bluuebunny/crossref_metadata_2025 huggingface dataset:
Microstructural defects and their elimination in heteroepitaxial GaAs on silicon-on-sapphire and sapphire substrates
Authors: Posthill J.B. , Parikh N.R. , Das K. , Humphreys T.P. , Nemanich R.J. , Markunas R.J.
Published: June 2020

There is still great interest in developing device-quality GaAs heteroepitaxial systems for several technological applications and advantages. The majority of research effort in this field is currently directed towards GaAs-on-silicon largely because of the strong desire for monolithic integration of GaAs optoelectronics with well-established Si integrated circuitry. But as is now well-known, a high density of microstructural defects has limited the electronic/optoelectronic performance of the grown GaAs layers. Also, the considerable difference in the respective coefficients of thermal expansion leads to the introduction of significant tensile strain in the GaAs epilayer upon cooling. Microcracking has even been observed in thicker (≥ 5μm) films. One way to reduce this phenomenon is to use a substrate with a coefficient of thermal expansion that is closer to GaAs - e.g. sapphire. This contribution reports on some of our recent microstructural studies of GaAs grown on nominal (0001), (), and () sapphire and silicon-on-sapphire (SOS) substrates by molecular beam epitaxy (MBE).
Evolution of dislocation substructures during monotonic deformation of low-carbon steel
Authors: Schmitt J. H. , Johnson P. E. , Morris J. W.
Published: June 2020

It has long been established that in many metals, dislocation substructures (cell structures) develop in the grains during plastic deformation. More recently, it has been shown that, in low carbon steel, the dislocation substructures are closely related to active slip planes, i.e. to the grain orientation and loading conditions. Such a correlation gives rise to an effect of the strain history. This correlation may have an influence on the plastic behavior of prestrained samples and on their plastic stability. In fact, depending on the amplitude of the change of loading conditions, the subsequent stress can be deduced by the use of a generalized latent hardening concept accounting qualitatively for the substructures. On the other hand, during the reloading, some microbands may develop in the grains ; and the appearance of the microbands may be connected with the early occurrence of plastic instability. It is not known however, whether or not the dislocation substructure has an inherent importance, that is whether a heterogeneous distribution of dislocations is required to induce these features. Up to now, this point has not yet been made clear since the models used have only assumed changes in the critical shear stress values or qualitatively described the occurrence of ‘avalanche’ processes. The role of the formation and presence of such substructures on the subsequent behavior of materials requires better understanding.
In situ fatigue of Al-Li-X alloys
Authors: Vecchio Kenneth S.
Published: June 2020

The introduction of Al-Li based alloys into commercial applications has not occurred without some major difficulties in alloy development. Poor ductility and low fracture toughness in these alloys has hindered their use in structures where they must serve as integral load-carrying members. On the other hand, superior fatigue and stiffness properties of these alloys has provided great incentive to pursue further development. The reason for the low ductility and poor fracture toughness has been attributed to several possible mechanisms including: i) shear band formation due to planar slip., and ii) strain localization resulting from large grain boundary precipitates. The rather good fatigue crack propagation characteristics have been attributed to significant macroscopic out-of-plane crack growth. However, little work has aimed at understanding the micromechanisms of this out-of-plane crack growth during cyclic loading. In addition, the role of the numerous precipitates that can exist in these complex Al-Li-X alloys to the fatigue crack growth resistance has not been examined closely.

In this research we have examined the microscopic evolution of fatigue deformation in two Al-Li-X alloys using in-situ fatigue experiments conducted within an intermediate voltage transmission electron microscope.
Dose rate and temperature dependence of the critical dose for amorphization of CuTi-irradiated by 1 MeV electrons
Authors: Xu Genbao , Koike Junichi , Meshii M. , Okamoto Paul
Published: June 2020

The dose rate and temperature dependence of critical dose for amorphization of CuTi under electron irradiation was investigated in a temperature range from 10 to 260K and in a dose rate range from 1.0 × 10−4 to 1.5 × 10−3 dpa/sec, using the Argonne HVEM and a helium coding specimen stage. An alloy button of Cu-54at%Ti was prepared by arc-melting and subsequently annealed at 1173K for two days. HVEM samples were spark-cut and thinned by jet polishing. The irradiation was carried out in HVEM with a fully focused beam. The Gaussian flux distribution of the beam was utilized to study the dose rate dependence of amorphization.

