bluuebunny/crossref_metadata_2025 huggingface dataset:
Visual limitations to image interpretation
Authors: Pelli Denis G.
Published: June 2020

Most images are interpreted solely by visual perception. What we can perceive in an image is limited by the quality of the image (e.g. the contrasts of the signal and noise) and by intrinsic limits of our visual systems. In this abstract I will describe the intrinsic noise in the visual system. In my presentation I will also demonstrate some limits to the complexity of patterns that may be perceived.

THE EQUIVALENT INPUT NOISE OF HUMAN VISION

In order to see, we must transduce the photons entering our eye into neural activity. The absorption of light by matter is a stochastic phenomenon. The intensity of the light determines only the probability of a photon absorption by a photoreceptor. Thus vision is intrinsically noisy, based as it is on the random absorption of photons.
Convergent-beam characterization of some oxides which undergo structural phase transformations
Authors: Tanaka M.
Published: June 2020

We have been concerned with the development of convergent-beam electron diffraction (CBED) techniques and their application to materials science. Our effort is now concentrated to identify the nature of crystal defects. Our application has been focussed on materials most of which undergo structural phase transformations: NdNbO4, La2Ti2O7, Sr2(Ta1-xNbx)2O7, Fe3O4, PbZrO3, PbZrO0-7Ti0.3O3, SrTiO3, BaPb1-xBixO3, hex.BaTiO3, Bi2.5Na0.5Nb2O9, MgAl2O4, MnSi, TaS3, NbS3+x, Mo2S3. In the present report, the results on four oxides will be reviewed.

a) La2Ti2O7

Gasperin determined the space group of the room temperature phase to be P21, but Scheunemann and Miüller-Buschbaum reported its space group to be Pna21. Ishizawa et al. studied the phase transformation between room temperature and 1173 K using X-ray diffraction. They determined the space group of the room temperature phase to be P21 and found the presence of twin domains.
The surface step: Its strain field and REM image contrast splitting
Authors: Peng L.-M. , Cowley J.M. , Hsu Tung
Published: June 2020

Experimental and theoretical considerations suggest that, due to the loss of two dimensional periodicity of an infinite surface at surface steps, some sort of atomic relaxation will be expected to occur around the step . A proposed surface step model is shown in Fig.l. A surface step (Fig.1c) may be decomposed into a perfect surface step (Fig.1a) and a distortion field around the step (Fig.1b). Since the perfect surface step produces phase contrast but does not produce diffraction contrast or contribute to the step contrast splitting, only the distortion field will be considered.

Shown in Fig.2 is the distortion field distribution associated with the surface step and the corresponding variation in its local incident angle. Fig.3 is the reflected intensity rocking curve around a specific Bragg angle. While the reflected beam from the undistorted surface area that is far from the step gives the background reflection intensity, the local variation in the incident angle, along with the dependence of the reflected beam intensities on the incident angle, will produce diffraction contrast in reflection.
Image simulation in scanning electron microscopy
Authors: Joy David C
Published: June 2020

Image simulation, as a means of image interpretation, has been an important component of high resolution transmission electron microscopy for many years. By contrast scanning electron microscopists have relied almost exclusively on simple visual analogies to understand the micrographs that they produce. However with the development of SEMs capable of near atomic levels of resolution, and with the requirement for accurate dimensional metrology of sub-micron features in semiconductor devices using the SEM, a more quantitative approach to image formation and analysis is needed.

Image simulation in the SEM encounters several special problems. Firstly, the signal displayed to form the micrograph is not well defined in terms of either the contrast mechanisms which give rise to it, or the energy and origin of the electrons actually collected to produce the signal.
Image processing for electron energy loss microanalysis
Authors: Ottensmeyer F.P. , Frankland B.W.
Published: June 2020

Over the years biology and materials science have profited greatly from the imaging potential of electron microscopy for structural and morphological analysis. Gross structures, grains, subgrains or subcellular organelles, mitochondria and the Golgi were fairly easy to interpret at a glance. However, as more high resolution detail was sought in the form of macromolecular structure, or dislocations or even atomic images, the wave nature, the finite number of electrons and specimen destruction from the high energy of the latter all made interpretation of the images more difficult. Image simulation, image analysis and image processing have helped greatly to extract relevant information and have assisted in our understanding not only of the structure per se but also of the interaction between the structure and the probing electron.
300kv high-performance analytical TEM (H-9000)
Authors: Kobayashi H. , Sato H. , Miyauchi K. , Onai T. , Shii K. , Isakozawa S. , Kubozoe M.
Published: June 2020

Higher voltage operation has many advantages for transmission electron microscopy.

These advantages include better TEM image resolution and ease of specimen imaging. For analytical microscopy, the higher voltage operation has advantages such as higher source brightness, and better spatial resolution.

We reported development of a 300kV ultra high resolution electron microscope. At this time, we would like to report an analytical type 300kV electron microscope.

