bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation of TEM samples by focused ion beams
Authors: Walker John F. , Reiner J C , Solenthaler C
Published: June 2020

The high spatial resolution available from TEM can be used with great advantage in the field of microelectronics to identify problems associated with the continually shrinking geometries of integrated circuit technology. In many cases the location of the problem can be the most problematic element of sample preparation. Focused ion beams (FIB) have previously been used to prepare TEM specimens, but not including using the ion beam imaging capabilities to locate a buried feature of interest. Here we describe how a defect has been located using the ability of a FIB to both mill a section and to search for a defect whose precise location is unknown. The defect is known from electrical leakage measurements to be a break in the gate oxide of a field effect transistor. The gate is a square of polycrystalline silicon, approximately 1μm×1μm, on a silicon dioxide barrier which is about 17nm thick. The break in the oxide can occur anywhere within that square and is expected to be less than 100nm in diameter.
Structural evolution of the aging martensite in duplex steel
Authors: Chu Hong-Yuan , Chen Fu-Rong , Wu Tai-Bor
Published: June 2020

In the past, the structure and crystallography of needle-like martensite in the as-quenched Fe-Al-Mn-C alloy were studied. The structure of the needle-like martensite was identified to be a structure between 18R(5)3 and 18R(4)3. The orientation relationship between the BCC matrix and martensite phase was determined to be and . The shape strain, habit plane and other crystallography of martensite were also determined. Comparison with the asquenched Fe-Mn-Al-C martensite, there is only little information about the aging behavior of the martensite phase. The purpose of this work is to study the structural evolution of the martensite phase after aged at high temperature.

The wrought Fe-29.4-Mn-8.03Al-0.3C alloy was heated at 1300°C for 1 hour in argon atmosphere, and followed by a quenched into water. Aging process were performed at 400 °C, 500 °C and 600 °C, respectively, for 1 hour in salt bath, and the specimens were then quenched into water. TEM specimens were examined with a JOEL-200CX STEM.
EM of mechanical plated Ti and tin coating on steel
Authors: Kashihara K. , Chiou W. A. , Lin C. S. , Meshii M.
Published: June 2020

Mechanical alloying, originally developed as a means to produce oxide dispersion strengthened alloys, has since been adopted for producing a wide variety of materials such as solid solution alloys, metal matrix composites, intermetallics, nanocrystalline and amorphous materials. It has also demonstrated as an efficient method for alloying gaseous elements by solid-gas reaction. Interest in using metal and metalloid nitrides as coating materials has been increased considerably in the past few years because of their special properties including extreme hardness, high temperature stability, high corrosion and wear resistance, and excellent conductivity. A new coating technique through the marriage of mechanical plating and nitriding has recently been developed in this laboratory. This paper presents an electron microscopic study of mechanically plated Ti and TiN coating.

Commercially produced (Johnson Matthey, Inc.) fine-grained (325 mesh, or 44 μm) Ti (99%) powders were used as the starting coating material. Interstitial-free steel sheet (provided by Inland Steel) were cut to a size of 1.5 cm × 1.5 cm as the substrate.
The first test on a magnetically focused mirror corrector
Authors: Crewe A.V. , Tsai F. , Korda P. , Ruan S.
Published: June 2020

In some recent publications we have discussed the possibility of correcting both spherical and chromatic aberrations using a magnetically focused mirror. If such a scheme could be made to work it would represent the simplest possible type of aberration corrector and for this reason we decided to test the principles of operation using a simple experimental system (Fig. 1). Electrons are obtained from a TFE source operating at 3.6kv which is immersed in a region of uniform magnetic field which refocuses them at a point 5 cm away. At this position we placed a simple specimen consisting of a copper grid with a holey plastic film. Electrons which pass through a hole in the specimen enter a region where the magnetic field is supplemented by a uniform electrostatic field which can act as a mirror. By a proper adjustment of the strength of this field the electrons can be refocused on the reverse side of the specimen where the total aberration should be zero.
Fresnel effect in high-resolution TEM imaging of small particles
Authors: Radmilovic Velimir , O'Keefe Michael A.
Published: June 2020

It is established that analysis of Fresnel contrast visible at the edges of nanoparticles in the high resolution electron microscope provides a valuable tool for determining the size of the particles with near atomic spacing accuracy.

Methods based on the Fresnel effect are enjoying increased use in the study of flat precipitates, multilayers, and grain boundaries. In profile imaging studies of solid surfaces, Fresnel effects have been observed by Marks, and by O'Keefe et al. who showed that image structure within Fresnel fringes from MgO cubes can produce “ghost” atoms far outside the specimen surface. However, consequences of the Fresnel effect appear not to have been included in the study of small particles at atomic resolution. In this work, we have used HRTEM image simulation to explore the changes in images of a nanoparticle under various imaging conditions, in particular to relate the model particle size and its apparent size as derived from the HRTEM image.
Refinement of rigid shift component normal to a Σ5 grain boundary in rutile by quantitative HREM
Authors: Paciornik S. , Michel D. , Dahmen U.
Published: June 2020

In contrast to the macroscopic parameters of a grain boundary, its microscopic parameters are difficult to determine with sufficient accuracy to make critical comparisons between models and experimental observations. Whereas the axis and angle of misorientation and the orientation of the boundary plane are easily measured from diffraction patterns or high resolution images, the rigid body shift and the localized atomic relaxation are far more complicated to determine and are more sensitive to image or sample artifacts. Yet it is only these microscopic parameters that differentiate the behavior of the same crystallographic interface in different materials. While the angle of misorientation and the plane of inclination are fixed macroscopically, the rigid body shift and localized atomic relaxations depend directly on the type of bonding that characterizes a given material.