Fig. 1 shows a few example photographs taken during a sequence of irradiation. The irradiation was always carried out on a low index bend contour. An interruption in the bend contour appears at the beam center where the dose rate is highest, as the irradiation proceeds. With further irradiation, the interruption grows outward into regions of lower dose rate. The critical dose for amorphization was calculated from each micrograph by multiplying the irradiation time with the predetermined dose rate. The length of the interruption in the bend contour was taken as the diameter of a completely amorphized volume at the accumulated dose. About ten micrographs were usually taken during a sequence of irradiation.
TEM characterization of MBE-grown interfaces for inverted-HEMT structures
Authors: Cole M.W. , Lehman J.L. , Newman P.G. , Dornath-Mohr M. , Smith D.D. , Guha S. , Madukar A.
Published: June 2020

With the advent of the high electron mobility transistor (HEMT), device mobilities in excess of 130,000 cm2/V-sec (77K) for an MBE-grown GaAs/AlxGa1-xAs (100) (0.25>x>0.30) heterojunction have become commonplace. However, the corresponding growth of high quality AlGaAs/GaAs inverted HEMT structures (Figure 1) has been difficult due to the high degree of disorder at the inverted heterointerface (maximum 77K mobility 80,000 cm2/V-sec). The AlGaAs growth front is subject to: 1) slower migration kinetics of Al with respect to Ga, 2) high reactivity of Al to the ambient impurities, and 3) outdiffusion of the silicon dopant along the growth front, which further compromises the uniformity and planarity of the growth. Past attempts to grow inverted HEMTs have been limited by the competing parameters of high substrate temperatures (T) prefered for achieving high migration of Al for smoother interfaces, and lower Ts to minimize silicon outdiffusion toward the critical interface. In this presentation we introduce TEM characterization of a novel MBE technique which optimizes interface growth conditions, achieving mobilities >120,000 cm2/V-sec, for the inverted HEMT structure.
Shear-induced changes in chain configuration of poly(acrylic acid) in dilute solution
Authors: Armistead J.P. , Price R.R. , Kim O.-K. , Choi L.-S.
Published: June 2020

Small amounts (less than 30 ppm) of polymer dissolved in solution may significantly reduce the work required to pump fluids through a pipe at a given rate. In other words, the drag of the solution along the pipe walls is reduced. Drag reduction by polymers has been well characterized, however the molecular origin of the phenomena is not fully understood. Polymers that exhibit drag reduction characteristics typically have high molecular weight, have predominantly linear, flexible chains, and have an expanded molecular configuration in solution.

Work in this laboratory has focused on the drag reduction behavior of poly(acrylic acid), PAA, in recent years. This polymer is one of the most shear stable water-soluble polymers and due to the ionic groups in the polymer chain its conformation in solution changes with pH and ionic strength. In a recent work, PAA solutions of 18 ppm, pH=8.1, showed an initial drag reduction of over thirty-five percent in rotating disc experiments. Over four minutes of shearing the drag reduction decreased to ten percent. This was surprising because of the known shear stability of PAA. When the sheared solution was left undisturbed for two weeks, it did not recover its drag reduction performance. However, the addition of NaCl to the solution during the shearing immediately restored drag reduction to its initial level. It was hypothesized that the shear flow induced interchain association that was possibly stabilized by hydrogen bonding and that the addition of the NaCl caused dissociation and drag reduction recovery. In additional work, fluorescence probe studies showed that shear flow induced local chain rigidity in the originally flexible polymer chains. In this study, the drag reduction experiments were repeated and the configurations of the sheared and unsheared polymer chains were viewed using electron microscopy.
Reduction of contamination using a specimen heating holder in an ultra high resolution Scanning Electron Microscope
Authors: Ogura K. , Ono A. , Kersker M. M.
Published: June 2020

In general, various improvements have been made to SEM vacuum systems, and clean high vacuum specimen chambers are now routinely available. However, in the ultra high resolution scanning electron microscope, the prevention or reduction of contamination on the specimen surface has recently become an important subject when SEM imaging is done at higher than 200,000x magnification using a very fine electron probe. Typically, the specimen carries hydrocarbon gas molecules which are the source of the contamination, into the SEM. They adhere not only to the specimen surface but may also incorporated in the specimen, most typically in biological specimens, and cannot be reduced by the anti-contamination device of the SEM. Recently, a specimen heating holder was used in a JSM-890 ultra high resolution SEM, to reduce the contamination deposition on the specimen surface during SEM imaging. Using this holder, the specimen can be heated up to 300°C inside the SEM. Images 1 to 4 in Fig. 1 are the secondary electron images showing the cone-shaped deposition of contamination on a platinum-coated carbon film at different heating temperatures. This platinum-coated film, which had been kept in wet and oily atmosphere for several weeks to insure it was well covered with hydro carbon gas molecules, was irradiated by an electron probe in a spot mode for 30sec. with 1×10−11 Amp. of probe current at 20kV. After the electron probe irradiation, the platinum-coated carbon film was tilted 45° for imaging. Image 1 in Fig. 1 shows the cone-shaped deposition of contamination when the specimen was not heated. Image 2 was at 35°C, Image 3 was at 55°C, and Image 4 in Fig. 1 was at 115°C. At higher than 120°C specimen heating temperature, the cone-shaped deposition of contamination could not be observed any more. On the other hand, we can heat up the specimen outside the SEM before we put the specimen into the SEM. Image 5 in Fig. 1 shows the results of specimen heating by a hair dryer. The same platinum- coated carbon film was heated by a hair dryer for 1 minute before it was intro- duced into the SEM, and was irradiated by the electron probe for 15, 30, and 45sec. in a spot mode. This 1 min. heating by a hair dryer shows almost same result as 55°C specimen heating in the SEM.
Microchannel plate-based detection systems for Scanning Electron Microscopy
Authors: Gray J. , Corey D. , Ellis G. , Sokol R.
Published: June 2020