We have incorporated a side entry specimen stage which permits ±45° specimen tilt and is convenient for characteristic x-ray detection. We have also incorporated an analytical objective polepiece which has Cs of 2. 5mm, Cc of 2. 3mm and theoretical TEM image resolution of 0.23nm.
Low-workfunction field-emission source for high-resolution EELS
Authors: Batson P. E.
Published: June 2020

In recent years,instrumentation for electron energy loss spectroscopy (EELS) has been steadily improved to increase energy resolution and collection efficiency. At present 0.40eV at 10mR collection half angle is available with commercial magnetic sectors (e.g. Gatan, Inc. and VG Microscopes, Ltd.), and 70meV at 10mR has been demonstrated by use of a Wien filter within a large deceleration field. When these high resolution spectrometers are coupled to the modern small electron probe instrument, we obtain a tool which promises to reveal local changes in bandstructure and bonding near defects and interfaces in heterogeneous materials.

Unfortunately, typical electron sources have intrinsic energy widths which limit attainable spectroscopic resolution in the absence of some monochromation system. For instance, the W thermal emitter has a half width of about 1eV.
New Side-Entry Environmental Cell Equipment for Dynamic Observation
Authors: Fukami A. , Fukushima K. , Ishikawa A. , Ohi K.
Published: June 2020

Since 1970, we have been developing wet cell microscopy to observe hydrated materials using film-sealed environmental cell (or hydration chamber; abbreviated as EC). Specimen stages in which the EC is set have been manufactured, four top-entry and one side-entry types, for 100 kV electron microscope in series with improvement. We have carried out in-situ observations on the behaviour of hydrated biological or non-biological materials according to the environmental changes; morphological and/or structural change caused by evacuation around the specimens after observing them in wet gas environment. chemical or biochemical reaction processes between specimens and liquid chemicals injected into the EC during observation.

Based on these experiences, we manufactured a new EC holder of side-entry type and its related equipments for dynamic observation with high resolution This new holder was designed for JEM-2000EX electron microscope, whose evacuation system was modified for EC operation.
Method for determining surface step heights and its application to α-Al2O3
Authors: Susnitzky D.W. , Carter C.B.
Published: June 2020

The free surfaces of ceramic materials control many of the important properties of these materials including, for example, their sinterability and catalytic activity. Ceramic surfaces are currently the subject of a range of theoretical and experimental treatments. This study is part of a broader program which monitors ceramic surfaces under experimental conditions which more closely parallel those encountered during the commercial processing of ceramic materials than the conditions utilized in conventional surface-sensitive UHV techniques. TEM foils of single crystal α-Al2O3 were prepared in the usual way and annealed in air for 24h at 1400°C . The microstructure which typically results from this heat treatment is shown in Fig.1A, a bright-field (BF) image from a (1120) α-Al2O3 foil with the electron beam parallel to the [1120] direction.
Scanning Electron Microscopy with Polarization Analysis – SEMPA
Authors: Celotta R. J. , Unguris J. , Pierce D. T.
Published: June 2020

The advancement of magnetics science and technology requires the ability to image magnetic microstructure, i.e. domains, below the 1 micron optical resolution limit. As an alternative to Lorentz microscopy, SEMPA was proposed in 1982 after it was realized that the secondary electrons ejected from a ferromagnetic surface are spin polarized. That is, they retain both the degree and direction of polarization characteristic of the spin alignment within the ferromagnet. By replacing the secondary electron detector in a UHV SEM with a detector capable of measuring both the number and polarization of the secondary electrons, it has been possible to simultaneously image both physical and magnetic microstructure. A conceptually similar microscope has been under development by Koike et al

SEMPA depends on the availability of a efficient way to measure the spin polarization of a free electron beam. Traditionally, Mott scattering has been used. Mott detection is based on the small spatial asymmetry that occurs when electrons are scattered from a high-Z material at 120KeV.
MgO Passivation of Vanadium on Cracking Catalyst
Authors: Hall J. B. , Hirschherg E. H.
Published: June 2020

Vanadium, which is present in crude oil as metal porphyrins, builds up on cracking catalyst during use and is believed to cause decomposition of the faujasite component. The addition of magnesia (MgO) to cracking catalyst is thought to passivate the vanadium thereby prolonging catalyst life. An analytical electron microscope (AEM) investigation was undertaken to gain an understanding of the passivation mechanism.

The base catalyst used in this study was composed of faujasite and an amorphous silica-alumina. Test samples were prepared by mixing steamed cracking catalyst with MgO, impregnating the mixture with vanadium and nickel naphthenates, drying at 250°F, calcining at 1000°F, and steaming at 1400°F. The two samples used for the AEM study each had final compositions of 1000 ppm Ni and 3000 ppm V, but contained levels of 30 and 3% MgO.
Z-Contrast Imaging of Heavy Metal Particles Supported in A Zeolitic Matrix Via High-Resolution Stem
Authors: Butler J. H. , Brown G. M.
Published: June 2020

High resolution Imaging of zeolites is difficult because these materials are very susceptible to Irradiation damage. It is now well known that dehydrated samples are more stable under the electron beam. Thus the most successful high resolution studies of zeolites to date have been on samples which were freeze-fractured and subsequently dehydrated via heating in a vacuum oven. Electron microscopy was then performed using a combination of low Incident beam currents and sensitive detectors. One problem with this method is that zeolites fracture along cleavage planes and therefore are deposited on microscope grids In a particular orientation. This limits the range of viewing angles. Here we describe a method of sample preparation via ultramlctrotomy as well as the establishment of suitable FEG/STEM Imaging conditions which permit the observation of small (7-14 A diameter) Pt particles within Individual zeolite channels using the method of Z-contrast as applied with a high-angle annular dark field detector. This method allows observation over all crystalline orientations for relatively long exposures to the beam.
A tem study of amorphization in Niti irradiated with Ni2+ ions at room temperature
Authors: Maziasz P. J. , Packan N. H. , Pedraza D. F. , Lee E. H.
Published: June 2020

Irradiation of a crystalline solid solution can cause the decomposition of the matrix into enhanced, modified thermal, or irradiation-induced precipitate phases. Irradiation can also transform the matrix into a new crystalline phase. Alternatively, the heavily irradiated matrix can become amorphous. Alloys of NiTi are intermetallic compounds with shape-memory applications whose amorphization is of both technological and basic scientific interest. Our purpose is to observe some details of the intermediate stages of amorphization to provide further insight into the mechanisms of the phenomenon.

