bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron microscopy investigation of Si and Mo field emitters coated with diamond powder by dielectrophoresis
Authors: Myers A. F. , Choi W. B. , Cuomo J. J. , Hren J. J.
Published: June 2020

Silicon and molybdenum are commonly used to fabricate field emission devices, which have promisingapplications in the flat panel display and vacuum microelectronics industries. However, uncoated Si and Mo field emitters usually exhibit oscillating currents during extended operation due to surface contaminant adsorption and desorption. Coating these cathodes with diamond by microwave plasma chemicalvapor deposition (MPCVD) has been shown to considerably improve the total electron emission current as well as to enhance the emission current stability. A very simple, inexpensive technique for depositing high pressure, high temperature (HPHT) synthetic diamond powder on Si and Mo emitters was recently developed and also shows greatly enhanced current-voltage characteristics. Since field electron emission from a cathode strongly depends on the emitter morphology, the diamond coating morphology and the diamond/Si and diamond/Mo interface microstructures were analyzed by electron microscopy.

Single-crystal <111>-oriented Si emitters 100-300μm in height were grown by aSi-Aueutectic vaporliquid- solid technique, then chemically sharpened to form tips with a radius of curvature ≤ 30nm.
Partitioning behavior of alloying elements in PWA 1484
Authors: Miller M. K. , Lin L. S. , Cetel A. D. , Harada H. , Murakami H.
Published: June 2020

In order to achieve a desired set of properties, such as high creep strength and oxidation resistance, a variety of alloying elements are typically added to commercial nickel-based superalloys. Since single crystal superalloys do not require the inclusion of alloying elements to improve the grain boundary properties, there is significant scope in optimizing the composition and heat treatment of these alloys. The effectiveness of these alloying elements depends on their level and location in the different phases present in the microstructure. Therefore, it is desirable to be able to accurately measure and theoretically predict their distribution within the microstructure.

The partitioning behavior of the alloying elements in nickel-based superalloy PWA 1484 has been determined with the atom probe field ion microscope. These results have been compared with predictions generated from the “Alloy Design Program”. This computer program is based on regression analysis.
TEM studies of solid-state reactions in sputter-deposited Nb/Al multilayer thin films
Authors: Ma F. , Vivekanand S. , Barmak K. , Michaelsen C.
Published: June 2020

Solid state reactions in sputter-deposited Nb/Al multilayer thin films have been studied by transmission and analytical electron microscopy (TEM/AEM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The Nb/Al multilayer thin films for TEM studies were sputter-deposited on (1102)sapphire substrates. The periodicity of the films is in the range 10-500 nm. The overall composition of the films are 1/3, 2/1, and 3/1 Nb/Al, corresponding to the stoichiometric composition of the three intermetallic phases in this system.

Figure 1 is a TEM micrograph of an as-deposited film with periodicity A = dA1 + dNb = 72 nm, where d's are layer thicknesses. The polycrystalline nature of the Al and Nb layers with their columnar grain structure is evident in the figure. Both Nb and Al layers exhibit crystallographic texture, with the electron diffraction pattern for this film showing stronger diffraction spots in the direction normal to the multilayer. The X-ray diffraction patterns of all films are dominated by the Al(l 11) and Nb(l 10) peaks and show a merging of these two peaks with decreasing periodicity.
Enhanced control of electropolishing for the preparation of thin foils for TEM: Artificial and multiple - phase micro-electropolishing
Authors: Lee Peter J.
Published: June 2020

By rapidly changing voltage and current settings during electropolishing conditions can be set up for polishing otherwise difficult to polish multi-phase material. In addition a wider a more flexible range of polishing conditions can be obtained by manipulating the film build-up condition in micro-electropolishing that is normally outside the useful range of electropolishing.

