bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycerol Permeabilization of Fluorescently-Labeled Phalloidin Improves Actin Visualization in Vibratome-Sectioned Roots
Authors: Blancaflor EB , Hasenstein KH
Published: July 2020

The actin cytoskeleton is involved in various cellular functions including cell shape determination, organelle transport, and cell polarity establishment. In order to study the function and dynamics of actin in plant cells, methods to image its orientation have to be optimized. Although, green fluorescent proteins (GFP) from the jellyfish Aequoria victoria have been used for visualizing actin filaments in plant cells, the technique is still in its infancy and has some limitations. Actin in plant cells is difficult to preserve and hence visualize mainly because of the problems associated with specimen fixation and processing. This is particularly true for bulky specimens such as plant organs because of the additional requirement for embedding and sectioning after the tissues are fixed and dehydrated. In order to overcome these difficulties, we have modified a method previously used for onion epidermal cells and suspension cells, and applied this to fresh Vibratome sectioned root tissues.
Visualization of Uptake of High-Density Lipoprotein by Rat Aortic Endothelial Cells and Smooth Mus.Cle Cells in Vitro
Authors: Chao W T , Yang V C
Published: July 2020

The high concentration of low-density lipoprotein in the plasma is the major risk factor of atherosclerosis. On the other hand, another plasma lipoprotein—high-density lipoprotein (HDL) — is inversely correlated with atherosclerosis. Recent studies have demonstrated that HDL mediates the transport of cholesterol from peripheral tissues to the liver through “reverse cholesterol transport” pathway. However there is considerable debate about the mechanisms by which HDL removes excess cholesterol from cells. Two different pathways were suggested: (i) a docking receptor promoting cholesterol translocation, or (ii) a receptor mediated intracellular endosomal pathway termed “retroendocytosis”. In the present study, we performed fluorescence and electron microscopy to visualize the uptake of HDL using cholesterol-load rat aortic endothelial and smooth muscle cells in vitro.

Endothelial and smooth muscle cells were obtained from rat aorta and cultured in medium under 5% CO2/ 95% air atmosphere. Then confluent monolayers of endothelial and smooth muscle cells were incubated in cholesterol or fluorescence-labeled cholesterol DMEM medium for 48 hr.
The Role of Superoxide and Nitric Oxide in the Development of Myocardial Injury in Rat Myocarditis.
Authors: Nishikawa T , Ishiyama S , Suzuki A , Masuda A , Kasajima T
Published: July 2020

The mechanism of myocardial injury in myocarditis is still unclear. Recently, nitric oxide (NO), massively produced in the inflammation, has been suggested to be harmful for tissue at excess amount as well as superoxide. However, it is still unclear the relationship between production of NO with superoxide and myocardial damage in myocarditis. In this study, we investigated whether NO and/or superoxide play an important role in the pathogenesis of myocardial injury in myocarditis.

The heart tissue specimens were taken from Lewis rats with experimentally induced myocarditis. Immunohistochemical staining was performed using polyclonal antibody for inducible NO synthase by avidin-biotin peroxidase complex method in paraffin sections of the tissue. Histochemical study for ultrastructural H2O2-producing site in the heart tissue was carried out by Brigg’ s method using CeCl3; briefly, the heart tissues were incubated in 0.1M Tris-malate buffer with ImM CeCl3 and 10mM aminotriazole. Then tissues were fixed in 2% glutaraldehyde, postfixed with 1% OsO4 and embedded in resin for electron microscopy.
Preparation of 3D Atom Probe Samples of Multilayered Film Structures using a Focused Ion Beam
Authors: Martens R L , Larson D J , Kelly T F , Cerezo A , Clifton P’H , Tabat N
Published: July 2020

Focused ion beam (FIB) instruments have become essential for the preparation of atom probe samples from heterogeneous materials. Previous sample preparation methods such as electropolishing are limited in their application due to either geometrical or electrochemical constraints. Recent developments in sample preparation using a FIB have enabled the production of AP samples from various materials and, more importantly, from non-traditional sample geometries that contain multilayered thin film structures (MLF).

Most sample preparation using a FIB first involves a sample that has been reduced in size through some manual sample preparation technique like tripod polishing or cutting. Smaller, thinner samples require less milling time in the FIB. A silicon wafer etched with the “Bosch” process was used to produce a surface that contains millions of 20, 16, 12, 8, and 4 μm square by -180 μrn long “posts”, Fig. 1. A multilayer film structure is deposited on the flat surface of the silicon posts in a standard deposition process.
Effects of Ion Species and Energy on the Amorphization of Si During FIB TEM Sample Preparation as Determined by Computational and Experimental Methods
Authors: Jamison R B , Mardinly A J , Susnitzky D W , Gronsky R
Published: July 2020

Gallium focused ion beam (FIB) milling is the method of choice to prepare cross sections of selected features from microelectronic devices for transmission electron microscope (TEM) imaging and analysis. The FIB milling technique is unsurpassed in producing an ultra-thin cross section accurately located through the feature of interest. While much effort has been invested in the development and refinement of Ga+ FIB techniques and instrumentation, there are problems due to the physics of the ion-solid interaction. The problem of surface amorphization limits the quality of the TEM samples and its significance increases as the feature size and the specimen thickness decrease.