An alloy of 50.5 at.% Ni/49.5% Ti was obtained from Raychem Co. as 0.76-mm- thick sheet and then rolled to 0.5 mm sheet. Disks 3 mm in diameter were punched and annealed for 0.5 h at 850°C. This results in an ordered austenitic (B2) structure, which partially transforms to martensite after air quenching.
Stress anisotropy and delamination of evaporated chromium films
Authors: Fisher Robert M.
Published: June 2020

Vapour deposited thin films are widely used for interconnections, insulators and other components in microelectronic semiconductor devices. Their mechanical and electrical properties as well as their adherence to substrates are strongly influenced by high internal streses that result from a pronounced columnar growth pattern of most thin films when deposition is at relatively low temperatures ie. 0.4 Tmp so that very little atom migration takes place. These stresses can cause cracking during deposition or during subsequent thermal or stress-activated relaxation and also can cause spontaneous delamination from the substrate. Effects clearly indicative of significant anisotropy in residual stresses in evaporated chromium films were observed during on-going studies of the primary factors determining interfacial adhesion.
TEM study of gold precipitation on defects in silicon
Authors: Kim M.J. , Carpenter R.W.
Published: June 2020

For several decades efforts have been made to investigate the roles of gold in silicon-based solid state device technology. It has been recognized that gold acts as a carrier lifetime controller due to its ability to provide recombination centers when it was introduced into silicon from the surface at high temperature via the so-called kick-out diffusion mechanism. It also has been reported that gold is more soluble in a silicon wafer with high dislocation density, and it can be gettered by diffusing phosphorus or by creating an area of high defect density. However, even though all the previous investigations predict the segregation of gold on defects in silicon it is still unclear how it precipitates on defects. Furthermore, no direct observation of such gold preciptates on defects has been made yet.
Transmission electron microscopy in VLSI/ULSI materials and devices: specimen preparation and applications
Authors: Tsai H. L.
Published: June 2020

The performance of a VLSI/ULSI device can be affected by material defects generated during device processing, structural characteristics of the device, and reliability of metallization. In these areas, the transmission electron microscope has been an important tool in helping understand the effects of material defects and process-induced issues on the device properties. With high resolution electron microscopy, it has been possible to characterize the structures of device interfaces, which often play a decisive role in the device performance. Recently, we have applied TEM/HREM to the studies of oxygen precipitation in silicon materials, thermal oxide integrity in VLSI/ULSI devices, and metallization. In order to improve the TEM/HREM capability in VLSI/ULSI devices, a multilayered technique to prepare TEM cross sections has been developed.
Structure of spinel/sesquioxide phase boundaries
Authors: Tietz L. A. , Carter C. B.
Published: June 2020

The spinel/sesquioxide ((A1-x,Bx)3O4/(A1-y,By)2O3) interphase interface can be formed by either an internal reduction or oxidation process. Internal reduction produces a dispersed product phase in the parent matrix instead of a continuous layer of product phase at the surface (external reduction). The kinetics of and thermodynamic criteria for internal reduction have been described. An (Al1-yFey) 2O3 solid solution, where y≤0.1, can be internally reduced at moderately high temperatures (T≥1300°C) and low oxygen partial pressures (pO2< 10-8) to produce a dispersion of nearly stoichiometric FeAl2O4 in an almost pure matrix of α-Al2C3 as can be seen by referring to a phase diagram of the second kind (pO2 vs. composition) for this system. FeAl2O4 has a cubic spinel structure which is based on a face-centered cubic (fee) arrangement of oxygen anions while α-Al2O3 has the corundum structure which is based on an hexagonal close-packed (hcp) arrangement of the oxygen anions. Alternatively, the spinel/sesquioxide phase boundary can be produced by oxidation, as in the oxidation of Nil-zFe2+zO4 to α-Fe2C3 (hematite) in a NiFe2O4 matrix.
Antiphase boundaries in β-Sic thin films
Authors: More K. L. , Bentley J. , Davis R. F.
Published: June 2020

Beta-SiC thin films are currently being grown via chemical vapor deposition (CVD) at North Carolina State University for potential use as a semiconductor material. Silicon carbide is a wide bandgap semiconductor with a high, saturated electron drift velocity and, as such, is a primary candidate material for high-temperature, high-speed, and high-frequency electronic devices. The β-SiC thin films are epitaxially grown on {100} silicon substrates by CVD of silicon and carbon from vapors of SiH4 and C2H4entrained in H2at a growth temperature of 1633 K. Since there is a lattice mismatch of -20% and a difference in thermal expansion coefficients of ∼10% between the silicon substrate and β-SiC, the silicon surface is reacted with C2H4 at 1583 K. for 150 s to form a converted β-SiC surface layer, approximately 5 nm thick, which helps prevent the formation of cracks during the growth of the thin films. The films are grown at a rate of ∼2 μm/h and are grown as thick as 40 μm.
Liquid-Phase reaction zones in wurtzite SiC alloys
Authors: Carpenter R. W. , Braue W. , Cutler R.A.
Published: June 2020