MicroChannel plate (MCP)-based detectors have distinct advantages over scintillator or solid-state types for low voltage applications in scanning electron microscopy. MCPs exhibit excellent detection efficiencies for electrons of energy down to lOOev and are usable at even lower energies, making them ideal detection elements for non-destructive testing and low voltage contrast measurements on integrated circuits. Major advantages of MCP-based detection systems include the following
High-resolution, extended views of membrane surfaces revealed by fracture-flip
Authors: da Silva Pedro Pinto
Published: June 2020

I will describe a new method — fracture-flip — that uses commercially available equipment to produce extended views of cell and membrane surfaces. The resolution of this new method permits the routine identification of surface structures down to 5 nm diameter. Moreover, in contrast to freeze-etching/deep-etching, extended views are easily obtained.

Conceptally, fracture-flip derives from label-fracture, another method developed in my laboratory. With label-fracture we showed that, after freeze-fracture, the exoplasmic (E) halves of cell membranes are stabilized by, and remain attached to, their platinum/carbon replicas. This allows the observation of co-incident views of the Pt/C replica of the E face, and of the distribution of colloidal gold labeled receptors or antigens. This is the sequence of steps in fracture-flip:
Organization and composition of the cytoskeleton of giant algal cells: Its role in wound healing
Authors: Goddard R. H. , La Claire II J. W.
Published: June 2020

We have been using the giant algal cells of Ernodesmis verticillata as a model system to study the process of wound healing at the cellular level of organization. Using immuno-localization techniques we have observed changes in the distribution of tubulin-containing microtubules (MTs) and actin-containing microfilaments (MFs), that occur during wound healing. Based on these and new findings, we carried out correlative experiments with inhibitors to investigate the functional roles of the various cytoskeletal components in wound healing.

Emodesmis cells were cultured, Wounded and Fixed as described previously. The cytoplasm from fixed cells was adhered to polylysine-coated coverslips for light microscopy, or to formvar coated gold grids for transmission electron microscopy (TEM).The attached cells were labeled with primary antibodies (against tubulin ,actin,calmodulin [CAM], intermediate filament [If] proteins, or myosin)followed by incubation in appropriate secondary antibodies conjugated with FITC or TRITC for fluorescence, or conjugated to gold beads for TEM.
Traffic to the endocytic compartment of plants
Authors: Griffing L. R. , Record R. D. , Mollenhauer H. H.
Published: June 2020

The endocytic pathway of plants has been identified and partially characterized using nonspecific membrane-bound and fluid phase probes . The function of endocytosis in plants is, however, unknown. We shall describe how ultrastructural histochemistry, immunocytochemical analyses and fluorescence imaging have been used to explore the physiology and function of the endocytic pathway in plant protoplasts and whole cells.

Cationized ferritin (CF) can be used as a marker of plasma membrane uptake in plant protoplasts. Several different organelles become labeled upon exposure of protoplasts to CF: clathrin-coated vesicles (CV), the partially coated reticulum (PCR), the Golgi complex (GC), the multivesicular body (MVB), and the vacuole (V). These organelles also participate in the pathways of secretion and delivery of protein to the lysosome (vacuole). What are the sites of overlap/divergence among the secretory, endocytic and lysosomal pathways in these cells?
Morphogenetic plastid migration and spindle dynamics in simple land plants
Authors: Brown R.C. , Lemmon B. E.
Published: June 2020

In many of the simple land plants mitosis and/or meiosis occurs in cells that contain a single plastid. Preparation for division in these monoplastidic cells is especially obvious because of predictive migration and division of the single plastid. The plastids serve as foci for organization of spindle microtubules (Mts) resulting in infallible coordination of plastid and nuclear division. Thus unlike most plant cells where a distinct MTOC is difficult to distinguish, monoplastidic cells provide a system in which the material responsible for nucleating Mts is closely associated with the plastid envelope and can be followed throughout the cell cycle. The intimate association of plastids with spindle poles has suggested the concept of plastid polarity which holds that the behavior of plastids parallels that of animal centrosomes. This concept is supported by recent studies using correlated methods of modern botanical microscopy.

Mitosis was studied in the basal meristem of hornwort sporophytes and in root tips of Isoetes and Selaginella.
Freeze fracture analysis of cellulose terminal synthesizing complexes in Vaucheria
Authors: Mizuta Shun , Brown R. Malcolm
Published: June 2020

It is well known that cellulose is assembled by specialized enzyme complexes (known as terminal complexes = TCs) located on or within the plasma membrane.At least three types of TCs have been described: a single linear row of particles observed in the brown alga Pelvetia; linear multiple rows in Oocystis and in some green algae; and, hexagonally arranged particle “rosettes” in some algae and solitary rosettes in all land plants. A new linear type of TC has been found in a freshwater xanthophycean alga Vaucheria hamata. Fine structure of the TC has been analyzed by high resolution analytical techniques for freeze-replication. Further examination for its characteristics was carried out by the treatment with inhibitors for cellulose formation(2,6-dichlorobenzonitrile, DCB) and its crystallization (Tinopal LPW).