In Ga+ FIB milling of silicon, the amorphous damage layer consists of amorphous Si doped with implanted Ga. This damage layer is caused by ions that strike the surface of the silicon and are subsequently scattered laterally as a result of collisions with the Si atoms.
Microstructural Characterization of Automated Specimen Preparation for TEM Analysis
Authors: Shannon C Urbanik , Giannuzzi L A , Raz E M
Published: July 2020

Automated specimen preparation for transmission electron microscopy has the obvious advantage of saving personnel time. While some people may perform labor intensive specimen preparation techniques quickly, automated specimen preparation performed in a timely and reproducible fashion can significantly improve the throughput of specimens in an industrial laboratory. The advent of focused ion beam workstations for the preparation of electron transparent membranes has revolutionized TEM specimen preparation. The FIB lift-out technique is a powerful specimen preparation method. However, there are instances where the “traditional” FIB method of specimen preparation may be more suitable. The traditional FIB method requires that specimens must be prepared so that the area of interest is as thin as possible (preferably less than 50 μm) prior to FIB milling. Automating the initial specimen preparation for brittle materials (e.g., Si wafers) may be performed using the combination of cleaving and sawing techniques as described below.
Focused Ion Beam Induced Copper Artifact Dose Study
Authors: Rossie BB , Anderson SD , Stevie FA , Brown SR , Shofner TL
Published: July 2020

Reduced feature dimensions in microelectronic devices has led to a growing reliance on the focused ion beam (FIB) for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) specimen preparation. The introduction of copper for use in electrical interconnects will increase this reliance. Copper, much more so than aluminum, tends to smear when conventional polishing techniques are employed, rendering mechanical polishing unsuitable for quality specimen preparation.

The sputtering characteristics of copper differ greatly from the aluminum based materials currently in use. Copper exhibits a grain orientation dependent sputtering rate that tends to produce uneven FIB polished surfaces. The sputtering rate for copper is also much higher than the silicon and dielectric materials that must be simultaneously removed. These differences in sputtering rates lead to the formation of curtains and ledges that also affect specimen quality.

Another FIB induced artifact was observed when preparing SEM specimens (Figure 3).
TEM Specimen Preparation of Oxidized Ni-Base Alloys using the Focused Ion Beam (FIB) Technique
Authors: More KL , Coffey DW , Pint BA , Trent KS , Tortorelli PF
Published: July 2020

Microstructural characterization of thin alumina scales formed on oxidized Ni-base alloys using transmission electron microscopy (TEM) has long been a challenge as a result of the many problems encountered during the preparation of thin specimens. Successful and reproducible preparation of uniformly thin, cross-section TEM specimens from these multicomponent “layered” structures (alumina scale on a metallic substrate) is extremely difficult using standard ion beam thinning procedures for several reasons: (1) differential thinning of the various constituents in the system can occur, (2) a weak ceramic-metal interface may lead to separation during the harsh mechanical grinding and thinning procedure, (3) fully intact scales are rarely achieved since native surface structures are usually lost during ion milling, and (4) extensive damage can be created in the scale and alloy during rough grinding and polishing steps as well as during final ion beam thinning.
Dimensional and Angular Measurements from Microvascular Corrosion Casts: 2D vs. 3D
Authors: B. Minnich , R. Sommer , Lametschwandtner A
Published: July 2020

In microvascular network analysis on vascular corrosion casts (VCC) detection of vascular relations between distant regions within vascular territories deserves to operate the SEM at low magnification (< 500x). As a result in low powered SEM micrographs of VCCs vessels may remain in the wide zone of focal depth (Dƒ), but can approach upper and lower focal planes of this zone (Fig.l). Their lengths, thus may severely be underestimated when measuring in 2D. The differences in lengths in 2D (L3D) and in 3D (L3D) of a distance L between points p1 and p2 (Fig.3) is obvious, denoting

While data from 2D-morphometry (1, 2, 3) have to be corrected for dimensional measurements in the direction of tilt by expanding the dimensions by [l/cos(tilt angle)] (4), modern 3D-morphometry techniques (5, 6, 7) consider the perspective projections by calculating the parallax in their working algorithms.
Tridimensional Characteristics of Rat Testicles Submitted to Tumour Cells Implant (Walker 256)
Authors: CHOPARD RP , LOURENCO MG , OCAÑA RP
Published: July 2020

The intention of this paper is to comparatively demonstrate the tridimensional architecture of the microvascularization and the pattern of the microvascularization of testicles submitted to tumour cells implant, considering that supporting mechanisms of cell vitality depend on the vascular array.

Testicles of 25 Wistar male rats were used. To proceed the implant of the tumour cells it was used a 50cc insulin syringe with short needle witch was introduced into the inferior apex of the organ. The animals were anaesthetised with 50% ethylic ether solution and 50% alcohol 96° by inhalation in proper chamber. The weight of the rats was verified every day to evaluate alterations due to tumour development and malignant evolution so it was possible to determine the exact day of sacrifice.