A relatively new method for densification of SiC compacts to produce high strength structural ceramics involves solid solution formation between wurtzite structure type α(2H)SiC and Al2OC or AIN. The solid solutions have the wurtzite structure and extend from 2 to 100 mol. % of the Al compounds. It is also known that Al2O3, and Al4C3 form a eutectic near 2150K. In the present work analytical methods were used to examine the solid/liquid reaction path followed by a powder compact of composition

xSiC+y(Al2O3+A14C3)= xSiC.3yAl2OC,

where x=l and y=0.02, heat treated in nitrogen atmosphere near the eutectic temperature. At equilibrium one would expect complete solid solution of the Al2OC in SiC. For shorter times, as in the present case, a multiphase microstructure is to be expected.
Transmission Electron Microscopy studies in deformed GaxIn1-xAs
Authors: Rai R. S. , Faber K. T. , Guruswamy S. , Hirth J. P.
Published: June 2020

The control of dislocation density during the growth of GaAs and related compounds is highly desirable for obtaining improved performance and reliability of opto-electronic devices. Doping of single crystal GaAs grown by the LEC process with Indium has been known to reduce the dislocation density significantly. Substitutional solid-solution strengthening of GaAs as an InAs, unit has been suggested to be responsible for reduction of dislocation density. To understand the mechanism involved in dislocation density reduction, deformation tests have been performed on [001] oriented Gax In1-xAs single crystals in the temperature range 700-1100°C and this paper reports some results of the TEM characterization of dislocations in these deformed single crystals.

Specimens of GaAs and Ga0.99In0.01As For TEM observations were made by cutting thin slices in a desired orientation, followed by mechanical polishing and ion-milling. These specimens were examined in a JEOL JEM 200CX transmission electron microscope at 200KeV equipped with a double-tilt goniometer stage.
Electronic properties of semiconductor interfaces
Authors: Woodall Jerry
Published: June 2020

Over the past decade III-V materials have been successfully commercialized for optoelectronic applications requiring LED's lasers and photodetectors. The success of these materials for these applications is based primarily on the use of heterojunction structures formed by epitaxial techniques in a manufacturing environment. More recently, III-V materials, notably GaAs, have been studied in the R&D environment as possible materials for use in high speed devices and circuits including VLSI. Even though the use of epitaxially grown structures has played a significant role in the success of laboratory scale devices and circuits, there are still several technology problems which will need to be solved before affordable manufacturing can be done. Two important challenges facing the commercialization of these materials for this application are metal contacts, and dielectrics for control and passivation. Both of these challenges are rooted in a common problem. Stated simply, the problem is that at nearly all GaAs/metal or dielectric interfaces the Fermi level is pinned near mid-gap.
Effects of substrate quality and annealing on defect structures at GaAs/Si interfaces
Authors: Tsai H. L. , Lee J. W.
Published: June 2020

Growth of GaAs on Si using epitaxial techniques has been receiving considerable attention for its potential application in device fabrication. However, because of the 4% lattice misfit between GaAs and Si, defect generation at the GaAs/Si interface and its propagation to the top portion of the GaAs film occur during the growth process. The performance of a device fabricated in the GaAs-on-Si film can be degraded because of the presence of these defects. This paper describes a HREM study of the effects of both the substrate surface quality and postannealing on the defect propagation and elimination.

The silicon substrates used for this work were 3-4 degrees off [100] orientation. GaAs was grown on the silicon substrate by molecular beam epitaxy (MBE).
Study of dislocation loops in Arsenic-Rich as-grown GaAs
Authors: Lee Byung-Teak
Published: June 2020

Grown-in dislocations in GaAs have been a major obstacle in utilizing this material for the potential electronic devices. Although it has been proposed in many reports that supersaturation of point defects can generate dislocation loops in growing crystals and can be a main formation mechanism of grown-in dislocations, there are very few reports on either the observation or the structural analysis of the stoichiometry-generated loops. In this work, dislocation loops in an arsenic-rich GaAs crystal have been studied by transmission electron microscopy.

The single crystal with high arsenic concentration was grown using the Horizontal Bridgman method. The arsenic source temperature during the crystal growth was about 630°C whereas 617±1°C is normally believed to be optimum one to grow a stoichiometric compound. Samples with various orientations were prepared either by chemical thinning or ion milling and examined in both a JEOL JEM 200CX and a Siemens Elmiskop 102.
A magnetic super lattice
Authors: Bentzon M.D. , Wonterghem J. v. , Thölén A.
Published: June 2020

We report on the oxidation of a magnetic fluid. The oxidation results in magnetic super lattice crystals. The “atoms” are hematite (α-Fe2O3) particles with a diameter ø = 6.9 nm and they are covered with a 1-2 nm thick layer of surfactant molecules.