Electropolishing may be broken down into two distinct processes: Macro-electropolishing or “smoothing” whereby large scale asperities are removed and Mcro-electropolishing or “brightening” in which smaller (<lμm) irregularities are removed. In “bath” (immersion) electropolishing both processes take place whereas in “jet” electropolishing the polishing mechanism is primarily micro-electropolishing. For micro-polishing to occur a thin solid film must be produced at the specimen surface. In order for micro-electropolishing (sometimes called brightening or brilliance) there must be random removal of metal from the surface irrespective of features such as grain-boundaries, grain orientation and defects.
Material analysis techniques using the ion mill
Authors: Benedict J. P. , Anderson R. M. , Klepeis S. J.
Published: June 2020

Ion mills equipped with flood guns can perform two important functions in material analysis; they can either remove material or deposit material. The ion mill holder shown in Fig. 1 is used to remove material from the polished surface of a sample for further optical inspection or SEM ( Scanning Electron Microscopy ) analysis. The sample is attached to a pohshing stud type SEM mount and placed in the ion mill holder with the polished surface of the sample pointing straight up, as shown in Fig 2. As the holder is rotating in the ion mill, Argon ions from the flood gun are directed down at the top of the sample. The impact of Argon ions against the surface of the sample causes some of the surface material to leave the sample at a material dependent, nonuniform rate. As a result, the polished surface will begin to develop topography during milling as fast sputtering materials leave behind depressions in the polished surface.
Sputter etching for microstructure evaluation of small-diameter corrosion-resistant MP35N alloy wire
Authors: Hanke L. D. , Schenk K.
Published: June 2020

Metal alloy microstructures are characteristic of the material’s mechanical and physical properties, including the key properties of strength and corrosion resistance. Microstructural evaluations typically use chemical etching to reveal the material’s structure. For corrosion-resistant alloys, chemical etching can be difficult due to the inherent chemical resistance of the material. This is especially true for active-passive alloys, where the etching reaction is highly dependent on the final polishing and even the time delay between polishing and etching.

Chemical etching is further complicated for extremely fine microstructures and when two or more metals are joined in the sample. These factors are concerns for fine wires, such as those used in implantable medical devices. A common alloy for wires in many medical applications is a Co-Ni-Cr-Mo alloy, designated as MP35N. Wires with extremely small diameters are produced by severe drawing processes that result in very fine microstructures. The alloy is often used in composite wire products, such as an MP35N outer sheath containing a silver core.
Molecule-specific imaging of biomaterials
Authors: Winograd N.
Published: June 2020

The development of focused ion beams and time-of-flight SMS have opened new possibilities for characterization of biomaterials. For some cases, laser postionization is important for further increasing the sensitivity of the analysis. Our group has been involved in finding methodologies and applications which could add to the repetoire of imaging methods available to biologists. Presently, these methods include fluorescence microscopy, confocal microscopy, scanning probe microscopies and autoradiography, to name just a few.

Sample preparation is a major issue for these materials. To achieve meaningful data it is essential to prepare an uncontaminated surface in vacuum that retains the original spatial composition of the target molecules. Although many elegant sample preparation methods have been developed in the electron microscopy community, the rules of the game for TOFSIMS imaging are quite different since we are after chemical information rather than morphological information. To solve this problem we have developed several cryogenic sample handling techniques whereby a biomaterial (e.g. a single cell) is rapidly frozen in the laboratory.
Prospects for scanned-probe microscopy
Authors: Revel Jean-Paul
Published: June 2020

In the last 50+ years the electron microscope and allied instruments have led the way as means to acquire spatially resolved information about very small objects. For the material scientist and the biologist both, imaging using the information derived from the interaction of electrons with the objects of their concern, has had limitations. Material scientists have been handicapped by the fact that their samples are often too thick for penetration without using million volt instruments. Biologists have been handicapped both by the problem of contrast since most biological objects are composed of elements of low Z, and also by the requirement that sample be placed in high vacuum. Cells consist of 90% water, so elaborate precautions have to be taken to remove the water without losing the structure altogether. We are now poised to make another leap forwards because of the development of scanned probe microscopies, particularly the Atomic Force Microscope (AFM). The scanning probe instruments permit resolutions that electron microscopists still work very hard to achieve, if they have reached it yet. Probably the most interesting feature of the AFM technology, for the biologist in any case, is that it has opened the dream of high resolution in an aqueous environment. There are few restrictions on where the instrument can be used. AFMs can be made to work in high vacuum, allowing the material scientist to avoid contamination. The biologist can be made happy as well. The tips used for detection are made of silicon nitride,(Si3N4), and are essentially unaffected by exposure to physiological saline (about which more below). So here is an instrument which can look at living whole cells and at atoms as well.
Granular cells: A new indicator of neuronal cell death in the spinal ganglia of the rat
Authors: Imel Kevin M. , Bailey J. Franklin , DeSantis Mark
Published: June 2020