The perfusion was performed trough the abdominal part of the aorta using a needle connected to a silicon tube.
Electron Microscopic Analysis of Cerebral Aneurysms
Authors: Grove-Sullivan M , Yonas H , Watkins S
Published: July 2020

A cerebral aneurysm is a dome or sac-like structure budding from weakened arterial walls in the brain. Cerebral aneurysms have no known specific cause currently. Considering the risks of surgery, when surgery should be performed on unruptured aneurysms is a very controversial issue. Some clinicians feel that an aneurysm should be surgically removed immediately upon discovery regardless of the size, if surgery is possible. While others feel that small aneurysms should be watched or treated with endovascular insertion of soft platinum coils, avoiding the risks associated with any brain surgery. To further study this question examination was done of specimens from surgical clip ligation of middle cerebral artery aneurysms and normal specimens.

Specimens of ruptured and unruptured aneurysm as well as normal artery removed from surgical patients were immediately preserved in cold 2.5% glutaraldehyde in 0.1 M PBS. The specimens were rinsed in PBS, post-fixed in 1% Osmium Tetroxide with 0.1% potassium ferricyanide,
Effects of 2,2',4,4',5-Pentachlorobiphenyl on Mitochondria and Glycogen Mean Volume Fractions in Rat Hepatocytes
Authors: Gallant TL , Singh A
Published: July 2020

Detection of polychlorinated biphenyls (PCBs) in the environment in the late 1960s along with increasing scientific data on their detrimental health effects led to a voluntary ban on these chemicals for industrial uses except in closed systems such as dielectric fluids. Commercial mixtures of the chemicals were manufactured extensively from the mid-1950s to the 1970s for use as flame retardants, lubricants, and dielectric fluids. It was not until the late 1970s that the manufacture of PCBs ceased. Environmental contamination of PCBs, however, continues to be a pervasive problem because of their lipophilic nature and resultant bioaccumulation in the food chain. PCB 118 (2,3',4,4',5-pentachlorobiphenyi) is one member of the chemically inert group of compounds composed of 209 congeners, effects of which are described in current work as a quantitative analysis of hepatocyte ultrastructure using stereology techniques. We determined the mean volume fractions of mitochondria and glycogen particles in the liver cells following exposure of PCB 118 to weanling Sprague-Dawley rats.
Expression and Co-Localization of C-Type Natriuretic Peptide and Endothelin in Human Kidney
Authors: Bailey GW , McKernan S , Price RL , Walck SD , Charest P-M , Gauvin R , Wei C , Burnett J C
Published: July 2020

C-type natriuretic peptide (CNP) is an endothelium-drived vasorelaxing factor which is present in human plasma, urine and renal epithelial cells. Endothelin (ET) is a potent renal and systemic vasoconstrictor and sodium regulating peptide. To date, the localization of C-type natriuretic peptide and endothelin gene expression in human kidney remains poorly defined.

We determined the co-localization of C-type natriuretic peptide and endothelin mRNA in normal human kidney using in situ hybridization (ISH) with synthesized human C-type natriuretic peptide or human endothelin oligonucleotide probes together with immunohistochemical staining (IHCS) with human CNP-22 and ET-1 antibodies with no crossreactivity. The sections treated with nonimmune rabbit serum (NRS) demonstrated no immunoperoxidase activity. Human renal tissue (n=7) was obtained from biopsy of normal cadaveric donor kidneys and normal autopsy specimens.

In situ hybridization (Figure 1) demonstrated that C-type natriuretic peptide and endothelin mRNA presence in human kidney which was localized to proximal and distal tubes with similar intensities.
Enhancement of Natriuretic Peptides in Rejected Human Renal Graft
Authors: Song H , Papadimitriou J , Drachenberg C , Weir M R , Wei C
Published: July 2020

Natriuretic peptides include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). ANP, BNP and CNP are structure related but genetic distinct 1 natriuretic peptide family. ANP and BNP are cardiac cell origin and CNP are endothelial cell and kidney origin. Natriuretic peptides have potent vasoactive and natriuretic actions through generation of cGMP. On the other hand, renal graft rejection is major problem after kidney transplantation with severe renal damage and renal vasoconstriction. We hypothesized that renal tissue level of natriuretic peptides increase in renal graft rejection through compensatory mechanism. Therefore, the present study was designed to determine the expression of natriuretic peptides by immunohistochemical staining (IHCS) in human renal tissue with rejection and compare with normal renal tissue.

Human renal biopsy (n=5) were obtained after kidney transplantation with mild and moderate renal rejection. Normal kidney biopsy was obtained during nephrectomy. ANP, BNP and CNP levels in renal tissue were determined by IHCS.
High Resolution Low Voltage Field Emission Scanning Electron Microscopy on De-Embedded Thick Sections Reveals Structural Interactions of the Surface of the Apicomplexan Parasite Toxoplasma Gondii with the Host Cell in the Parasitophorous Vacuole
Authors: Schatten Heide , Sibley David , Ris Hans
Published: July 2020