Magnetic fluids are homogeneous suspensions of small magnetic particles in a carrier liquid. To prevent agglomeration, the particles are coated with surfactant molecules. The magnetic fluid studied in this work was produced by thermal decomposition of Fe(CO)5 in Declin (carrier liquid) in the presence of oleic acid (surfactant). The magnetic particles consist of an amorphous iron-carbon alloy. For TEM investigation a droplet of the fluid was added to benzine and a carbon film on a copper net was immersed. When exposed to air the sample starts burning. The oxidation and electron irradiation transform the magnetic particles into hematite (α-Fe2O3) particles with a median diameter ø = 6.9 nm.
Filler material in one-component room-temperature-vulcanizing (RTV) silicone rubber
Authors: Greer R. T. , Zhang D. T.
Published: June 2020

Silicone rubber adhesive is widely used in medical applications. Recent observations show that silica filler reduces the blood compatibility of silicone rubber significantly. It is of interest to characterize the nature of the filler (size and structure) and its removal from the adhesive so that the silicone rubber that is left can be used in dip coating applications and subsequently be polymerized by the moisture in the air. The one-component RTV's contain the silica filler for improved strength compared with a filler free silicone polymer; however, in coating applications the tissue and blood compatibility are generally important features of interest. The one-component RTV's contain a relatively low molecular weight hydroxy end-blocked silicone polymer, methyl triacetoxy silane to cross-link the polymer, and a filler similar to that used in heat vulcanizing silicone. The filler content is about 14%. On exposure to moisture in air, the soft silicone paste cures, and has the same chemical composition as other medical-grade elastomers.
A Modulated quasi-crystalline structure in an Ai-Li-Cu-Mg-Zr alloy
Authors: Zhang J. P. , Ye H. Q. , Kuo K. H. , Jiang Z. Z. , Marks L. D.
Published: June 2020

Sainfort et. al. have observed a quasi-crystalline phase in normal equilibrium precipitates in Al-Li-Cu and Al-Li-Cu-Mg alloys produced by classical casting rather than rapid solidification. Hence the intergrowth of the icosahedral phase (I-phase) and the related crystalline structures and formation mechanism is a subject of some interest. By means of electron microscopy we have studied the I-phase precipitates extracted from an Al-Li-Cu-Mg-Zr alloy.

The presence of I-phase was verified by selected area diffraction patterns shown in Fig. 1, showing the typical 5-fold and 3-fold symmetry of the point group 3 m 5, instead of ordinary translation symmetry, in (a) and (b) respectively. Fig. 2 is a high resolution image in a 2-fold axis of I-phase with the corresponding EM pattern inserted. The configuration of Fig. 2 shows not only the quasi-per iodic arrangement, but also a modulation in two directions perpendicular to this axis.
Effect of long-range order on craze microstructure in deformed PS/PB block copolymers
Authors: Henkee Chris S. , Thomas Edwin L.
Published: June 2020

The morphology present in block copolymers is determined by the volume percent of each of the components present. When the minority component ranges from 0-20 vol%, spherical micelles of the minority component are found. From 20-35 vol%, cylindrical microdomains are observed; at volume fractions slightly greater than this (35-38%), the ordered bicontinuous double diamond structure is observed. Finally from 38-≃60 vol%, regular alternating lamellae are found. It is now well known that for polystyrene-polybutadiene (PS/PB) diblock copolymers in which the PB is the minority component forming either spheres or cylinders, that deformation takes place via crazing. The mechanism for craze growth in these polymers appears to be a two stage process; the cavitation of the PB phase, followed by the drawing of the PS matrix into fibrils spanning the region of local deformation.
Integrated microscopy resource for biomedical research at the university of wisconsin at madison
Authors: Schatten G. , Pawley J. , Ris H.
Published: June 2020

The High Voltage Electron Microscopy Laboratory [HVEM] at the University of Wisconsin-Madison, a National Institutes of Health Biomedical Research Technology Resource, has recently been renamed the Integrated Microscopy Resource for Biomedical Research [IMR]. This change is designed to highlight both our increasing abilities to provide sophisticated microscopes for biomedical investigators, and the expansion of our mission beyond furnishing access to a million-volt transmission electron microscope. This abstract will describe the current status of the IMR, some preliminary results, our upcoming plans, and the current procedures for applying for microscope time.

The IMR has five principal facilities:

1.

High Voltage Electron Microscopy

2.

Computer-Based Motion Analysis

3.

Low Voltage High-Resolution Scanning Electron Microscopy

4.

Tandem Scanning Reflected Light Microscopy

5.

Computer-Enhanced Video Microscopy

The IMR houses an AEI-EM7 one million-volt transmission electron microscope.
Recent developments and controversies in the ultrastructural pathology of tumors
Authors: Henderson Douglas W.
Published: June 2020

Fundamental aspects of the diagnostic fine structure of tumors are covered in detail in standard works on this topic, and will not be reiterated here. Instead, this presentation concentrates on selected recent developments and controversies in tumor ultrastructure.