Several methods for the measurement of rigid shifts at interfaces have been given recently. Their accuracy is limited by instrumental parameters such as beam tilt, sample tilt, residual two-and threefold astigmatism, specimen noise or twist components.
Practical aspects about hollow-cone imaging
Authors: Geipel T. , Mader W.
Published: June 2020

Hollow-cone imaging (HCI) as a possibility to improve the resolution of a TEM has already been proposed in the late 40ties and besides others, there have been extensive hollow-cone experiments 10 years back using a low resolution TEM with a non-tilt specimen holder. In a recent paper the optimum imaging parameters for HCI were determined leading to an improvement of the resolution by a factor of two. However, there are contrast limitations and experimental problems for HCI which were only partly considered in Ref. and which will be discussed in this paper for a modern electron microscope. Preliminary experiments were performed which are not shown in the abstract.

In Fig. 1 ∫ ct f(u)du is plotted versus defocus Δ f for different cone radii Θc and a fixed aperture radius Θo = 1/δ = 5 nm-1 (ct f is the phase contrast transfer function (PCTF) for HCI and δ = 0.2 nm is the resolution of a CM30 supertwin microscope).
Structure determination of the incommensurate intercalation compound HgxTiS2 using TEM
Authors: Sidorov M.
Published: June 2020

Although metal intercalated transition-metal dichalcogenides have been generally viewed as ionic compounds with guests occupying discrete lattice sites, the HgxTiS2 intercalates (1.24≥×≥0.00) have been recently discovered to have vastly different character, including essentially-neutral covalently-bound guests, superstoichiometric Hg uptake (x>1.00), complete thermal reversibility of the intercalation process and unusually complex structures. A combination of HREM and electron diffraction was used to determine the structure of the intercalates formed during the in situ deintercalation process. Digital Fourier image processing provided HREM image analysis and contrast enhancement.

HgxTiS2 has a remarkable in-plane guest-layer structure, which is composition independent. It results from the presence of partially incommensurate monoclinic Hg and TiS2 sublattices, with common a and c axes and incommensurate b axes (a= 5.92 Å; bTis2 = 3.41 Å; bHg= 2.76 Å; c= 8.70 Å, and β= 96.5°). Figure 1a shows a typical c-axis SAD pattern from a thin crystal indicating the presence of two subsets of reflections incommensurate along b* direction. In the corresponding HREM images this gives rise to the 14.5Å structure modulation along b (Fig. 1b).
1 nm resolution x-ray elemental mapping using a 300kV field emission TEM
Authors: Murakoshi H. , Tsuchiya T. , Kakibayashi H.
Published: June 2020

The characteristics of advanced semiconductor devices are affected by contamination due to impurity atoms with sizes at the nanometer level, so it is very important to evaluate such contamination with nanometer-level resolution. In order to cope with this task, we have developed an x-ray elemental mapping system, attached to a 300-kV FE-TEM, with an information limit of 0.11 nm.

Both small spot size and sufficient probe current are necessary for high-resolution and highsensitivity x-ray elemental mapping. The elemental mapping is performed with the scanning transmission electron microscope(STEM) mode. For high-resolution STEM imaging, the objective lens works with high excitation as a condenser-objective single lens where the aberrations of the other illuminating lenses are demagnified to negligible level, allowing the extremely small spot size to be obtained. The probe current, however, decreases in proportion to the square of the demagnification of the illuminating system. In this study, the optimum illuminating condition for elemental mapping is examined.
State of the development of multipole correctors for a probe-forming system and a high-resolution 200kV TEM
Authors: Haider M. , Zach J.
Published: June 2020

The development of modern high resolution electron microscopes has shown the emergence of new modern microscopes which are easy to operate and, more importantly, with which one can attain high resolution on a routine basis. However, the physical limit set by the inherent aberrations of electron lenses could not be overcome by simply optimising the geometry of the pole pieces. The only direct way to get rid of the aberrations of the round lenses is to use a corrector with which the resolution limiting aberrations can be compensated.