v. hamata was cultured under appropriate light conditions and single cells were cut into small pieces and recultured for two days. They were plasmolyzed in 0.2M NaCl, washed with fresh medium, and then recultured with or without inhibitors for 4-10 h. The cells were placed on Balzers double replica supports and quickly frozen in liquid propane. The supports were inserted in a Balzers double replica holder cooled by liquid nitrogen and placed in the chamber of a Balzers BA 360. Cells were fractured at 2x10-4 Pa and etched for 1 min. Most of the specimens were rotary shadowed (1 rps) with Pt-C at an angle of 30° and coated with carbon. Replicas were cleaned with 50% sulfuric acid containing 5% sodium dichromate and distilled water, and observed at 80Kv on a Philips EM 420.
Scanning tunneling microscopy and atomic force microscopy: New tools for biology
Authors: Bracker CE , Hansma P. K.
Published: June 2020

A new family of scanning probe microscopes has emerged that is opening new horizons for investigating the fine structure of matter. The earliest and best known of these instruments is the scanning tunneling microscope (STM). First published in 1982, the STM earned the 1986 Nobel Prize in Physics for two of its inventors, G. Binnig and H. Rohrer. They shared the prize with E. Ruska for his work that had led to the development of the transmission electron microscope half a century earlier. It seems appropriate that the award embodied this particular blend of the old and the new because it demonstrated to the world a long overdue respect for the enormous contributions electron microscopy has made to the understanding of matter, and at the same time it signalled the dawn of a new age in microscopy. What we are seeing is a revolution in microscopy and a redefinition of the concept of a microscope.

Several kinds of scanning probe microscopes now exist, and the number is increasing. What they share in common is a small probe that is scanned over the surface of a specimen and measures a physical property on a very small scale, at or near the surface. Scanning probes can measure temperature, magnetic fields, tunneling currents, voltage, force, and ion currents, among others.
Fluorescence microscopy and image analysis studies showing how plastids grow
Authors: Coleman A.W. , Maguire M.J.
Published: June 2020

Biochemical analyses have shown that leaf plastid material/cell, both genomes and other materials, increase during growth and in response to light and other cues. However, the biochemical methods inevitably express results as an average per cell, leaving the precise timing of events in the cell cycle and course of ontogeny uncertain, and the variation in guantity/cell largely unknown. The power of quantitative microscopy lies in the capability of determining information on the single cell or single plastid level.

To test the feasibility of using microscopic methods to study plastid growth and variability, we chose as an analog of the plant meristematic cell, the photosynthetic alga Ochromonas danica. This unicellular Chrysophyte combines a number of advantageous features. It typically has but 1-2 plastids and these occupy only a small proportion of the cell volume. Secondly, Ochromonas grows rapidly both in light and in darkness to high cell densities. Thirdly, the cell lacks a wall, so that its contents can be examined in detail in living cells flattened beneath a coverslip. Thirdly, the organism synthesizes chlorophyll both in light and darkness, so that plastids in living cells, however small, can be distinguished by virtue of the autofluorescence of their chlorophyll. Finally, for measurement of DNA per plastid, the arrangement of plastid DNA in Ochromonas plastids, as a necklace closely underlying the perimeter of the plastid, ensures that when one has isolated, stained intact necklaces, one is measuring the entire DNA content of the plastid.
In situ analysis of cell lineage and axonal guidance in the developing nervous system
Authors: Fraser S.E.
Published: June 2020

The development of the nervous system involves the same classes of cell movements and differentiations as seen in the development of other tissues; however, the nervous system adds the complication of axonal processes that can travel over large distances to establish selective connections. Typically, the lineages of the neuronal precursors, their migrations, or the migrations of neuronal growth cones are studied in tissues fixed at different stages. Such timed samples can be used to infer some aspect of the developmental processes, but cannot offer any direct information on the dynamic aspects of these processes. This is a severe limitation because it is becoming ever-apparent that cellular dynamics provide the driving force behind many developmental phenomena.

This presentation will discuss techniques that permit individual cells to be uniquely identified and followed over time as development proceeds in intact vertebrate embryos. There are two challenges to be met in performing an in situ analysis of neural development: unique labeling of individual cells, and observation of the cells. Labeling of individual cells is accomplished by microinjection of the vital dyes lysinated fluorescein dextran (LFD) or lysinated rhodamine dextran (LRD). Observation of the labeled cells is simplified by the recent strides in computer enhanced video microscopy. Much of the work has utilized the embryos of the frog, Xenopus laevis. The embryos of this species are hardy and have been the subject of a large body of experimental work. An important feature for these experiments is that the tissues surrounding the brain become progressively more transparent as development proceeds, offering the possibility of directly observing the labeled cells non-invasively in an intact, living embryo. Other experiments utilize the chicken embryo, because of the careful experimental embryology of the past decades provides a fertile backdrop for these new experimental approaches.
Applications of post-embedding immunogold labelling in the study of human tumors
Authors: Hearn Stephen , Kontozoglou Theodosios , Cramer Harvey
Published: June 2020

Post-embedding immunogold labelling (IEM) can provide an exact correlation between the ultrastructure of tumor cells and the location of specific antigens. This technology links the molecular features of tumor cells derived from immunohistochemistry with our knowledge of tumor ultrastructure from electron microscopy. Criteria for the use of IEM in the study of human tissues include: the ability to localize many different antigens with commercially available antibodies; specimen preparation that is rapid, standardized and effective for different biopsies and tissue culture; results should compare with immunoperoxidase labelling on formalin-fixed, paraffin-embedded tissue.