The protozoan parasite Toxoplasma gondii represents a large group of Apicomplexan parasites with a highly unusual motility system that is crucial for cell locomotion and host cell invasion. Studying the motility system and interactions with host cells will increase our knowledge on how to prevent infection. Apicomplexan parasites can cause considerable health problems to animals including sheep, goats, pigs, and chicken. Eimeria is known as a pathogen of coccidiosis in chicken, and Cryptosporidium causes cryptospiridiosis in cattle and other farm animals. Toxoplasma gondii can cause infection that results in abortion, central nervous disorders and death of stressed and immunocompromised farm animals. Common to all Apicomplexan parasites is an unconventional motility system that is thought to be actively involved in host-cell invasion. However, the structural and biochemical characterization of Apicomplexan parasites has proven more difficult than previously thought because of genetic and behavioral differences of the parasites’ cytoskeletal proteins that differ from those in well studied mammalian systems.
Bacterial Attachment to Oral Epithelium
Authors: Vitkov L , Krautgartner W D , Hannig M , Fuchs K
Published: July 2020

The purpose of this work was to examine, by electron microscopy, the mechanism of bacterial binding to oral epithelium in patients with stomatitis using ruthenium red in combination with osmium tetroxide.

Bacteriological semi-quantitative estimation of smear from the palatal mucosa was performed in patients with stomatitis and only patients with viridans Streptococci were further included in the present investigation. Biopsies were taken from the inflamed keratinizing mucosa under regional block anesthesia in agreement with Vancouver guidelines 1978. Control samples were fixed with 1.2% glutaraldehyde (buffered at pH 6.5 with 0.1 M sodium cacodylate) for 2 h at 4° C. Postfixation was performed with 1% osmium tetroxide (buffered at pH 6.5 with 0.1 M sodium cacodylate) for 2 h at 4° C. The other samples were fixed and postfixed by the fixatives mentioned before with addition of 1.6 % ruthenium red in each case.
Localization and Visualization of Specific Gene Products in Fungal Plant Pathogens
Authors: Bourett T M , Czymmek K J , Dezwaan T M , Sweigard J A , Howard R J
Published: July 2020

Specific gene products of both pathogens and hosts have been implicated as decisive elements during plant pathogenesis. While expression of some of these genes is constitutive, that of others is likely ephemeral and activated only during a particular stage of the interaction. Because the relative timing of expression may be critical, transcription and translation have often been addressed by extracting mRNA and proteins from infected plant tissue. This approach, however, cannot readily detect proteins of low abundance in bulk samples nor offer much useful information on cell-cell interaction. Only a cytological analysis that employs microscopy can resolve the temporal and spatial details of gene expression. Typically, such protein localization studies have required specific antibodies, but these large probe molecules do not diffuse into living or conventionally fixed cells of either fungal pathogens or plant hosts. For TEM analysis, these permeability-imposed limitations have been reduced by thin sectioning to render accessible antibody binding sites.
High-Pressure Freezing to Study Structure and Function of the Host Parasite Interface
Authors: Mendgen K
Published: July 2020

The high-pressure freezing instrument exposes a sample to a pressure of 2100 bar, which lowers the freezing point and, as a result, reduces the rate of ice nucleation and ice-crystal growth. The reduced critical cooling rate allows adequate freezing of samples up to 0,3 mm in thickness without using cryoprotectants. Before pressure application, the sample is sandwiched between specimen holders. To optimize heat conductivity and to avoid damage by the high pressure impact, the free space inside the specimen holders and within the sample has to be filled with liquid. This means that plant leaves need to be infiltrated to remove gas from the intercellular space. We have used water, 3-8% methanol in water, 1-hexadecene or heptane as infiltration medium. Subsequently, samples were freeze substituted in unhydrous acetone with 2% Os04 for 24 h at -90°C. Samples were slowly warmed up to 4°C and embedded in Unicryl, or warmed up to room temperature and embedded in epoxide resin.
Use of Freeze Substitution for TEM Studies of Fungal Spores
Authors: Minis C W , Richardson E A
Published: July 2020

Since its initial use to fix fungal hyphae (1), plunge freezing followed by freeze substitution has become the “gold standard” for TEM studies of fungal hyphae and spores. In this presentation we discuss results we have obtained using plunge freezing and freeze substitution to fix various types of spores produced by plant pathogenic fungi. Examples include basidiospores and aeciospores of rust fungi, teliospores of rust and smut fungi and conidia of a variety of ascomycetes and deuteromycetes.

Generally speaking, plunge freezing followed by freeze substitution usually yields best results when spore diameter does not exceed 10-15 μm. However, in a number of cases we (2) have obtained good to excellent results with much larger spores. A key to good freezing appears to be direct exposure of spores to liquid propane during plunging. We have had best results by placing spores on small pieces of dialysis membrane and then plunging these membranes. In most cases the spores remain attached to membranes throughout the freezing, substitution, infiltration and embedment procedures.
Abnormal STEM Development in Maize nal/nal Mutant - A Confocal Microscopy Study
Authors: Cheng Wayne Y , Cheng Ping-chin , Walden David B
Published: July 2020

There are number of dwarf mutants in maize such as d8, dl and nal. The nana plant 1 (nal/nal) is characterized as short erect dwarf; the phenotype does not respond to treatment of giberellins. The recessive nal allele is located on the long arm of chromosome 3 (3L-113). In this study, we report the abnormal development of the stem vasculature and the lack of clearly defined nodes.