For the observation of uncoated biological surfaces we started a project to develop a Low Voltage Scanning Electron Microscope (LVSEM). The main problem with using low energies is to design electron optics with which one can achieve high resolution in the range of 1 nm at energies of 1 keV and below. Hence, the two main axial aberrations of such a probe forming system: the chromatic and the spherical aberration Cc and C3, respectively, have to be considered as the limiting factors of the probe diameter. Therefore, the compensation of these two aberrations is the best choice as one does not want to run into other limitations if, for example, the geometry of the objective lens is scaled down in order to obtain small aberration coefficients.
Diffracted beam interferometry for EM and materials research
Authors: Herring Rodney A.
Published: June 2020

Diffracted beam interferometry, DBI, is a practical method of interferometry which removes the strict requirement for a bright, coherent source and extends the availability of holography to a wider community of electron microscopists. The only alteration to the microscope is the installation of an electron biprism into a post-objective lens position. DBI is very flexible as the interferogram can be produced from the conditions of the infocus diffraction (Fraunhofer plane), through the Fresnel (de)focus region to the infocus specimen plane by varying the strength of the post-objective lenses, Fig. 1. As well the convergence of the interfering beams can vary from highly convergent (Fig. 1) to pseudoparallel (Fig. 2). Although the phase in the interferogram due to beam tilt and the contrast transfer function of the prefield objective lens cancel out when the interfering beams are exactly overlaid, the phase due to the spatial frequency of the biprism, KB, the contrast transfer function, uL,R, of the postfield objective lens for each beam, L,R, and the inherent material phase, FL,R, remain.
Experimental study on the restoration from a tilt-azimuth series for improvement in resolution
Authors: Baba Norio , Ichise Norihiko , Watanabe Syunya
Published: June 2020

The tilted beam illumination method is used to improve the resolution comparing with the axial illumination mode. Using this advantage, a restoration method of several tilted beam images covering the full azimuthal range was proposed by Saxton, and experimentally examined. To make this technique more reliable it seems that some practical problems still remain. In this report the restoration was attempted and the problems were considered. In our study, four problems were pointed out for the experiment of the restoration. (1) Accurate beam tilt adjustment to fit the incident beam to the coma-free axis for the symmetrical beam tilting over the full azimuthal range. (2) Accurate measurements of the optical parameters which are necessary to design the restoration filter. Even if the spherical aberration coefficient Cs is known with accuracy and the axial astigmatism is sufficiently compensated, at least the defocus value must be measured. (3) Accurate alignment of the tilt-azimuth series images.
Modeling and simulation of octahedral Pb inclusions in Al
Authors: Xiao S.Q. , Paciornik S. , Kilaas R. , Johnson E. , Dahmen U.
Published: June 2020

Pb inclusions in Al have been extensively studied for their unusual melting/solidification behavior. Pb inclusions have a cube on cube parallel orientation relationship with the Al matrix and assume cuboctahedral shapes faceted on {111} and {100}. Al and Pb are both fcc structures but with very different lattice parameters: aAl = 0.405 nm, apb = 0.495 nm. Thus 5 Al spacings match approximately 4 Pb spacings giving rise to a moire pattern visible in HREM images.

High resolution electron micrographs in the <110> zone axis orientation were recorded on the Berkeley ARM at an accelerating voltage of 800 kV. In this orientation the cuboctahedra project as truncated parallelograms as shown in Fig. 1. Although the four (111) interfaces revealed in Fig. 1 are imaged edge-on, the Al lattice overlaps the Pb lattice above and below, because the other four (111) interfaces are inclined. Therefore, even though the (111)Al lattice is clearly resolved, the determination of inclusion size is not straightforward because the contrast depends on defocus (Δf), particle size (s), depth of the inclusion in the matrix (z) and total sample thickness (t).
A hitchhiker's guide to microscopy and microanalysis using telecommunications, e-mail, and the Internet: part II
Authors: Zaluzec Nestor J.
Published: June 2020

In this tutorial, we will update, review and demonstrate the means of finding and accessing resources of electronic information which are maintained by the Microscopy Society of America and related national/international organizations. During the course of this live demonstration over the Internet, we illustrate the use of Electronic Mail, Newsgroups, File Transfer Protocols, Gophers, and Information Servers using the World Wide Web protocols. Although the MSA electronic bulletin board system (BBS) has be recently discontinued, MSA has recently decided expanded it's role in other telecommunications areas. The procedures needed by individuals or organizations to access this wealth of information will be presented in detail. The following list of topics summarizes some of the areas which will be covered.

Electronic Mail: (EMail) is textual communications which is sent over networks from one individual to another. Each user is assigned a unique address by the computer system administrator. The message composed of normal ASCII (text) characters which is sent from one computer to another.
Atom-probe analysis of the solid-liquid interface
Authors: Panitz J.A.
Published: June 2020

The first few atomic layers of a solid can form a barrier between its interior and an often hostile environment. Although adsorption at the vacuum-solid interface has been studied in great detail, little is known about adsorption at the liquid-solid interface. Adsorption at a liquid-solid interface is of intrinsic interest, and is of technological importance because it provides a way to coat a surface with monolayer or multilayer structures. A pinhole free monolayer (with a reasonable dielectric constant) could lead to the development of nanoscale capacitors with unique characteristics and lithographic resists that surpass the resolution of their conventional counterparts. Chemically selective adsorption is of particular interest because it can be used to passivate a surface from external modification or change the wear and the lubrication properties of a surface to reflect new and useful properties. Immunochemical adsorption could be used to fabricate novel molecular electronic devices or to construct small, “smart”, unobtrusive sensors with the potential to detect a wide variety of preselected species at the molecular level. These might include a particular carcinogen in the environment, a specific type of explosive, a chemical agent, a virus, or even a tumor in the human body.
Scanning Electron Microscopy and x-ray analysis of microparticles and early hydration effects
Authors: Lu Yun , Joy David C.
Published: June 2020