Damage to axons in the neonatal mammal results in the death of the affected neurons rather than survival and axonal regeneration as generally seen in the adult. This phenomenon has been well documented using indirect methods such as counting cells in histological sections of the experimentally damaged versus control tissue. Recent experiments have shown that cells with osmiophilic dense bodies, which have been termed “granules”, become apparent at the light microscopic level in ipsilateral lumbar dorsal root ganglia after destruction of the sciatic nerve on that side in neonatal rats. Within 84 - 96 hours after the lesion, the rat pups were anesthetized, perfused with 4% paraformaldehyde and the ganglia post-fixed in 1% unbuffered osmium tetroxide for at least 72 hours. Such a lengthy exposure to osmium was found to be necessary to visualize the granules.

This offers the possibility of using these granules as a direct indicator of neuronal death in the lumbar dorsal root ganglia. The cells containing these granules were usually chromatolytic neuronal somata which had an eccentrically positioned nucleus (when a nucleus or nuclear remnant was still present) and peripheral aggregation of the rough endoplasmic reticulum (i.e. Nissl bodies).
Immunoelectron microscopic verification of a resident central-nervous-system macrophage in the rat that is not a pericyte, microglia, or type of mast cell
Authors: Dimlich Ruth V. W.
Published: June 2020

One type of perivascular cell in the brains of all species so far examined has been given many different names. This has introduced a great deal of confusion into the literature. Using light microscopy this cell is autofluorescent, stains with periodic acid-Schiff (PAS), and cannot be identified by H&E that is used routinely in neuropathological analysis. Ultrastructurally this cell is on the luminal side of the basal lamina and has a non-lobulated nucleus, few mitochondria, a well defined Golgi area, and numerous lysosomes, phagosomes, and multivesicular bodies. Because of these features, the most common names for these cells have been “yellow fluorescing or Mato cells”, “perivascular or pericytal microglia”, “globular or phagocytosing pericytes” and “type II brain mast cell or neurolipomastocytes”.

Application of a variety of histochemical techniques verified that these cells did not contain heparin or histamine, and identified for the first time that they also did not contain serotonin. Although granular and autofluorescent, the data confirmed that these cells were not mast cells. Immunohistochemically these cells also did not stain with OX42, an antibody specific for resting or activated microglia, or for actin as expected for pericytes.
X-ray microprobe for materials science
Authors: Ice G.E.
Published: June 2020

The increasing availability of synchrotron x-ray sources has stimulated the development of advanced hard x-ray (E≥5 keV) microprobes. With new x-ray optics these microprobes can achieve micron and submicron spatial resolutions. The inherent elemental and crystallographic sensitivity of an x-ray microprobe and its inherently nondestructive and penetrating nature will have important applications to materials science. For example, x-ray fluorescent microanalysis of materials can reveal elemental distributions with greater sensitivity than alternative nondestructive probes. In materials, segregation and nonuniform distributions are the rule rather than the exception. Common interfaces to whichsegregation occurs are surfaces, grain and precipitate boundaries, dislocations, and surfaces formed by defects such as vacancy and interstitial configurations. In addition to chemical information, an x-ray diffraction microprobe can reveal the local structure of a material by detecting its phase, crystallographic orientation and strain.

Demonstration experiments have already exploited the penetrating nature of an x-ray microprobe and its inherent elemental sensitivity to provide new information about elemental distributions in novel materials.
Chemical and orientational imaging of polymeric samples
Authors: Ade H. , Hsiao B. , Mitchell G. , Rightor E. , Smith A. P. , Cieslinski R.
Published: June 2020

We have used the Scanning Transmission X-ray Microscope at beamline X1A (X1-STXM) at Brookhaven National Laboratory (BNL) to acquire high resolution, chemical and orientation sensitive images of polymeric samples as well as point spectra from 0.1 μm areas. This sensitivity is achieved by exploiting the X-ray Absorption Near Edge Structure (XANES) of the carbon K edge. One of the most illustrative example of the chemical sensitivity achievable is provided by images of a polycarbonate/pol(ethylene terephthalate) (70/30 PC/PET) blend. Contrast reversal at high overall contrast is observed between images acquired at 285.36 and 285.69 eV (Fig. 1). Contrast in these images is achieved by exploring subtle differences between resonances associated with the π bonds (sp hybridization) of the aromatic groups of each polymer. PET has a split peak associated with these aromatic groups, due to the proximity of its carbonyl groups to its aromatic rings, whereas PC has only a single peak.
Automated fluorescence-imaging cytometry for the detection of cervical cancer
Authors: Herman Brian , Lockett Stephen , Ayscue Andrea , Periasamy Ammasi , Wodnicki Pawel , Siadat-Pajouh Majid
Published: June 2020