The distinction of epithelial mesothelioma from adenocarcinoma and of sarcomatoid mesothelioma from fibrohistiocytic sarcomas is a well-known problem in surgical pathology. In contrast to the short microvilli found in adenocarcinomas, mesothelial cells are typified by numerous elongated complex microvilli (MV), and a characteristic interdigitation of the MV with stromal collagen fibres may be seen in some cases - which I have never encountered in an adenocarcinoma. Estimation of the microvillus length:diameter ratio (MLDR) is now widely used in the evaluation of suspected mesotheliomas; values exceeding 11 are considered suggestive of mesothelioma, whereas lower values are usually found in adenocarcinomas.
Technical notes on routine TEM procedures
Authors: Chien K. , Van de Velde R.L. , Heusser R.C.
Published: June 2020

Sectioning quality of epoxy resins can be improved by the addition of a 1% silicone 200 fluid (Dow Corning), however this produces a softer block. To compensate, a harder plastic has been used for embedding various tissues encountered in our pathology laboratory. Exact amounts of the plastic mixture can be directly made up for embedding as shown:

The chart reveals a Poly/Bed 812 (WPE 145) to anhydride ratio of 1:0.7 and a NMA to DDSA ratio of 7:3. 1% silicone fluid is added to above mixtures.

Due to impurities within the DDSA and NMA, the polymerized epoxy blocks vary in darkness and appear to affect sectioning quality. After discussing this problem with Polysciences Inc., they have agreed to purify their anhydrides in an effort to standardize the consistency of the plastic.
Video microscopy in cell biology
Authors: Inoué Shinya
Published: June 2020

The application of video improves the image quality and extends the applicability of the light microscope to an unprecedented degree. Rectified, Plan Apochromatic high N.A. objectives can be used at full condenser N.A. in polarized light, D.I.C., single sideband edge enhanced, brightfield, or fluorescence, etc., microscopy to yield exceptionally well-corrected images with high resolution and shallow depth of field. Contrast and image features barely visible through the ocular are clearly displayed after analog and digital video enhancement. Stationary image blemishes and random noise due to low light levels can both be removed, and either recorded- or current-dynamic images can be projected for pseudocolor, monochrome, and stereo display. Serial optical sections recorded on laser disk recorders can be played back at different through-focusing speeds, or as through-focal stereo pairs, to reveal extraordinarily fine cellular details. Several examples of such applications in cell biology will be demonstrated at the meetings.
Ultrarapid cooling and molecular distillation drying for sample preparation
Authors: Linner J.G. , Livesey S.A. , Harrison D.S. , Schifani T.A.
Published: June 2020

The efficacy of electron microscopy as a tool to answer biochemical structure-function questions relates directly to the changes induced during sample preparation. Limitations of conventional methods include: slow penetration and crosslinking
by fixative, aqueous extraction, solvent extraction and chemical modification by crosslinking agent. Advantages include: stability of resin embedded sections and the
opportunity for repeated analysis. The aim of this study has been to develop and apply a technique of sample preparation which minimizes the limitations while maintaining the advantages of a resin embedded section. The method is divided into four components i.e., ultrarapid cooling, molecular distillation drying, vapor phase osmication and direct resin infiltration in vacuo.

Ultrarapid cooling was obtained by the use of the metal mirror technique. Tissue samples (1mm3) or cultured cells grown on Therminox plastic were mounted on sample holders designed to minimize compression.
Artefacts in the x-ray microanalysis of frozen-hydrated biological samples
Authors: Echlin Patrick
Published: June 2020

A number of papers have appeared recently which purport to have carried out x-ray microanalysis on fully frozen hydrated samples. It is important to establish reliable criteria to be certain that a sample is in a fully hydrated state. The morphological appearance of the sample is an obvious parameter because fully hydrated samples lack the detailed structure seen in their freeze dried counterparts. The electron scattering by ice within a frozen-hydrated section and from the surface of a frozen-hydrated fracture face obscures cellular detail. (Fig. 1G and 1H.) However, the morphological appearance alone can be quite deceptive for as Figures 1E and 1F show, parts of frozen-dried samples may also have the poor morphology normally associated with fully hydrated samples. It is only when one examines the x-ray spectra that an assurance can be given that the sample is fully hydrated.
A time comparison circuit in the electric fish midbrain
Authors: Carr C. E.
Published: June 2020

The weakly electric fish Eigenmannia is able to detect temporal disparities as small as 400 nanoseconds between two signals from different parts of the body surface. The elements of this time comparison circuit have been identified by EM reconstruction of its component cells.

Information about the timing of the zero-crossings of signals on each area of the body surface is coded by phase coder receptors, a subset of tuberous electroreceptors. Electroreceptors on the body surface are innervated by primary afferents with a central termination on the spherical cells of the medullary electrosensory lateral line lobe. These cells project to lamina IV of the midbrain torus, a structure similar to the inferior colliculus. Afferents entering lamina VI form a very restricted terminal arbor in which they synapse upon the three cell types of this lamina.
Microcircuitry of the cat retina: Connections between arrays of neurons
Authors: Freed Michael A. , Sterling Peter
Published: June 2020

One of the most basic of structure-function relationships in the mammalian visual system is the relationship between the size of a ganglion cell's receptive field and the number of rod photoreceptors which are connected to it. There is also the flip side of the coin: how many ganglion cells does a single photoreceptor connect to? We have estimated the number of rods which converge upon an on-beta type of retinal ganglion cell; we have also estimated the number of on-beta ganglion cells which a single rod diverges to. Our method is to extract a three-dimensional circuit from a series of two-dimensional electron microscope sections. The results have implications for the preservation of the signal/noise ratio in the ganglion cell.