High resolution scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDXA) were performed to investigate microparticles in blended cements and their hydration products containing sodium-rich chemical wastes. The physical appearance of powder particles and the morphological development at different hydration stages were characterized by using high resolution SEM Hitachi S-900 and by SEM S-800 with a EDX spectrometer. Microparticles were dispersed on the sample holder and glued by 1% palomino solution. Hydrated bulk samples were dehydrated by acetone and mounted on the holder by silver paste. Both fracture surfaces and flat cutting sections of hydrating samples were prepared and examined. Some specimens were coated with an 3 nm thick Au-Pd or Cr layer to provide good conducting surfaces. For high resolution SEM S-900 observations the accelerating voltage of electrons was 1-2 KeV to protect the electron charging. Microchemical analyses were carried out by S800/EDS equipped with a LINK detector of take-off angle =40°.
Development of a scanning tunnelling atom probe
Authors: Spence J.C.H. , Weierstall U. , Lo W.
Published: June 2020

An STM has been constructed which will allow atomic clusters of interest to be transferred into a time-of-flight spectrometer for species identification. Atoms will first be transferred onto the tip, using a small voltage pulse. The sample is then removed, and these atoms ejected into a time-of-flight (TOF) analyser for mass identification (see for an excellent review of TOF methods). Figure 1 shows the experimental arrangement. The STM is based on a miniature Burleigh inchworm, to which a piezo tube is directly attached. This is used in bending mode to provide scan motion of the stage plate in two lateral dimensions, and fine motion in z. The inchworm accounts for the coarse approach. The sample holder sits on three balls, (to one of which the tunnel current amplifier is connected) and may be removed using a wobble-stick. Electrical insulation for 15 kV at the tip is provided, which does not scan. The tip is not cooled. A 75mm chevron dual channel plate is positioned L = 200 mm above the tip.
Use of fluorescence in situ hybridization to study the arrangement of DNA sequences in normal and disease-related chromosomes
Authors: Trask Barbara J. F. , Massa Hillary , Friedman Cynthia , Esposito Richard , van den Engh Ger , Yokota Hiroki
Published: June 2020

The sites of specific DNA sequences can be fluorescently tagged by fluorescence in situ hybridization (FISH). Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using epifluorescence microscopy. The distances between points on the same or different chromosomes can be determined easily in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed. Our work has focussed on the analysis of hybridization patterns in two dimensions using conventional fluorescence microscopy.

We have used FISH to study various aspects of genome organization that are difficult to study using other techniques. Examples of these applications will be presented.

FISH is now the method of choice for determining the chromosomal location of DNA sequences. DNA sequences can be positioned in the genome with <1:1000 accuracy (to a 3-Mbp region within a 3000-Mbp genome). Through FISH, the cytogenetic, physical and genetic maps of chromosomes can be linked.
Application on in situ PCR methods in histochemistry
Authors: Bagasra Omar
Published: June 2020

Since the publication of the first report regarding the in situ amplification of HIV-1 gag gene in a HIV-l infected cell line in 1990, there has been an explosion of research in the area of in situ PCR. There are over 200 publications describing various forms of in situ gene, identifying various infectious agents, tumor marker genes and other genetic elements of interest, in peer reviewed journals (reviewed in 2-15). The polymerase chain reaction (PCR) method for amplification of defined gene sequences has proved a valuable tool not only for basic researchers but also for clinical scientists. Using even a minute amount of DNA or RNA and choosing a thermostable enzyme from a large variety of sources, one can enlarge the amount of the gene of interest, which can be analyzed and/or sequenced. Thus genes or portions of gene sequences present only in a small sample of cells or small fraction of mixed cellular populations can be examined.
Use of calcium imaging technologies to dissect mechanisms underlying neuronal growth cone responses to environmental cues
Authors: Williams C.V. , Kater S.B.
Published: June 2020

Since calcium is a key second messenger in both the developmental formation and adult function of the nervous system, the ability to rapidly image changes in this molecule has added greatly to our understanding of how development of the nervous system is regulated. The nervous system is comprised of billions of neurons and glial cells that establish characteristic patterns of connections during development. Neurons extend processes that often must grow long distances to establish appropriate synaptic connections. Neurons perform a pathfinding behavior largely via the highly dynamic behavior of the neuronal growth cone at the distal tip of elongating processes. The motile behavior characteristic of growth cones allows the growth cone to survey the local environment, read local cues and respond to those cues with a change in behavior. A variety of cues are now known to direct growth cones (e.g. electrical activity, depolarization, growth factors, mechanical factors, neurotransmitters, substrate factors). This collection of factors includes both growth promoting and growth inhibitory influences.
Time-lapse confocal calcium imaging in an intact unrolled hippocampus preparation
Authors: Cornell-Bell A. H. , Villalba R M. , Selinfreund R.H. , Stein B. D. , Cornell J.L. , Riblet L.A.
Published: June 2020