Accumulating evidence strongly associates human papillomavirus infection with the development of cervical cancers. Approximately 70 distinct HPV genotypes have been discovered and a subclass ofthese have been classified as "high" (e.g. 16 and 18) or "low risk" (e.g. 6 and 11) types depending on their association with benign or malignant cervical disease. Numerous studies have suggested that the"high-risk" types of HPV are causally involved in the pathogenesis of cervical cancer. For example, "high-risk" HPVs have been found in 84% of cervical carcinoma. However, it has also become increasingly clear that HPV infections of the cervix span a wide clinical spectrum from benign lesions to precancerous lesions, with only a minority of infections resulting in invasive cancers , although the reasons for this are not clear. Longitudinal epidemiologic studies using cytologic methods to detect HPV infection have shown that the majority of women infected with HPV will regress spontaneously. In addition, age-stratified data for rates of HPV positivity from crosssectional studies also suggest that many women clear the infection spontaneously.
Refinement of the three-dimensional structure of PhoE porin at 3.2Å resolution as determined by electron crystallography
Authors: Jap Bing K. , Walian Peter , Earnest Thomas
Published: June 2020

Density modification methods were used to improve the three-dimensional map of PhoE porin which has been obtained by electron crystallographic techniques. The electron crystallographic data set consists of diffraction amplitudes that extend to a resolution of∽ 2.8 Å and phase information derived from 22 tilted and 8 untilted images at 3.5 Å resolution. However the data set was restricted to tilt angles less than ∽ 60 degrees, and therefore there is a missing cone regionin our 3-D data set. Non-crystallographic symmetry averaging and solvent flattening techniques were used to improve and extend the phase information, allowing for the calculation of high resolution Coulomb potential maps of membrane-embedded PhoE. These maps were used as a basis for building a partial molecular model, which was subsequently used to supply phase information to be combined with the experimentally determined phases. The combined phases were then used to calculate improved maps. As the maps continue to improve, more of the backbone and some of the side chains were built into the maps.
Spot-scan imaging of ice-embedded catalase crystal on a slow-scan CCD camera in a 400-kV electron cryomicroscope
Authors: Chiu Wah , Sherman Michael , Brink Jaap
Published: June 2020

In protein electron crystallography, both low dose electron diffraction patterns and images are needed to provide accurate amplitudes and phases respectively for a 3-dimensional reconstruction. We have demonstrated that the Gatan 1024x1024 model 679 slow-scan CCD camera is useful to record electron diffraction intensities of glucose-embedded crotoxin complex crystal to 3 Å resolution. The quality of the electron diffraction intensities is high on the basis of the measured intensity equivalence ofthe Friedel-related reflections. Moreover, the number of patterns recorded from a single crystal can be as high as 120 under the constraints of radiation damage and electron statistics for the reflections in each pattern.

A limitation of the slow-scan CCD camera for recording electron images of protein crystal arises from the relatively large pixel size, i.e. 24 μm (provided by Gatan). The modulation transfer function of our camera with a P43 scintillator has been determined for 400 keV electrons and shows an amplitude fall-off to 0.25 at 1/60 μm−1.
Time-resolved cryo-electron microscopy using spray mixing
Authors: Berriman John , Unwin Nigel
Published: June 2020

It is often necessary to investigate the three-dimensional structures of biological molecules, or complexes, in short-lived, transitional states in order to obtain a clear understanding of how they work. A simple method is described to this end for cryo-electron microscopy. The two reactants (e.g. protein complex and ligand) are mixed by the coalescence of spray droplets containing one component with a thin, grid-supported aqueous film containing the other. The transitional structure is then trapped by adjusting the delay between the droplet impact and freezing. As an alternative to the photolytic release of caged compounds, this method does not require special chemicals and avoids any heating or photolysis of the sample. It should be possible to catch reaction times as short as 1 millisecond.