There are two well-documented routes from the rods to the on-beta ganglion cell.
Polychlorinated biphenyl induced hepatocyte alterations
Authors: Collins D.N. , Turner J.N. , Brosch K.O. , Seegal R.F.
Published: June 2020

Polychlorinated biphenyls (PCBs) are a ubiquitous class of environmental pollutants with toxic and hepatocellular effects, including accumulation of fat, proliferated smooth endoplasmic recticulum (SER), and concentric membrane arrays (CMAs) (1-3). The CMAs appear to be a membrane storage and degeneration organelle composed of a large number of concentric membrane layers usually surrounding one or more lipid droplets often with internalized membrane fragments (3). The present study documents liver alteration after a short term single dose exposure to PCBs with high chlorine content, and correlates them with reported animal weights and central nervous system (CNS) measures. In the brain PCB congeners were concentrated in particular regions (4) while catecholamine concentrations were decreased (4-6). Urinary levels of homovanillic acid a dopamine metabolite were evaluated (7).

Wistar rats were gavaged with corn oil (6 controls), or with a 1:1 mixture of Aroclor 1254 and 1260 in corn oil at 500 or 1000 mg total PCB/kg (6 at each level).
Visualization of a small polypeptide within an adenovirus capsid fragment by subtracting the hexon x-ray structure from stem images
Authors: Furcinitti P. S. , van Oostrum J. , Wall J. S. , Burnett R. M.
Published: June 2020

Planar groups-of-nine hexons (GONs) comprise the central region of each facet of the icosahedral capsid of adenovirus, and are released intact under mild dissociation conditions. Each facet is a small p3 array of 12 hexons. GONs consist of just two proteins: polypeptide II (Mr 109,077), which combines in trimers to form hexons; and polypeptide IX (Mr 14,339), which is present in 12 copies and stabilizes the GON. A model for the location of polypeptide IX within the capsid has recently been proposed that explains both the non-random dissociation of GONs and the disruption pattern of the virion capsid. The STEM was used to examine negatively stained GONs, and an artificial GON without polypeptide IX was created from the 6 Å x-ray model of hexon in order to perform difference microscopy.
The use of immunocytochemical techniques in studying the ultrastructure of elastin in vascular smooth muscle cell cultures: I. Anti-alpha-elastin staining pattern
Authors: Toselli P. A. , Faris B. , Moscaritolo R. , Franzblau C.
Published: June 2020

We have used cell cultures to localize elastin in the extracellular matrix (ECM) by ultrastructural immunocytochemistry. An ideal model for such studies are neonatal rat aortic smooth muscle cell (SMC) cultures described by Oakes et al. As much as fifty percent of the total protein in the ECM of these cultures is elastin. During recent years, an alternative and most useful application of the antibody staining “sandwich” technique has evolved to indirectly localize tissue antigens on ultrathin sections. The sandwich technique involves the use of a marker employing colloidal gold-labelled IgG intermediate layer acting to detect the presence of primary antibody. In this case, the primary antibody (anti-alpha-elastin) attaches to the epitope(s) of the elastin molecule present on the cut surface of the sectioned elastic fiber. The value of the immunogold (IMG) antibody staining technique lies in its extraordinary ability for detecting the 10-40 nm diameter colloidal gold particle.
Nerve terminal plasma membrane localization of Ca++-Mg++ ATPase
Authors: Pappas G. D. , Kriho V.
Published: June 2020

Depolarization of the frog neuromuscular junction results In an Influx of calcium from the extracellular space. The cell calcium concentration is increased several fold and release of neurotransmitter results. Following depolarization, a necessity exists for an outwardly directed calcium pump to return the intracellular Ca++ concentration to resting levels. A calcium pump such as this has been described In the erthrocyte plasma membrane as well as In synaptosomal plasma membrane from guinea pig, synaptic membrane fractions from rat cerebral cortex and the plasma membrane of mouse neuroblastoma X rat glioma hybrid cell line 108CC5. Jorgensen has shown a Ca+++Mg++-dependent ATPase in the sarcoplasmic reticulum of rat skeletal muscle using Indirect Immunoferritin labelling of ultra thin frozen sections.
Extracellular filaments in the perineurium of the florida lobster,Panulirus argus
Authors: Baerwald Roy J. , Williamson Lura C.
Published: June 2020

In arthropods the perineurium surrounds the neuropile, consists of modified glial cells, and is the morphological basis for the blood-brain barrier. The perineurium is surrounded by an acellular neural lamella, sometimes containing scattered collagen-like fibrils. This perineurial-neural lamellar complex is thought to occur ubiquitously throughout the arthropods. This report describes a SEM and TEM study of the sheath surrounding the ventral nerve cord ofPanulirus argus.

JuvenileP. arguswere collected from the Florida Keys and maintained in marine aquaria. Nerve cords were fixed for TEM in Karnovsky's fixative and saturated tannic acid in 0.1 M Na-cacodylate buffer, pH = 7.4; post-fixed in 1.0% OsO4in the same buffer; dehydrated through a graded series of ethanols; embedded in Epon-Araldite; and examined in a Philips 200 TEM. Nerve cords were fixed for SEM in a similar manner except that tannic acid was not used.
Visualization in the EM of the intermediates in the actin-myosin ATPase cycle prepared by a rapid-freezing/stopped-flow method
Authors: Pollard T.D. , Bhandari D. , Maupin P. , Weeds A.G. , Huxley H.E.
Published: June 2020

Motion and force are produced by myosin and actin in muscle and nonmuscle cells when myosin heads move along the surface of actin filaments. The energy is provided by the hydrolysis of ATP on the head of the myosin molecule. The details of the molecular motion in the myosin crossbridge have resisted characterization, in part because the crossbridge act asynchronously, even in the semicrystalline array of filaments found in striated muscles. Consequently, averaging techniques, including spectroscopic methods, tend to record a blurred signal from a large population of crossbridges.