The excitatory neurotransmitter glutamate (100 μM) induces intracellular calcium transients in cultured hippocampal astrocytes that can be imaged using the calcium indicator (Fluo3AM) and time-lapse microscopy. In response to glutamate (Fig. 1A), cultured astrocytes exhibit distinct patterns of intracellular Ca2+ oscillations and long-distance intercellular waves. Two distinct types of intercellular Ca2+ waves are attributed to excitation by different agonists of the glutamate receptor subtypes. A long-distance regenerative intercellular wave is induced by the ionotropic glutamate receptor, kainate (Fig. 1B). This is a true wave lasting for 50-125 sec with a constant velocity of 10-20 μm/sec. This wave requires extracellular Ca2+ and Na+ and is driven by the Na+/Ca2+ exchanger. A fast Ca2+ wave which travels at speeds from 10 to 200 μm/sec is dependent upon the metabotropic glutamate receptor, is inducible by t-ACPD and is dependent upon cytoplasmic release of Ca2+ regulated by IP3 (Fig. 1C). This wave is not dependent upon extracellular Ca2+ and is stopped by MCPG, a specific inhibitor of IP3-mediated intracellular Ca2+ release.
Changes in rat pituitary gonadotropes after immunization with peptides derived from the receptor for gonadotropon-releasing hormone
Authors: Petrusz P. , Grossman G. , Weaver C.
Published: June 2020

Gonadotropin-releasing hormone (GnRH) is a neuropeptide which regulates hormone release from pituitary gonadotropes. The recently cloned pituitary GnRH receptor consists of a linear chain of amino acids in which seven transmembrane segments separate 4 extracellular and 3 intracellular (cytoplasmic) sequences. The rat receptor is approximately 90% homologous with that of the human.

Observations made in several autoimmune diseases and experimental models suggest that anti-receptor antibodies can interfere with receptor function. The present studies were designed to test this hypothesis in female rats (n=9×3) after immunization with synthetic peptides whose overlapping sequences replicated all putative extracellular domains of the cloned human GnRH receptor (Table 1). The peptides (A-H) were conjugated to keyhole limpet hemocyanin and the conjugates were injected i.m. at monthly intervals for four months. A control group (K) received adjuvant only. Anti-peptide antibodies in the sera of immunized rats were detected by ELISA. Recognition of the “native” GnRH receptor was tested in immunocytochemistry on normal pituitary and brain tissue.
Oblique section 3-D reconstruction from thin sectioned biological specimens
Authors: Taylor K. A. , Winkler H.
Published: June 2020

Three dimensional (3-D) image reconstruction of plastic embedded and sectioned tissue has become an important tool for imaging complex biological structures in situ. Most 3-D imaging methods combine multiple views of tilted specimens which leads to radiation damage and mass loss. On the other hand oblique section 3-D reconstruction (OSR) can produce a 3-D image from a single view because an oblique section through a 2- or 3-D periodic specimen contains 3-D information which can be folded into a 3-D image. The resolution achievable in an OSR depends on section thickness and is lowest in the direction perpendicular to the section plane. In OSR there is no missing cone or wedge in the 3-D image because of tilt angle restrictions, but finite section thickness limits the vertical resolution. Two OSR methodologies have been developed, one called Crystallographic Serial Section Reconstruction or CSSR, and another called Super Lattice Reconstruction or SLR.
Formation of polyribosomal crystals in Entamoeba histolytica by hyperosmotic solution of NaCl
Authors: Arguello C. , Reyes J.L.
Published: June 2020

Entamoeba histolytica is a parasitic protozoan that during its life cycle presents two different biological forms; the trophozoite that is a pleomorphic, motile and phagocytic organism, and the cyst that is small, nonmotile and rounded form covered by an external fibrous coat. Differentiation of trophozoites into the cyst involves a series of complex events including the organization of helical polyribosomes within a crystal known as the chromatoid body. Induction of this differentiation has been achieved experimentally in hypoosmotic medium composed of Mg SO4 and bovine serum (100 mOs/kg) in Entamoeba invadens, a reptilian parasite. However, Entamoeba histolytica is unable to survive when treated on the above conditions. Due to the biological importance of understanding which are the physiological conditions that may elicit ency station of this organism, we cultured trophozoites in TYI-S-33 medium containing NaCl to a final osmolality of 400, 500, 600 and 800 mOs/kg. Parasites were observed directly through a videosystem and noticed that at 600 mOs the trophozoites stop moving and rounded in about 3 min, similar results were obtained at 800 mOs.
An enhanced cellular organelle visualization procedure when staining for peroxisomes
Authors: Horn J. W. , Dovey-Hartman B.J.
Published: June 2020

Reliable visual identification of peroxisomes is important in developmental, clinical, and investigational research. The current technique employed in most laboratories uses a specific electron dense label for the demonstration of peroxisomes by transmission electron microscopy by applying 3,3'- Diaminobenzidine Tetrahydrochloride (DAB) directly to freshly fixed tissue samples to react with endogenous peroxisomal catalase. After routine processing, ultrastructural examination of tissue sections is conducted either with light staining or without post-staining of grids. While peroxisomes are easily identified using this method, remaining tissue architecture is difficult to visualize due to the opacity of the tissue. Additionally, if grids are post-stained with heavy metal solutions, they must be modified to allow for enough staining to visualize cellular components without compromising the quality of the peroxisome label. We will describe a technique whereby DAB-reacted tissues are stained with a postfixative solution including potassium ferricyanide that imparts density to cell membranes and cellular components thereby enhancing identification and interpretion of data.
Suitability of zinc-based bioceramic for repair of bone defects
Authors: Taylor C. , Mehling B. , Longo A. , Snead D. , Reynolds D. , Bajpai P. , Barre P.
Published: June 2020