The freezing device was a standard guillotine-type plunger in which the grid, mounted on forceps, is allowed to drop by free-fall from a height of several centimeters above the ethane surface.
Silver enhancement of nanogold and undecagold
Authors: Hainfeld James F. , Furuya Frederic R.
Published: June 2020

Silver enhancement of colloidal gold is commonly used to improve visibility of small gold particles in the electron microscope, or to visualize immunogold labeling in the light or confocal microscope. Silver enhancement can also produce a signal visible by eye in such applications as immunodot blotsor staining of gels or transfers with gold reagents. A recent advance in gold technology has been theuse of gold clusters, which are gold compounds, and can be covalently linked to antibodies or other molecules. Two gold clusters developed for such uses are undecagold (Au11 ), which has a 0.8 nm gold core, and a larger, 1.4 nm gold core cluster, •Nanogold•. Because of their small size, many uses requirefurther enlargement via silver deposition.

Here we report quantitative studies of the silver reaction rates for Nanogold and undecagold. These were followed by measuring the UV-visible (UV-vis) spectra at 1 min intervals of solutions of gold clusters mixed with a light insensitive silver enhancer (LI Silver). Solutions were in a 1 cm path length disposable cuvette and spectra were recorded using a Hewlett Packard 8452A diode array spectrophotometer.
Cryo-scanning Electron Microscopy of reovirus structure
Authors: Chen Ya , Centonze Victoria E. , Nibert Max L. , Borisy Gary
Published: June 2020

The introduction of the "in lens" type field emission gun has revolutionized SEM technology. Usingtest specimens, it has been shown that it is now possible to achieve nanometer resolution with the advantages of cryotechniques. Topographical views similar to replicas can also be obtained without having to remove biological material prior to microscopical inspection. Using these techniques we have been able to image the 5.5nm subunit repeat and the 37nm cross-over repeat of actin filaments in vitro and in situ and the 4nm repeat of tubulin subunits in both axonemal microtubules and in vitro polymerized microtubules. We are currently using cryo-SEM to study the reoviruses. Reoviruses are large,nonenveloped viruses with segmented RNA genomes. Understanding the molecular basis of reovirus pathogenesis requires characterization of the structure of reovirus particles and protein components through which they interact with cells early in infection. Recent cryo-TEM studies on reovirus characterized 3 particle forms - virions, ISVPs, and cores - at ≤ 3 nm resolution.
Characterization of cryosectioned cell suspensions by STEM
Authors: Shi S. L. , Sun S. Q. , Nash T. E. , Leapman R. D.
Published: June 2020

To exploit the improved resolution and sensitivity that is now available for biological microanalysis it is crucial to optimize the specimen preparation techniques. This requires rapid freezing and ultrathin cryosectioning to a thickness of around 100 nm. Recently, it has been shown that low-dose scanning transmission electron microscopy (STEM) when applied to such specimens can yield structural detail which is comparable to that from conventional thin plastic sections. After the water is removed from the cryosections by freeze drying in vacuum, the dark-field STEM signal provides a high contrast image of the dry mass distributions of the specimen. In this paper we demonstrate the quality of ultrastructure that is achievable for suspensions of cultured cells which are ideal systems to study since the culture medium can be readily controlled. The method is illustrated by application to the protozoan parasite, Giardia lamblia.

G. lamblia trophozoites, cultured in TYI-S-33 growth medium, were centrifuged at low speed to obtain a very high density suspension of cells.
Synaptic connectivity in hamster circumvallate taste buds
Authors: Zubrzycki K. J.
Published: June 2020

The hamster is an interesting animal model for studying gustation because of its remarkable responsiveness to sweet stimuli. Previous investigators of hamster circumvallate taste have categorized cells into type I, II, and basal cells. The goals of the present study were to determine the ultrastructure of the various cell types and to analyze synaptic contacts in circumvallate taste buds of the hamster.

Adult female Syrian hamsters were prepared for electron microscopy and thick Epon sections (2-4μm) were collected on microscope slides. Selected sections were then reembedded for thin sectioning. Thin sections were sliced and mounted onto Formvar-coated slot grids and poststained in uranyl acetate and lead citrate. The sections were examined with a Hitachi H-7000 transmission electron microscope at 100 kV.