The tumors examined in this study included: squamous carcinomas, melanomas, multiple myelomas, schwannomas, medullary carcinomas of the thyroid, adenocarcinomas, leiomyomas, germ-cell carcinoma, myeloid leukemia, lymphomas, merkel cell carcinoma, and carcinoid tumors. Fine-needle aspiration biopsies (FNAB), core biopsies of bone marrow, peripheral blood, cell culture monolayers and surgically removed tissues were fixed in 1.6% glutaraldehyde. Samples were dehydrated, infiltrated in Lowicryl K4M and UV polymerized (total preparation time: 3 days). Technique for FNAB samples used mesh filtration to purify tumor cells which were then entrapped in albumin prior to embedding to prevent loss of cells. Glass coverslips (4x2mm) bearing cell monolayers were fixed in situ, infiltrated, placed in gelatin capsules and the cells separated from the coverslips after polymerization for en face sectioning. Thin sections were immunolabelled with readily available monoclonal and polyclonal antibodies using the protein A-gold technique. Samples of normal tissues were labelled for each antigen as positive controls and the negative controls used had appropriate low levels of non-specific labelling.
Structure of agarose gels: Mean pore radius
Authors: Griess Gary A. , Serwer Philip
Published: June 2020

To use an agarose gel's sieving for the biophysical characterization of macromolecules and supramolecular complexes, the effective gel's pore radius (PE) is determined as a function of the agarose percentage (A). In previous studies, performed by use of agarose gel electrophoresis, the sieving of spheres of known radius (R) was used, together with two different (unproven) theories of sieving, to obtain PE. The PE values obtained were self-consistent and independent of R. The PE vs. A relationship did not agree with that predicted by a model that represents the gel as straight, randomly-oriented fibers (random fiber model). To test the accuracy of both the empirical PE vs. A relationship and the previously assumed model of gel structure, in the present study thin sections of agarose gels have been examined by use of electron microscopy.

Gels of the agarose previously used to quantify sieving (SeaKem LE, Marine Colloids) were cast in the buffer previously used: 0.025 M sodium phosphate, pH 7.4, 0.001M MgCl2. Pieces of gel were fixed with osmium tetroxide, dehydrated and embedded in Epon. Dark gold (120 nm) sections were examined with a JEM-100CX transmission electron microscope. Electron micrographs were captured with a DataTranslation QuickCapture video digitizer and were processed on a Macintosh II computer using the program, Image.4
Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment
Authors: Aggarwal S.K.
Published: June 2020

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.

Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.
Focal adhesions to the synaptic matrix at mouse neuromuscular junctions
Authors: Robbins Norman , Polak Joseph
Published: June 2020

Studies of identified synapses in living animals have revealed ongoing synaptic remodelling in the mature nervous system. We recently demonstrated the existence of filopodia and lamellipodia (structures associated with nerve outgrowth in developing or tissue culture systems) at the mature mouse neuromuscular junction (NMJ) . Evidence of motor nerve terminal retraction, regeneration and perisynaptic outgrowth indicated that even established synaptic contacts were not permanent. It now appears that variation in the stability of synaptic contacts, not the amount of nerve terminal outgrowth,is the basis for synaptic remodelling and extension. Although it has been documented that regenerating motor nerve terminals adhere to former synaptic matrix,and that the matrix contains information for inducing and aligning presynaptic and postsynaptic specializations,it was not clear whether the synaptic matrix was uniformly adhesive, or formed specialized adhesive foci with the preterminal membrane. To determine if there are specific sites of synaptic adhesion we utilized hypertonic fixatives to induce shrinkage at the NMJ.

Male mice were anaesthetized, the soleus muscle was exposed, and the overlying skin was retracted to create a pool into which either 2% glutaraldehyde in HEPES Krebs (normal fixative) or 2% glutaraldehyde in HEPES Krebs with a 2X NaCl concentration (hypertonic fixative) was added continually for 30 minutes. The surface layer of each muscle was then removed and submerged in the same fixative for an additional 1.5 hours. Tissue was washed in buffer (pH 7.2) and immersed in cholinesterase stain for 5 minutes to localize endplate regions . Endplate regions were cut into 1 mm blocks, washed in buffer (pH 7.2) and post-fixed for 1 hour in HEPES Krebs buffered 2% osmium tetroxide (pH 7.2). Following fixation, specimens were dehydrated in a graded ethanol series and embedded in Polybed 812 (Polysciences). Mouse soleus muscle was stained for immunocytochemical localization of actin with a rabbit polyclonal antiserum against chicken gizzard actin (from Dr. James Lessard, University of Cincinnati) as previously described .
Structural and elemental characterization of heart cells grown in a collagen matrix
Authors: LeFurgey A. , Hawkey L.A. , Carney M.C. , Ingram P. , Lieberman M.
Published: June 2020