The advantages of a synthetic bone-graft substitute include reduction of risks due to immunological rejection and transmission of diseases, such as AIDS. A satisfactory synthetic material must be non-toxic, allow adequate re-vascularization, and be capable of bearing weight and tolerating strain. Our experiments using a resorpable, zinccalcium- phosphorous-ceramic oxide (ZCAP) in composite with malic acid, vitamin E, and gentamicin sulfate, indicate that this zincbased bioceramic implant fulfills the requirements of an osteoconductive filler between two fractured ends of rat femurs.

Assessment of the suitability of ZCAP involved the following techniques :
1) Radiological examination showing the position of the implant, bridging of the defect with bone, and dissolution of the bioceramic.
2) Histology of serial sections indicating resorption of the ZCAP (a few granules remain after 10 weeks) and replacement by endochondral ossification.
3) Scanning electron microscopy revealing that the intact ZCAP first becomes surrounded by fibrous tissue and then infiltrated by cell processes.
Microstructural morphology of sphingolipid dispersions as investigated by freeze-fracture EM
Authors: Kulkarni Vitthal S. , Anderson Wayne H. , Brown Rhoderick E.
Published: June 2020

The biological significance of the sphingomyelins (SM) and monoglycosylated sphingolipids like galactosylceramides (GalCer) are well documented Our recent investigation showed tubular bilayers in the aqueous dispersions of N-nervonoyl GalCer [N-(24:lΔ15,cls) GalCer] (a major fatty acyl moiety of natural GalCer). To determine the influence of lipid head groups on the resulting mesophasic morphology, we investigated microstructural self-assemblies of N-nervonoyl-SM [N-(24:1 Δ15,cls) SM; the second most abundant sphingomyelin in mammalian cell membranes], 1- palmitoyl-2-nervonoyl phosphatidylcholine [PNPC] (the lipid species with the same acyl chain configuration as in N-(24: 1) GalCer) and also compared it with egg-SM by freeze-fracture EM.

Procedures for synthesizing and purifying N-(24:1) GalCer, N-(24:1) SM, and PNPC have been reported . Egg-SM was purchased from Avanti Polar Lipids, Alabaster AL. All lipids were >99% pure as checked by thin layer chromatography. Lipid dispersions were prepared by hydrating dry lipid with phosphate buffer (pH 6.6) at 80-90°C (3-5 min), vigorously vortexing (1 min) and repeating this procedure for three times prior to three freeze-thaw cycles.
Immunocytochemical studies on the renl fate of circulating advanced glycated products
Authors: Gugliucci A. , Bendayan M.
Published: June 2020

A growing body of evidence points to a causal relationship between advanced glycation end products (AGE) accumulation in tissues and the development of chronic diabetic complications. Particularly impressive are data showing a dramatic increase in the levels of circulating AGE peptides in diabetic end stage renal disease that correlates with the severity of the nephropathy. We have recently shown that AGE-BSA probes tagged with colloidal gold display great reactivity towards glomerular structures. In the same way, a recent study has shown that chronic injection of AGEBSA but not BSA produces the classical picture of diabetic glomerulopathy in otherwise euglycemic animals. It is apparent from these and other studies that small metabolic fragments of AGE proteins are implicated in these phenomena. It is certain that the kidneys play an important role in the clearance of circulating AGE products from the bloodstream. However, so far little is known about the fate of AGE proteins and AGE peptides in renal tissue.
Ultrastructural analysis redefines light microscopic findings relative to morphological relationships among cells of the cerebral vasculature in the rat
Authors: Dimlich Ruth V. W.
Published: June 2020

A previous light microscopic study determined that ~70% of all connective tissue mast cells (CTMCs) in the thalamus of the rat were adjacent to periodic acid-Schiff staining cells which also were ED2 positive therefore identified as a type of macrophage. Using transmission electron microscopy, the goal of the present study was to examine that association at the ultrastructural level and to determine the relationship of each of these cells to other components of the cerebrovasculature.

Brains of anesthetized adult male Sprague-Dawley rats were fixed by intracardiac perfusion with paraformaldehyde and glutaraldehyde [2%:2.5% (phosphate buffer 0.1 M, pH 7.40 or 2%:2.5% for 5 min followed by 1%: 1.25% (phosphate buffer 80 mM, pH 7.4) for the remainder of the fixation]. Brains were sliced into ~1 mm sections and samples of thalamus were postfixed in OSO4 (1%, 0.1 M phosphate buffer), dehydrated, and flat embedded in LX112. Semi-thin (1 μm) and thin (60-90 nm) sections were cut and stained with toluidine blue and uranyl acetate/lead citrate respectively. Grids were viewed on a JOEL 100CXII electron microscope at 80kV.
Immunogold localization of HMB-45 antigen in pulmonary lymphangiomyomatosis
Authors: Minda J.M. , Dessy E. , Pietra G. G.
Published: June 2020

Pulmonary lymphangiomyomatosis (PLAM) is a rare disease occurring exclusively in women of reproductive age. It involves the lungs, lymph nodes and lymphatic ducts. In the lungs, it is characterized by the proliferation of smooth muscle cells around lymphatics in the bronchovascular bundles, lobular septa and pleura The nature of smooth muscle proliferation in PLAM is still unclear. Recently, reactivity of the smooth muscle cells for HMB-45, a melanoma-related antigen has been reported by immunohistochemistry. The purpose of this study was the ultrastructural localization of HMB-45 immunoreactivity in these cells using gold-labeled antibodies.