Maize (Zea mays L., var. Ohio 43 and nal mutant) plants were grown in 1999 summer nursery at the fieldstation of University of Western Ontario, London Ontario, Canada. Plants at approximately 2 months old were fixed in 1:3 acetic acid/ EtOH. Multiple changes of fixative were made for a period of a month to remove as much pigmentation as possible. The specimen was then washed thoroughly, stained in diluted Schiffs reagent (10%), washed in 0.1M metabisulphate solution (with 1 ml of 1 N HC1 per liter), dehydrated in EtOH and cleared in methyl salicylate.
Microscopic Characterization of a Sperm Mutant Line of the Fern Ceratopteris Richardii
Authors: Davidson Kelly A , Hickok Leslie G , Renzaglia Karen S
Published: July 2020

Mature sperm cells of Ceratopteris richardii are spiraled over three revolutions and contain a locomotory apparatus with approximately 70 flagella attached at the cell anterior (Fig. 1). Abundant organelles are dispersed along the inner surface of an elongated, coiled nucleus (Figs. 1, 3, 8). The cytoskeleton comprises a network of microfilaments that encases the nucleus (Fig. 6) and two distinct microtubule arrays: a microtubular ribbon (spline) and flagella with anchoring basal bodies (Fig. 3). This study uses light, fluorescence, transmission electron (TEM), and scanning electron microscopy (SEM) to characterize cell organization in a motility impaired sperm mutant line of Ceratopteris. Because motility mutations likely involve the cytoskeleton, emphasis was placed on observing microtubule and actin arrays in the mutant compared to wild-type sperm cells. Protocols follow Hoffman and Vaughn for TEM and Schmitt and Renzaglia for SEM. Microfilament arrays in relation to the nucleus were examined by rhodamine-phalloidin and DAPI fluorescence.
Characterization of Dislocation Reactivity and Dynamics in Thin Metal Films Using Scanning Tunneling Microscopy
Authors: Hwang RQ
Published: July 2020

The accommodation of strain in metal films can lead to complex phenomena, both structurally and chemically. It can generically lead to the formation of dislocations. These dislocations play an important role in defining the evolution and response of the film to further growth and external forces. The structures of the dislocations are directly analogous to bulk dislocations, but their properties are very different due to the presence of the surface. In addition, the strain can greatly affect the chemical properties of the film, leading to alternative strain relief mechanisms involving vacancy formation resulting from reaction with gases such as sulfur and oxygen. Using STM and LEEM, we have investigated these phenomena in the prototype systems of Cu and Ag on Ru(0001).

For example, figure la - d shows a sequence of STM images of a 2 layer Cu film on Ru(0001) during exposure to oxygen. Dislocations present in the film are altered by reaction with oxygen.
Evaporation Spirals on {111} and {001} Surfaces of MgAl2O4 Spinel
Authors: Bailey GW , McKernan S , Price RL , Walck SD , Charest P-M , Gauvin R , Yanina Svetlana V , Barry Carter C
Published: July 2020

Processes at dislocation sites are frequently predominant mechanisms of surface evaporation or growth and thus determine resulting surface morphologies. Dislocation-induced evaporation patterns are also a convenient object for observation of dynamics of step motion on crystal surfaces, as such patterns have “fixed” origins, being pinned at the point of dislocation emergence. In the present work, observations of evaporation spirals formed on ﹛111﹜ and ﹛001﹜ surfaces of MgAl2O4 spinel are reported.

2x2x1 mm samples of single-crystal MgAl2O4 spinel of ﹛111﹜ and ﹛001﹜ orientation were annealed in a spinel crucible in vacuum (10-4—10-5 Torr) in a Centorr™ furnace at 1800°C for 8 hours. SPM images were collected in air in contact mode on a Nanoscope III (Digital Instruments, Santa Barbara, CA), using Si3N4 cantilevers (Ultralevers, Park Inst., Sunnyvale, CA) with a nominal spring constant of 0.12 N/m. Selected sample surfaces were examined for impurities by energy-dispersive X-ray spectroscopy (EDS) and X-ray photoemission spectroscopy (XPS).
Surfaces of Gadolinium Gallium Garnet
Authors: Gilliss S R , Yanina S V , Ravishankar N , Carter C B
Published: July 2020

Sintering and many problems in adsorption and catalysis require an understanding of the surface structures of crystals, which have been subjected to various types of heat treatment. For example, heat treatment of m-plane sapphire surfaces can cause a transformation from a flat vicinal surface into those of a faceted hill-and-valley structure. Many technologically interesting materials form with the garnet structure, including YIG, YAG, GGG and many silicates. However, there has been relatively little discussion of the structure and behavior of such surfaces. This paper illustrates the use of visible-light microscopy (VLM), atomic-force microscopy (AFM) and scanning electron microscopy (SEM) for studying the ﹛111﹜ surface of gadolinium gallium garnet (GGG or Gd3Ga5O12).