One way to circumvent this problem is :to examine individual myosin heads as they go through their cycle of ATP hydrolysis on the surface of the actin filament. The heads of myosin are large enough to image by EM, but the low affinity of active crossbridges for actin filaments complicates the situation.
Psammomatous duodenal carcinoid producing somatostatin: Ultrastructural investigation
Authors: Ilse G. , Kovacs K. , Gardiner G. , Moore T.
Published: June 2020

Somatostatin producing duodenal psammomatous carcinoids, described first by Kaneko et al. in 1979, are rare. We have studied such a tumor by histology, immunohistochemistry, transmission electron microscopy, immunoelectron microscopy and energy dispersive x-ray microanalysis.

A 60-year-old woman with attacks of biliary pain underwent cholecystectomy. At surgery, a tumor measuring 3 cm in diameter was discovered in the wall of the duodenum. Transduodenal biopsy revealed a psammomatous carcinoid exhibiting strong immunopositivity for somatostatin. The tumor was removed 4 months later. By histology, the tumor showed a tubular pattern as well as a cribriform arrangement, contained amorphous hematoxylinophil deposits and psammoma bodies which were positive with the von Kossa method and alizarin red. The avidin-biotin-peroxidase complex technique demonstrated positivity for somatostatin, mAB lu-5 (a monoclonal panepithelial antibody), keratin, neuron specific enolase and chromogranin. No immunoreactivity was obtained for ACTH, calcitonin, endorphin, FSH, gastrin, glucagon, GH, insulin, LH, neurophysin, PP, PRL, serotonin, α-subunit, VIP and vasopressin.
A freeze fracture technique for examining human hair medulla with Scanning and Transmission Electron Microscopy
Authors: Coble D. W. , Kairinen E. O.
Published: June 2020

Examination of hair medulla by transmission electron microscopy (TEM) is difficult because of the keratinous composition of hair and because of sectioning problems that result from insufficient infiltration and nonmiscibility of hair with embedding resins, even those of low viscosity. Although longitudinally cutting or tearing fibers will expose the medulla for embedment or direct viewing, considerable disruption occurs in its structure. Less disruption results from the use of freeze fracture techniques for either transmission or scanning electron microscopy (SEM).

Freshly plucked human scalp and beard hairs were submersed in liquid nitrogen for a minimum of three minutes, held at proximal and distal ends with Dumont #10 tweezers, and slowly bent to an arc until the specimens broke at the apex. Customarily, clean bevelled fractures occurred along the tips of the arcs and exposed not only the medulla but also the cortex and cuticle. The fractured specimens were then removed from liquid nitrogen.
Ultrastructural studies of the effect of cystamine on primary rat proximal tubular cell (PTC) cultures
Authors: Koizumi K. , Nishihira T. , Elliget K. A. , Phelps P. C. , Berezesky I. K. , Trump B. F.
Published: June 2020

Cystamine has been shown to alter intracellular Ca2+ homeostasis in isolated perfused rat liver and isolated hepatocytes by inhibiting Ca2+ efflux. This inhibition of the active extrusion of Ca2+ from hepatocytes is associated with the formation of mixed disulfides between cystamine and plasma membrane thiols and a subsequent inactivation of Ca2+-ATPase. The resulting sustained increase in cytosolic free Ca2+ is followed by both proteolysis and phospholipid hydrolysis, the proposed mechanism of hepatotoxicity.

We have investigated cystamine-induced nephrotoxicity in PTC primary cultures. This study reports ultrastructural changes in kidney cells resulting from exposure to 10 mM cystamine for 15, 60, 120 and 180 min.

Primary tubular cells were isolated from male Fischer 344 rat kidney cortex and maintained in a 50:50 mixture of DME and Ham's F12. Four day-old cultures were treated with cystamine, washed twice with HBSS, fixed in 4% phosphate-buffered formaldehyde:1% glutaraldehyde, post-fixed is OsO4, dehydrated through an ethanol series and embedded in Polybed 812. Ultrathin sections were examined in a JEOL 100B electron microscope.
Hexamethyldisilazane drying for rapid detection of bacteria in implant specimens
Authors: Giammara B. , DeVries W. , Baker R. , Dobbins J. , Hanker J.
Published: June 2020

Detection of bacteria in implants can be time consuming and equivocal. Routine histology of biopsy specimens from implant sites requires approximately 24 hours and is frequently indecisive for the demonstration of bacteria. Culture requires a minimum of 24-48 hours and may require repetitive cultures or subcultures which result in inhibition of the growth of commensals as well as conditions which favor the growth of suspected pathogens. Direct examination of implant specimens with the scanning electron microscope can show bacteria but the time, special equipment, and hazards associated with preparative techniques such as critical point drying can be prohibitive. Another limitation is the small size of the drying chamber which restricts the specimen size. Nation showed that a rapid procedure for drying insect soft tissues utilizes stepwise dehydration with graded ethanols followed by very brief treatment with hexamethyldisilazane (HMDS). It did not require specialized equipment.