Lung tissue from three cases of PLAM, referred to our Institution for lung transplantation, was embedded in either Poly/Bed 812 post-fixed in 1% osmium tetroxide, or in LR White, without osmication. For the immunogold technique, thin sections were placed on Nickel grids and incubated with affinity purified, monoclonal anti-melanoma antibody HMB-45 (1:1) (Enzo Diag. Co) overnight at 4°C. After extensive washing with PBS, grids were treated with Goat-anti-mouse-IgG-Gold (5nm) (1:10) (Amersham Life Sci) for 1 hour, at room temperature.
Ultrastructural changes in the mouse placenta after administration of trichosanthin
Authors: Ooi V.E.C. , Lau C.F. , Yeung H.W.
Published: June 2020

The root tuber ofTrichosanthes kirlowiiMaxim (Family curcurbitaceae) has long been used in Chinese traditional medicine as an abortifacient and in therapy for choriocarcinoma. The active principle of the root tuber extract was recently purified and characterized. It is a basic protein of 24 KDa and is named trichosanthin. Investigations on trichosanthin action showed that it affected directly on the placenta, selectively causing necrosis of the syncytiotrophoblasts of placental villi. The present study attempts to determine the initial action of trichosanthin and follow through from the earliest sign of histopathological and ultrastructural changes of the mouse placentae after administration of trichosanthin.

Mature ICR female mice were used. On the tenth day of gestation, a single dose of trichosanthin was injected intraperitoneally. The dosage used was 0.1 mg of trichosanthin in physiological saline per 25 g body weight of the mouse. At various prescribed intervals after injection, the mice were sacrificed by cervical dislocation.
Adherence of enterohemorrhagic Escherichia coli in a beef system
Authors: Schwach T. S. , Zottola E. A.
Published: June 2020

Microbiologists have traditionally studied pure cultures growing in laboratory media. Food microbiologists, interested in interactions between the food system, pathogen, and host have tried to extrapolate information in the same manner. However, clinical isolates readily lose virulence characteristics when repeatedly subcultured into laboratory media. Expression of many virulence factors, such as fimbriae, extracellular polymers and outer membrane adhesins are affected by growth environment; an environment which encompasses not only nutrient availability and growth temperature, but also physical surroundings, moisture, oxygen and pH levels, and the presence of competitive flora. The ability of an organism to adhere to a surface is a major virulence factor.

Enterohemorrhagic Escherichia coli (EHEC) are one of five groups of enteropathic E. coli which cause intestinal disease world-wide. The most well known of this group, serotype O157:H7, has been implicated in major food-borne illness outbreaks primarily due to the consumption of undercooked hamburgers. They are found in the intestinal tracts of cattle, feces, hanging carcasses, and frozen ground beef. E. coli O157:H7 adheres to large intestinal epithelium with characteristic lesions and produces verocytoxins which damage internal organs, such as the kidney.
Microscopic study of the plerocercoid larva of Otobothrium (Trypanorhyncha)
Authors: Abdou N. , Heckmann R. A. , Gardner J. S. , Ashour A. A.
Published: June 2020

This study represents the first scanning electron microscope (SEM) evaluation of cestode larvae infecting Red Sea fish. General structures of the larvae and different types of microtriches on the integument are described.

Plerocercoids of Trypanorhyncha were collected from the mesenteries and liver of Cephalopholis oligostricta. The capsule of each larva was opened, the plerocercoid was removed and fixed in 10% formalin. Following buffer (pH. 7.3) wash, they were fixed in OSO4 12 hr, washed in buffer, immersed in 2% tannic acid 8 hr, washed in buffer, fixed again in OSO4 2 hr, buffer washed, dehydrated, critical point dried, and sputter coated with gold.