2x2x1 mm samples of monocrystalline GGG with ﹛111﹜ surface orientation were heat treated in air at 1575°C for 24 hours in order to study the processes a surface undertakes while reaching its equilibrium state.
Characterization of Large-Area Silicon Drift Detectors at High Count Rates
Authors: Patt BE , Iwanczyk JS , Tull CR
Published: July 2020

Silicon Drift Detectors (SDD) are being developed for analytical x-ray spectrometry having large active area, high-energy resolution and capability of operating at high counting rates. The development derives from the charged coupled device (CCD) for light-signal imaging, utilizing the extremely low capacitance of the detector and readout electronics and subsequent developments of silicon drift detectors for high-energy physics applications and more recently, x-ray spectroscopy applications. The now well-known advantage of the drift detector design is that, unlike traditional planar detectors, it allows for relatively large active area while still maintaining a very low anode capacitance (60 fF). This low value of detector capacitance results in a lowering of the series-noise component and hence the overall inherent electronic noise. Additionally, the reduction of the series noise leads to faster optimal shaping time, and as a consequence this provides for extremely high count rates.
Distribution and Behaviour of Positive Ions in the Environmental Scanning Electron Microscope (ESEM)
Authors: Craven John P , Baker Frank S , Thiel Bradley L
Published: July 2020

Electron detection in the environmental scanning electron microscope relies upon the presence of a small pressure of a gaseous phase inside the microscope chamber. The presence of this gas controls two important mechanisms by which the microscope functions. The conventional gaseous secondary electron detector (GSED) applies a variable positive bias (0 to +600 V) directly above the specimen. Electrons ejected from the specimen surface due to the incident scanning probe are accelerated by this field towards the positively biased detector. Whilst traversing the gap between specimen and detector the electrons undergo ionizing collisions with gas molecules. These result in an amplification of the incident electron signal through the production of ejected ‘daughter’ electrons and leave behind positively charged ions. After production these ions are repelled by the detector bias and drift back towards the microscope stage where they aid charge neutralization on the surface of the specimen.
Applying Dynamic Experiments and Stereo Measurements to Evaluation of Dentin Specimen Preparation Techniques for Environmental and Conventional SEM
Authors: Dusevich VM , Eick JD
Published: July 2020

The acid etching process is the primary stage for preparation of dental composite restorations. Dentin specimens from extracted teeth are usually prepared by fracturing etched pieces of dentin. Etching creates a demineralized layer on the dentin surface with the thickness of a few microns. The layer consists of wet collagen and dissolved proteins; its drying process is of special interest in dentistry since it affects the bonding of the resin adhesives to dentin. Observations of fractured surfaces can be performed in either a conventional or an environmental SEM.

Three wet dentin samples were observed (XL30 FEG ESEM, FEI/Philips) in wet mode at 5°C Peltier cooling stage temperature and 5.8 MPa water vapor pressure. Samples with excess water on their top surface were put in an ESEM. Thickness measurements of the demineralized zone were begun immediately after the evaporation of this water layer with subsequent measurements taken after 5, 10, 20, 40, 60 and 100 minutes of drying.
Introducing a Method for In Situ Electro-Rheology & Electrophoresis of Complex Fluids by Electron Microscopy.
Authors: Stokes DJ , Baker FS
Published: July 2020

It has previously been shown that liquid microstructures can be observed, in their natural state, by use of Environmental SEM (ESEM). In order to take full advantage of the ability to observe the dynamics of complex fluids, efforts are now being directed at developing in situ experimental methodologies. For example, we have recently shown that the coalescence of oil droplets in emulsion systems can be observed in real time. The purpose of the present study is to tackle the challenge of making EM observations of complex fluids under electrical stress (and ultimately electrical & mechanical stress combined) by subjecting particle dispersions to an electric field.

The technique of attracting charged particles to one of two electrodes (electrophoresis) is commonly used as a means of determining the nature and density of charges on such particles. Another technique, using uncharged particle systems, involves applying an electric field across a sample as well as, for example, a shear strain (electro-rheology).
X-Ray Microanalysis of Insulators in the ESEM
Authors: Phillips MR , Toth M , Griffin BJ
Published: July 2020

We have investigated the affect of accelerating voltage (E0), pressure (P), GSED bias (VGSED), field line geometry and specimen conductivity on the landing energy of the primary beam in an XL30 ESEM. The landing energy of the primary beam can be measured by using the Duane-Hunt cut-off energy, EDH, in EDS X-ray spectra. We present experimental evidence that the landing energy of the primary beam can be significantly altered when analyzing insulators in an ESEM. Shifts in EDH result from electric fields produced by the presence of positive ions in the ESEM chamber and electrons trapped in the bulk. No shift in EDH was observed with conductive specimens at all E0 and P. Changes in the landing energy of the primary beam can significantly effect the relative intensity of characteristic peaks in EDS spectra measured in the ESEM (Figure 1).

The magnitude and sign of the shift in EDH is determined by E0 and P for a specific insulator specimen and stage geometry.
Factors Affecting Quantitative Analysis Error in ESEM-EDS
Authors: Carlton R A , Lyman C E , Roberts J E
Published: July 2020

Standard Reference Material (SRM) 482 of the National Institute of Standards and Technology is a set of 6 gold/copper wires, ranging in concentration from 0 to 100% Cu in 20% steps, intended for calibration studies of electron beam microanalyzers. This is an appropriate standard to test the accuracy of energy dispersive x-ray spectrometry (EDS) in the Environmental Scanning Electron Microscope (ESEM). While the presence of the gas in the sample chamber gives the ESEM its unique capabilities, it also is the source of complications to x-ray spectrometry. The gas can spread the primary electron beam into a wide skirt of electrons with the consequent production of x-rays many micrometers from the target location of the beam.