Cultured embryonic chick heart cells have been utilized as a model system for characterization of various membrane transport mechanisms. One advantage of this system is that the cells may be grown with differing geometries to minimize diffusion limitations and to optimize the growth configuration for particular techniques, such as ion-selective microelectrode measurements, fluorescent dye indicators, patch clamp, etc. A spontaneously contracting strand of cells embedded in a collagen matrix has recently been developed for measurements of cytoplasmic free ions by nuclear magnetic resonance (NMR) spectroscopy. These same strands, which provide the large numbers of cells needed for NMR, can be subdivided into small fragments ideal for cryopreservation prior to electron probe X-ray microanalysis (EPXMA). The aims in this study were to characterize the ultrastructure of cardiac cells within the strand, to demonstrate the quality of preservation obtainable by quick freezing methods, and to quantitatively map with EPXMA the distribution of physiologically relevant elements in thin, freeze-dried cryosections of the cells.

Cells were isolated by serial trypsinization of 11-day old embryonic chick hearts. Strands of cells approximately 100 cm in length and 0.2 mm in diameter were obtained by extrusion of a cell-collagen mixture through polyethylene tubing into media within a culture dish. Three to five millimeter segments of 1-day old strands which contracted spontaneously were preserved by rapid immersion in liquid nitrogen-cooled liquid propane at 〜-185°C and stored in liquid nitrogen prior to being (a) cryosectioned for subsequent EPXMA or (b) freeze-substituted for conventional transmission electron microscopy (CTEM). Segments of strands were also chemically preserved in 2.5% glutaraldehyde in 0.1 M sodium cacodylate and processed as above for comparative CTEM. Cryosections of the frozen strands were cut at <-140°C with a dry glass knife and placed directly onto pre-cooled, carbon-coated, 200 mesh, fine bar nickel grids with a precooled implement. The grids were transferred to a liquid nitrogen cooled copper well for freeze drying at 10‒3 Torr over 24 to 48 hours. Prior to EPXMA, the grids were coated with 〜1OOÅ carbon.
Patterns of mucilage production and secretion in pennate diatoms
Authors: Sicko-Goad L. , Kociolek J. P , Stoermer E. F.
Published: June 2020

Although mucilage production is ubiquitous among diatoms, there have been few studies documenting its production and secretion. Several studies have identified a regular arrangement of Golgi, endoplamic reticulum, and mitochondria as being responsible for mucilage production in centric diatoms. However, such an arrangraent has not been identified for pennate diatoms, although fibrillar vesicles have been described in the vicinity of Golgi and mitochondria and are believed to be involved in mucilage production. The production and secretion of mucilage in certain taxa of pennate diatoms is especially important because it provides various types of growth forms including stalks, tubes and encapsulated cells. The following report compares ultrastructural differences among araphid, monoraphid and biraphid diatoms.

Collections of mucilage-producing diatoms exhibiting different growth forms were taken from rocks along the shores of Lakes Michigan and Superior. Samples were field-fixed in 1% paraformaldehyde-glutaraldehyde in cacodylate buffer and stored on ice until samples were returned to the lab. Samples were then postfixed in 1% OsO4, dehydrated in a graded ethanol-propylene oxide series, embedded in Embed 812 and sectioned with a diamond knife. Sections were collected on cleaned formvar- coated 100 mesh copper grids and examined with a Philips 400 TEM.
Two structural states of the Z-band in cardiac and skeletal muscle
Authors: Schroeter J.P. , Goldstein M.A. , Bretaudiere J.P. , Michael L.H. , Sass R.L.
Published: June 2020

We have recently established the existence of two structural states of the Z band lattice in cross section in cardiac as well as in skeletal muscle. The two structural states are related to the contractile state of the muscle. In skeletal muscle at rest, the Z band is in the small square (ss) lattice form, but tetanized muscle exhibits the basket weave (bw) form. In contrast, unstimu- lated cardiac muscle exhibits the bw form, but cardiac muscles exposed to EGTA show the ss form.

We have used two-dimensional computer enhancement techniques on digitized electron micrographs to compare each lattice form as it appears in both cardiac and skeletal muscle. Both real space averaging and fourier filtering methods were used. Enhanced images were displayed as grey-scale projections, as contour maps, and in false color.

There is only a slight difference between the lattices produced by the two different enhancement techniques. Thus the information presented in these images is not likely to be an artifact of the enhancement algorithm.
Transmission and Scanning Electron Microscopic studies of ocular leprosy in nine-banded armadillos
Authors: Malaty R. , Pedroza L. , Beuerman R.W.
Published: June 2020

Leprosy is one of the leading causes of preventable blindness in the world, yet It is largely ignored in blindness prevention programs. To date, little is known about the pathogenesis of ocular leprosy in humans. Histopathological studies have been necessarily 1imited to eyes removed at autopsy or enucleated from patients with advanced lepromatous leprosy. In an attempt to define the pathogenesis of the ocular complications of leprosy, we have studied the eyes of animal models namely, nine-banded armadillos with 1epromatous leprosy and injectedMycobacterium leprae(M. leprae) directly in the corneal stroma of armadillos.

