preparations may be normal or show only nonspecific changes. Central core disease shows areas of pallor evident in the central areas of many myofibers in histochemical preparations.
Macrophage Targeted Photodynamic Regulation of Wound Healing
Authors: Coleno Mariah , Wallace Vincent P , Tromberg Bruce J , Hasan Tayyaba
Published: July 2020

Wound healing is coordinated by the diverse activities of cells of the monocyte/macrophage lineage.1 Macrophage depletion impairs wound healing, while overexpression of macrophage products is associated with chronic inflammatory states. If light-activatable pharmacologic agents could be selectively targeted to macrophages, clinical conditions resulting from overexpression such as postsurgical adhesions, hypertrophic scarring, and the formation of intimal hyperplasia could be suppressed. Similarly, adjusting light dose parameters to provide a stimulatory effect could selectively enhance wound repair processes.

To test these hypothesis, photoactivatable cytotoxins (photosensitizers, PS) coupled to specific receptor ligands have been evaluated for localization in cells found in cutaneous tissue using a non-invasive imaging method, two-photon microscopy.

The two-photon microscope (TPM) has been found to be a useful tool for imaging cells with submicron resolution thus allowing for the determination of photosensitizer localization within cells. Additional uses of the TPM will include imaging into epithelial tissue grown in culture and tracking cells in wounds.
Subcellular Localization of an Oncoprotein using Immunofluorescence, Confocal Imaging and Immuno Electron Microscopy
Authors: Murti K , Caslini C , Domer PH , Korsmeyer S J , Boer J , Grosveld G , Look AT
Published: July 2020

The mixed-lineage leukemia (MLL) gene is associated with more than 25 translocations of chromosome band 1 lq23 in the human leukemias. In certain unusually aggressive leukemias, the N-terminal portion of MLL fuses in frame to a portion of the transcription factor AF4. As a part of our investigation of the role of the MLL-AF4 fusion oncoprotein in Leukemogenesis, we studied the cellular sites of distribution of MLL-AF4 in the Hela carcinoma cells and U937 myeloid cells by immunofluorscene, confocal imaging and immuno-electron microscopy. Hela and U937 cells were stably transfected with a FLAG epitope-tagged MLL-AF4 (F-MLL-AF4) under the control of a tetracycline-responsive promoter and the expression of F-MLL-AF4 detected by indirect immunofluorescence (IF) with an anti-FLAG monoclonal antibody. These studies revealed that in both cell types the antibodies labeled two nuclear components, the nucleoli in an intense manner and the nucleoplasm in a punctate (100 to 200 dots) pattern. No antibody staining was observed in control (in the presence of tetracycline) cells.
Gamma Tubulin is Localized to a New Organelle, The Multivesicular Aggregate, In Mammalian Oocytes
Authors: Calarco Patricia G
Published: July 2020

Oocyte meiotic maturation occurs without the aid of centrioles in the mouse; instead the division spindles are organized by a number of microtubule organizing centers (MTOC). Immunofluorescence microscopy using anti-γ tubulin shows that multiple MTOCs migrate to surround the central nucleus (germinal vesicle, GV) during early maturation.. However, at the EM level, recognizable MTOCs have never been seen prior to the nucleation of microtubules (MT) which occurs near the time of nuclear envelope breakdown. Recently, I described multivesicular aggregates (MVAs) as the organelles most likely to represent immature MTOCs prior to MT nucleation4. These are first seen at time zero by EM as two large aggregates in the cortex, with sizes averaging 10um. MVA subsequently fragment into smaller units as they migrate centrally.

Materials and Methods: Female ICR mice were injected IP with 100 iu of chorionic gonadotropin (Sigma) to stimulate follicle growth, and ovaries were removed 48 hours later.
Investigation of DNA Loop Domains using Fluorescent in Situ Hybridization (FISH) and Epi-Fluorescence Microscopy
Authors: Klaus Angela V , McCarthy Siobhan , McCarrey John , Ward W Steven
Published: July 2020

The average sperm nucleus is only 8 micrometers long, but contains 1 meter of DNA. We have previously described how the sperm cell is able to package this DNA into the small volume of the sperm head. The only DNA structure that exists in both somatic cells and sperm nuclei is the organization of DNA loop domains that are attached at their bases to the nuclear skeleton, the nuclear matrix. We compared the structure of one gene in sperm nuclei to that of spermatogenic and adult cells.

Individual loop domains of the 5S rRNA gene cluster were visualized by fluorescent in situ hybridization (FISH). Nuclei were isolated from hamster spermatozoa, spermatogonia, pachytene spermatocytes, round spermatids, and adult liver cells. They were then extracted to remove the protamines or histones, fixed, hybridized to a biotinylated 5S rDNA probe, then viewed by epifluorescence microscopy. The 5S rDNA in liver nuclei was organized into a single large loop domain (Fig. 1),