The six wires (∼ 500 jam in diameter) were embedded and polished in one epoxy mount. The mount was carbon coated in one set of experiments. The coating was removed and the sample retested.
Phosphor Imaging Plate Measurement of Electron Scattering in the Environmental Scanning Electron Micrsoscope
Authors: Wight SA , Zeissler CJ
Published: July 2020

In this work, phosphor imaging plate technology is applied to measure electron scattering directly in the environmental scanning electron microscope (ESEM) specimen chamber. The scattering of electrons from the primary electron beam, under relatively high-pressure conditions (266 Pa) in the ESEM sample chamber, degrades the analytical accuracy of elemental analysis. The degree of this degradation is poorly known. To date, attempts to measure experimentally the spatial distribution of the scattered electrons have been limited to observing secondary effects such as the intensity of x-rays produced from copper targets positioned at various distances from the primary electron beam interaction point. A more accurate distribution of the scattered electron intensity can be obtained from a direct measurement of both the scattered and unscattered electrons over a large area with single electron sensitivity. Improvements to the accuracy of Monte Carlo models of the scattering process will be made possible by the direct measurement data.
Cell Boundary Detection and Volume Approximation of Confocal Microscope Images for Bioinformatics
Authors: Fernandez Cybelle , Habets Damiaan , Kremer Stefan C , Maguer Marc Le
Published: July 2020

Bioinformatics is the science of developing computer databases and algorithms for the purpose of speeding up and enhancing biological research. This paper will detail the application of a three-dimensional cell boundary detection algorithm to compute the volumes of osmotically dehydrated apple cells in an effort to better understand the effects of this treatment.

Osmotic dehydration is an efficient pre-treatment technique in food processing. However, the changes that occur in the food material at the cellular level have not been thoroughly understood and this has limited the full industrial application of the process. During osmotic dehydration, plant tissues when placed in hypertonic solutions undergo plasmolysis that can be directly observed under light microscopy. The usual phenomenon observed during plasmolysis is the separation of the cytoplasm from the cell wall, due to the removal of water from the protoplast (Frey-Wyssling and Muhlethaler, 1965). Although readily observed under the microscope, however, the quantitative changes that occur during the process are not easily measurable.
Analisis of A-Kinase Anchoring Protein Interactions With PKA Using Fluorescence Resonance Energy Transfer
Authors: Ruehr M L , Damron D S , Bond M
Published: July 2020

The clustering of components of a signaling pathway at a specific subcellular location raises the local concentration of the appropriate messengers and serves to amplify the signal. The cAMP dependent-protein kinase (PKA) pathway is regulated by compartmentalization of its components. A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular sites, thus presumably increasing substrate specificity. Phosphorylation of the type II regulatory subunit of PKA (RII) increases its affinity for AKAPs in vitro (1). The purpose of this study was to investigate whether altering the phosphorylation state of RII in live cells changes its affinity for an AKAP. Specifically, we investigated the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid (2), and RII, in response to PKA activators or inhibitors.

Fluorescence resonance energy transfer (FRET) was used to monitor binding events between RII and the catalytic subunit (C) of PKA, Ht31, or Ht31P, a mutated form of Ht31 which does not bind RII.
Oxidant Stress-Induced Alterations in Mitochondrial Physiology in the Presence and Absence of BCL-2
Authors: Takahashi Akiyuki , Herman Brian
Published: July 2020

Apoptosis is an actively regulated process of cell death necessary for proper control of tissue growth. In humans, ∼100,000 cells die every second by apoptosis and are replaced by new cells of the same number. Apoptosis was originally described by Kerr, Wyllie, and Currie in 1972 as an event characterized by plasma membrane blebbing, cell shrinkage, chromatin condensation, and degradation of DNA (1). Apoptotic death is a tightly controlled process, and the cellular pathways that regulate apoptosis have been well conserved in multicellular organisms from simple to complex animals.

One of the most reproducible inducers of apoptosis is mild oxidative stress. Oxidative modification of proteins and lipids has also been observed in cells undergoing apoptosis in response to nonoxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. Several incompletely reduced forms of oxygen are highly reactive and quite cytotoxic.
Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence
Authors: Levenson Richard M
Published: July 2020

Autofluorescence, also known as adventitious fluorescence or background fluorescence, ofter poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can swamp the desired signal. Particularly difficult samples to image include many pathology specimen that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most (green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels which can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal.However, in many cases, these approaches are either infeasible or ineffective.
Cationic Liposome-DNA Complexes: Polymorphism versus Transfection Activity
Authors: Sternberg-Papahadjopoulos B , Hong K , Zheng W , Papahadjopoulos D
Published: July 2020

Complexes formed during interaction of cationic liposomes with polynucleotides such as DNA (CLDC) self-assemble into a variety of polymorphic structures. They display bilayer (FIG. 1-5) and non-bilayer structures (FIG. 6). We have recorded bilayer structures such as spaghetti/meatball-type structures (FIG. I), map-pins (FIG. 2) spherical particles and invaginated liposomes (FIG. 3, 4) and oligolamellar structures (FIG. 5). The non-bilayer lipid arrangements include honeycombtype structure (Hn, FIG. 6) and cubic phase lipids.