Fifty armadillos with experimental or naturally-acquired lepromatous leprosy were studied. Six armadillos were injected in the corneal stroma with 1 ul of anM. lepraesuspension and examined two and four months later. Both eyes of each animal were enucleated at autopsy, fixed in paraformaldehyde-glutaraldehyde and processed for light and electron microscopy (EM). For light microscopy, the eyes were embedded in plastic methacrylate and sections stained with a modified acid-fast stain. For EM, tissues were post fixed in 1% OsO4dehydrated, and embedded in epon.
Ultrastrctural identification of viral infections in paraffin sections stained by immunohistochemistry and in situ hybridization
Authors: Chien K. , Shintaku I.P. , Wu G.D. , Van de Velde R.L. , Heusser R. , Danao F. , Geller S.A.
Published: June 2020

Confirmation of viral infections in surgical biopsies is sometimes limited to routine paraffin sections because a specimen may not have been set aside for culture or culture results are negative. Since viral infections are frequently focal and localized to only a few cells, definitive histopathological diagnosis can be difficult to achieve even with immunohistoGhemistry (IHC), in situ hybridization (ISH) or electron microscopy. In this paper, we would like to demonstrate a siirple and direct approach to confirming viral infections in paraffin sections , using two immuno-labelling techniques coupled with electron microscopy.

Paraffin sections of surgical specimens are deparaffinized in xylene and rehydrated through graded ethyl alcohol. Herpes simplex virus (Figure 1) and Toxoplasma gondii are localized immunohistochemically by the peroxidase antiperoxidase technique and Pneumocystis carinii (Figure 2) by 3 step ipdirect method, using 3,3'-diaminobenzidine tetrahydrochloride as the chrmogen, CMV ENA probe (ENZO Biochemicals) is used for in situ hybridization, following kit instructions (Figure 3 & 4). After the final chromogen reaction, wet sections are examined with a light microscope. Slides in vrfiich focal positive staining is difficult to interpret as specific are fixed in osmium tetroxide, dehydrated with graded acetone, and infiltrated with Poly/Bed 812. After polymerization, the epoxy blocks containing area of interest can be easily separated from the glass slides by heating on a 100 C hotplate for approximately 20 seconds . The area of interest should be positioned in the center of the block face for ultrathin sectioning. With careful adjustments of knife and microtcme, the area of interest can be precisely sectioned without loss of tissue. Ultrathin sections are collected on uncoated mesh grids for higher contrast and then directly examined by TEM. Peroxidase-labelled microorganisms or viral DHA can be easily localized and identified without heavy metal staining. Cellular details can be further increased by staining with uranyl acetate and lead citrate. Tissue remaining in block face after sectioning is available for IM comparison with the EM micrographs.
Quantification of cell-surface expressed antigenic sites with 5-nm gold markers: Getting close to biologically relevant data
Authors: de Harven Etienne , Christensen Hilary , Leung Richard , Ackerley Cameron
Published: June 2020

The T-derived subset of human peripheral blood normal lymphocytes has been selected as a model system to study the usefulness of 5 nm gold markers for quantification of single epitopes expressed on cell surfaces. The chosen epitopes are parts of the CD3 and CD5 molecules and can be specifically identified by hybridoma produced monoclonal antibodies (MoAbs; LEU-4 and LEU-1; Becton-Dick- inson, Mountain view, CA) . An indirect immunolabeling procedure, with goat anti-murine IgG adsorbed on the surface of 5 nm colloidal gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) has been used. Backscattered Electron Imaging (BEI) in a field emission scanning electronmicroscope (SEM) and transmission electron microscopy of thin sections of lymphocytes labeled before plastic embedding, were both used to identify and quantitate gold labeled cell surface sites, Estimating that the thickness of “silver” sections is approximately 60 nm and counting the number of gold particles on the entire cell perimeter, we calculated that, for LEU-4, the number of markers per um2of cell surface is in the 140-160 range (Fig.l). Cell contour length measurements indicated that the surface of one lymphocyte is approximately 130-160 um2that of a smooth sphere of identical diameter, reflecting the role of microvilli in expanding the surface area. The total number of gold labeled sites on the surface of one lymphocyte averages, therefore between 20,000 and 24,000 per cell.
Ultrastructural localization of acid phosphatase in lactating rat mammary gland
Authors: Farrell Harold M. , Maleeff Beverly E. , Wickham Edward D. , Leung Cecilia T.
Published: June 2020

Acid phosphatases occur in a variety of mammalian tissues. Generally these enzymes can be divided into degradative enzymes (lysosomal) which are tartrate inhibited and general acid phosphatases which are not tartrate sensitive. The metabolic function of the latter class of enzymes is uncertain, but many phosphatases reverse the regulatory phosphorylation of enzymes, carried out by specific protein kinases. Mammary tissue actively accumulates casein, calcium and inorganic phosphate in secretory vesicles; these components subsequently condense into the colloidal complexes (casein mice lies) found in skim milk. In addition, tartrate insensitive acid phosphatases are found in mammary secretory tissue and in milk We therefore investigated the cytochemical distribution of mammary acid phosphatase to determine if any activity is associated with the endomembrane system responsible for the secretion of casein micelles.