The plerocercoids were identified as belonging to the genus Otobothrium. Each organism was divided into a scolex and body. The scolex consisted of two large bothria, each bothrium was nearly round in outline and possessed two circular invaginations on its posterior margin. On the apex of the scolex, four extended tentacles covered with hooks were observed (Fig 1).
Methods for three-dimensional reconstruction of cellular components within a thick section
Authors: Frank J. , McEwen B. F. , Radermacher M. , Rieder C. L.
Published: June 2020

The tomographic reconstruction from multiple projections of cellular components, within a thick section, offers a way of visualizing and quantifying their three-dimensional (3D) structure. However, asymmetric objects require as many views from the widest tilt range as possible; otherwise the reconstruction may be uninterpretable. Even if not for geometric obstructions, the increasing pathway of electrons, as the tilt angle is increased, poses the ultimate upper limitation to the projection range. With the maximum tilt angle being fixed, the only way to improve the faithfulness of the reconstruction is by changing the mode of the tilting from single-axis to conical; a point within the object projected with a tilt angle of 60° and a full 360° azimuthal range is then reconstructed as a slightly elliptic (axis ratio 1.2 : 1) sphere.
Resolution and contrast enhancement on biological specimens by means of electron spectroscopic imaging (ESI)
Authors: Hezel U. B. , Bauer R. , Zellmann E. , Miller W. I.
Published: June 2020

The main elemental constituents of biological material - C,H,N,O - are the same elements found in typical embedding materials. Because of this the contrast of unstained biological material is very poor. Additionally, electron scattering by low Z atoms is mainly inelastic resulting in unsharp images from the concomitant chromatic aberration.

These effects have been delt with by employing stains of such heavy metals as Os, U, or Pb. These stains are for the most part located at the biological structures themselves and primarily scatter the electron beam elastically. Thus with ultra-thin (<80nm) heavy metal stained sections of biological material the contrast in the CTEM is very good and chromatic aberrations are negligable.
Electron crystal structure analysis of phospholipids and their solid solutions
Authors: Dorset Douglas L. , Massalski Andrew K. , Fryer John R.
Published: June 2020

Epitaxially crystallized phosphatidylethanolamines produce (Fig. 1) (001) electron diffraction data similar in resolution to X-ray data from oriented multilamellae. An attemped crystal structure analysis with such data based on a rigid body translation of the molecular conformation found in the X-ray crystal structure of the racemic dilauroyl homolog yielded a structural solution unlike that found in our re-analysis of the low angle X-ray data. The X-ray analysis, like other phospholipid crystal structure determinations, indicate that the z-component of the polar head group packing must place phosphorus atoms 4 to 5Å apart in the absence of solvent. The Patterson functions computed from electron or X-ray data are also consistent with this thesis.
Structure of clathrin cages and coats in ice
Authors: Vigers G.P.A. , Crowther R.A. , Pearse B.M.F.
Published: June 2020

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.
Scanning electron microscopy of the integument of schistosoma rodhaini
Authors: Evers P. , Schutte C. , Dettman C. D.
Published: June 2020

S.rodhaini (Brumpt 1931) is a parasite of East African rodents which may possibly hybridize with the human schistosome S. mansoni. The adult male at maturity measures approximately 3mm long and possesses both oral and ventral suckers and a marked gynaecophoric canal. The oral sucker is surrounded by a ring of sensory receptors with a large number of inwardly-pointing spines set into deep sockets occupying the bulk of the ventral surface of the sucker. Numbers of scattered sensory receptors are found on both dorsal and ventral surfaces of the head (Fig. 1) together with two conspicuous rows of receptors situated symmetrically on each side of the midline. One row extends along the dorsal surface of the head midway between the dorsal midline and the lateral margin.
Correlation averaging of stem images of negatively stained adenovirus groups-of-nine herons
Authors: Furcinitti P. S. , Oostrum J. van , Wall J. S. , Burnett R. M.
Published: June 2020

Adenovirus has an icosahedral capsid composed of 252 morphological units of two chemical types. There are 240 copies of the trimeric maior coat protein hexon (Mr 3 x 109,077) and 12 copies of the pentameric penton base and trimeric fibre vertex proteins. The X-ray crystal structure of hexon provides an accurate molecular model for the interpretation of electron micrographs of the virion and its fragments. Dissociation of the virions first releases the 60 peripentonal hexons surrounding the pentons, and then the remaining hexons as planar groups-of-nine hexons (GONs). The disruption pattern of the virion, and the non-random dissociation of the GONs, are at variance with the overall organization of the capsid as small p3 arrays of 12 hexons on each facet.
The effect of ionophore A23178 on the α-actinin crosslinks in the z-band of frog skeletal muscle: An EM study
Authors: Llados F.T. , Krlho V. , Pappas G.D.
Published: June 2020

It Is known that Ca++enters the muscle fiber at the junctional area during the action of the neurotransmitter, acetylcholine (ACh). Pappas and Rose demonstrated that following Intense stimulation, calcium deposits are found In the postsynaptic muscle membrane, Indicating the existence of calcium uptake In the postsynaptic area following ACh release. In addition to this calcium uptake, when mammal Ian skeletal muscles are exposed to a sustained action of the neurotransmitter, muscle damage develops. These same effects, l.e., Increased transmitter release, calcium uptake and finally muscle damage, can be obtained by Incubating the muscle with lonophore A23178.
3-D reconstruction of a regular bacterial surface layer: Effects of limited tilting and preparation conditions
Authors: Engelhardt H. , Saxton W.O. , Baumeister W.
Published: June 2020

A 3-D reconstruction of the regular surface layer of the bacterium Sporosarcina ureae was performed with negatively stained preparations. Particular attention was paid to two major sources of artifacts that may cause problems in the interpretion and comparison of 3-D reconstructions: effects created by missing information due to limited tilting ("missing cone"), and distortions of the protein due to adsorption to the carbon support.