We have chosen mainly freeze-fracture electron microscopy (FIG. 1-3, 5,6) but also cryo-electron microscopy (FIG.4) for recording polymorphic structures, and for studying factors and conditions triggering the formation and stabilization of specific structure types. Furthermore, we took microscopically snapshots of the interaction of specific structure types with cultured cells. In order to find out the “active” structure in terms of transfection, we investigated the transfection activity both in vivo and in vitro of CLDC, and studied in parallel their morphology in serum as well as in cell medium.
Ultrastructure of the Tertiary Envelope of the Cyst of the Tadpole Shrimp Triops longicaudatus (Branchiopoda; Notostraca)
Authors: Rosowski James R , Bartels Terry L , Colburn James F , Colton Jannell L , Belk Denton , Lee Kit W
Published: July 2020

Tadpole shrimp inhabit temporary freshwater pools and ponds where their occurrence is largely regulated by rainfall events and water temperature. When dry basins are flooded, cysts of Triops imbibe water and hatch to produce rapidly growing, carapaced larvae. While previous studies show anostracan (fairy shrimp) cyst-surface morphology often species specific, few studies illustrate shell ultrastructure of Triops and none has considered T. longicaudatus. Here we examine the shell of T. longicaudatus (Notostraca) and compare its fine structure to other species of Triops and to that of Artemiafranciscana(Anostraca), which we previously studied.

Cysts, produced in culture from Utah broodstock, were purchased from Triops, Inc., 1924 Creighton Rd., Pensacola, FL 32504. Thin sections of cysts were prepared for transmission electron microscopy (TEM) as previously described (Fig. 1). Cysts were also examined with scanning electron microscopy (SEM), dry, whole or fractured (Figs. 2,3), or after imbibition and/or hatching in oxygen saturated, double-distilled water, at 25 ° C.
A Freeze-Fracture Study of Nuclear Pore Complex Structure in Intact Dunaliella Cells
Authors: Parthasarathy MV , Daugherty CS
Published: July 2020

The nuclear envelope in all eukaryotic cells is perforated by nuclear pores, that consist of the nuclear pore complex (NPC). The NPC is a highly complex supramolecular structure that plays a pivotal role in the trafficking of macromolecules and particles between the nucleus and the cytoplasm. During the past two decades a large body of information has accumulated on both the structure and role of the NPC. The diameter of NPC is about 120 nm and is composed of approximately 100 proteins.

The NPC structure has been elucidated using various electron microscopy techniques. The basic structure of the NPC is comprised of three concentric rings, each displaying an 8-fold symmetry. The cytoplasmic and nuclear rings are vertically integrated by eight spokes that approximately span the width of the nuclear envelope. A set of eight fibers extends from the nucleoplasmic ring into the nucleus, and are joined at the distal ends by a ring to form the so called basket.
Induction of Apoptic Features in Human Colon Cancer Cells After Exposure to Specific Vitamin Combinations
Authors: Brown B , Garvin K R , Hughes B G , Standing M D , O'Neill K L , Murray B K
Published: July 2020

Proper nutrition and vitamin consumption have long been associated with lower risks for many human cancers. Data from recent studies have suggested that exposure to antioxidant vitamins and vitamin combinations can induce genetic programed cell death, apoptosis, in human cancer cells. ‘“3 In this study, we report on the induction of apoptosis in HT-29 human colon adenocarcinoma cells after exposure to low doses of 13- c/s-retinoic acid (RA) and vitamin E succinate (VES). Induction of apoptosis was seen only in cancerous colon cells and not in normal human colon cells when exposed to RA and VES in combination, but not when exposed to either vitamin alone.

Cultures of HT-29 cells were seeded in 6 well plates at 10,000 cells/well and grown at 37°C, 5% C02 in RPMI 1640 medium supplemented with 10% fetal bovine serum.
Nanomechanical Properties of a Biocomposite, Mollusk Shell Nacre
Authors: William N. Mercer  , Sopp J M , Fong H , Katti K S , Katti D R , Sarikaya M
Published: July 2020

Mechanical properties of the structural elements of mollusk hard tissues were determined using nanoindentation techniques. Red abalone (Haliotis refuscens) has a dual laminate ceramic/polymer biocomposite, evolved for protection against impact. The nacre (mother-of-pearl) section of the shell has excellent bending strength (180 MPa) and fracture toughness (12 MPa m1/2), orders of magnitude stronger and tougher than monolithic CaCO3, it's primary component. The outer prismatic structure is comprised of columnar calcite (rhombohedral CaCO3) crystallites oriented normal to the shell surface and the inner nacreous structure consists of layers of pseudo-hexagonal shaped aragonite (orthorhombic CaCO3) platelets arranged in a brick and mortar microarchitecture (Fig. 1). The platelets (0.25 μm thick and 5 μm edge length) are surrounded by a thin layer (10-25 nm) of organic phase within and between the individual layers. This sub-um layered structure results in excellent bulk properties, not present in man-made composites of similar phase composition.