Tissue from lactating female Sprague-Dawley rats, 8-10 days postpartum, was excised
in blocks of 3 to 5 mm, immediately placed in ice-cold fixative (2% formaldehyde,0.25% glutaraldehyde in 0.lM cacodylate buffer, pH 7.4) , and minced with a razor blade. After fixation for 15 minutes, the finely minced tissue was transferred to 0.lM cacodylate buffer containing 8% sucrose (w/v) and 10% (v/v) dimethyl sulfoxide (DMSO) and kept overnight at 40°C.
Electron Microscopy as an aid to diagnosis in pediatric hematology/oncology
Authors: Saving K. L. , Caughey R. C.
Published: June 2020

This presentation is designed to demonstrate how scanning and transmission electron microscopy techniques can be utilized to confirm or support a variety of unusual pediatric hematologic/oncologic disorders. Patients with the following diagnoses will be presented: (1) hereditary pyropoikilocytosis, (2) familial erythrophagocytic lymphohistiocytosis, (3) acute megakaryoblastic leukemia, and (4) pseudo-von Willebrand’s disease.

All transmission and scanning electron microscopy samples were fixed in 2.5% glutaraldehyde, rinsed in Millonig’s phosphate buffer, and post-fixed with 1% osmium tetroxide. The transmission samples were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole ’ s non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin. Ultramicrotomy thin sections were stained with uranyl acetate and lead citrate and scanned using a JEOL-JEM 100C, The scanning samples were dehydrated with graded series of ethanols, critical point dried with CO2, gold-coated, and scanned using a JEOL-JSM 35. The peroxidase samples were fixed in 3% glutaraldehyde, incubated in diaminobenzidine (DAB), dehydrated with ethanol, embedded with Epon 812, and scanned without post-staining using a JEOL-JEM 100C.
Ultrastructural study of pulmonary endothelium in mouse model of the IL-2-induced vascuiar leak syndrome
Authors: Valentine T. M. , Anderson T. D.
Published: June 2020

Interleukin-2 is being evaluated in cancer immunotherapy in people. rIL-2 is associated with vascular leak syndrome (VLS) characterized by peripheral edema, ascites and pulmonary edema. The histopathologic changes associated with rIL-2 administration have been described in mouse models, however, the ultrastructural changes in VLS are undefined. In these studies we have utilized a mouse model of VLS to characterize the pulmonary ultrastructural changes which are associated with pulmonary edema.

B6D2F1 (BDF) mice, (3M/3F/groups) from Charles River Lab., New York, were treated intraperitoneally for five days with vehicle for rIL-2 or with 30,000 U/gm rIL-2, BID as previously described. At necropsy, lungs were fixed by intratracheally at perfusion pressure of 30cm of H2O with modified Karnovskys fixative diluted with 0.2M sodium caccdylate buffer, ph7.4, for 24 hours. Transverse sections of the left lung (1mm thick) were post-fixed in osmium tetroxide (2hrs) and processed for electron microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Philips 300 electron microscope.
Positive stain for light and electron microscopic demonstration of spirochetes in subgingival plaque and crevicular fluid samples from periodontal diseased sites
Authors: Giammara B. , Burkes E.J. , Scruggs R. , Greco G. , Yates P. , Hanker J.
Published: June 2020

In a recent study of 400 subgingival plaque samples from over 110 adult periodontitis patients, spirochetes were the overwhelming microbial type, averaging about 45% of the microbial count. This finding supports earlier arguments that spirochetes are pathognomonic in periodontal disease. Other studies had shown clear-cut differences in the microbial composition of healthy and diseased subgingival sites — the proportion of spirochetes being significantly higher in the latter. Another study indicated that periodontal deterioration at these sites could be predicted better by increased proportions of motile rods and spirochetes than by clinical measurements. However, spirochetes of all sizes and species do not show the same degree of association with periodontal breakdown. Moreover, spirochetes are usually difficult to culture and stain; they are generally monitored by darkfield or phase contrast microscopy.

The PATS reaction, a modified periodic acid-Schiff(PAS) reaction which deposits silver for light and electron microscopy appears to stain Gram(-) bacteria positively as well as neutrophils and activated macrophages. When studying the stained Gram(-) bacteria on coverslip smears of subgingival plaque or crevicular fluid samples of patients by light microscopy, varying numbers of intensely stained spirochetes of different sizes were observed (Figs. 1,2). More spirochetes were usually seen in samples from diseased sites. After drying replicate PATS-stained coverslips with hexamethyldisilazane they were sputter coated with gold, and. then examined by the SEI and BEI modes of scanning electron microscopy (Figs. 3-6). A permanent record of the proportions of large, medium and small spirochetes at each site could thus be obtained. Generally, greater numbers of gram negative bacteria including some spirochetes were stained in samples from diseased sites. At some sites, however, spirochetes were the predominant microbes in both crevicular fluid and subgingival plaque (Fig. 1).