The circumvallate (=vallate) papilla of the hamster is located in the midline of the posterior portion of the tongue. Type I (dark cells), type II cells (light cells) and basal cells were identified within these taste buds.
Organization of primary afferent and local-circuit synapses in the olfactory glomerulus
Authors: Greer Charles A. , Bartolomei Juan C. , Dembner Jeffrey M.
Published: June 2020

Odorant molecules are transduced by olfactory receptor cells whose axons join to form the olfactory nerve which distributes across the surface of the olfactory bulb (OB). Axons exit the nerve layer to terminate within the glomerular neuropil of the OB. While there appears a gross topography between the epithelium and OB4, it is clear that extensive topographic reorganization of axons occurs within the olfactory nerve. To better understand the mechanisms that may contribute to the establishment of glomerular-specific fascicles and functional domains within the OB, we have investigated axonal organization within the nerve and the intraglomerular distribution of primary afferent synapses using light, confocal and electron microscopy.

Sprague-Dawley rats, 30 to 50 days postnatal, were anesthetized, lightly perfused with 0.9% NaCl and the OBs removed. Crystals of the lipophilic dye, Dil, were inserted into the olfactory nerve layer and the tissue placed in 4% paraformaldehyde at room temperature for 10 - 30 days.
Fluorescent potentiometric dyes for membrane-potential imaging
Authors: Loew Leslie M.
Published: June 2020

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.
An ultrastructural and biochemical study of the effects of a 5-lipoxygenase inhibitor on peroxisome proliferation in rat liver and kidney
Authors: Tahir S. K. , Gagne G. D. , Miller M.F.
Published: June 2020

The present study was undertaken to assess the effects of a drug candidate on the morphological and biochemical changes associated with peroxisome proliferation in the liver and kidneys of CD®BR rats. Zileuton, (Abbott-64077), a benzothiophene N-hydroxyurea, is a 5-lipoxygenase inhibitor which has potential therapeutic uses for conditions such as rheumatoid arthritis. To date, several hypolipidemic drugs, certain plasticizers, and a number of unrelated compounds are known to induce peroxisome proliferation in animals. Peroxisomes (also referred to as microbodies) are single membrane-bound organelles found in most tissues, however, their occurrence in hepatocytes is greater than in any other cell type. In the kidneys, peroxisomes are predominantly located in the cortex.

The prolonged proliferation of hepatocellular peroxisomes is of concern since chronic proliferation of peroxisomes has been associated with an increase in hepatic tumors in rodents. In general, peroxisome proliferation can be assessed by an increase in peroxisome number and an increase in fatty acid β-oxidation activity.
Automated counting and 3-D morphometry of nuclei in thick tissue slices from laser- scanning confocal microscopy data
Authors: Ancin H. , Roysam B. , Chestnut M.H. , Otte T.E. , Szarowski D.H. , Turner J.N.
Published: June 2020

This paper presents recent advances in automated three-dimensional (3-D) image analysis methods for cell counting, and quantitative measurement of various nuclear properties in thick (30-120 μm) tissue sections that are imaged by a laser-scanning confocal microscope. The technical advances reported here are: (i) improved 3-D nuclear separation methods for analyzing samples containing large numbers of nuclei per unit volume, and large connected clusters; (ii) methods for adapting the image analysis system to handle a much larger variety of specimens with greater variability in image parameters, such as intensities, nuclear shapes and sizes; and (iii) methods for assisting a user in selecting parameter inputs to the counting system.

Improved 3-D nuclear separation was achieved by computing image gradients from each optical slice. Perona and Malik’s algorithm was used to enhance the image gradient at true nuclear boundaries while suppressing the undesirable intra-nuclear image gradients. The result was used to compute a new proximity index for the partitional cluster analysis method.
Fast automated mosaic synthesis method for 2-D/3-D image analysis of specimens much wider than the field of view
Authors: Becker D.E. , Ancin H. , Roysam B. , Turner J.N.
Published: June 2020

We present an efficient, robust, and widely-applicable technique for computational synthesis of wide-area images from a series of overlapping partial views. The synthesized image is the set union of the areas covered by the partial views, and is called the “mosaic”. One application is the laser-scanning confocal microscopy of specimens that are much wider than the field of view of the microscope. Another is imaging of the retinal periphery using a standard fundus imager. This technique can also be used to combine the results of various forms of image analysis, such as cell counting and neuron tracing, to generate large representations that are equivalent to processing the total mosaic, rather than the individual partial views.

The synthesis begins by computing a concise set of landmark points for each partial view. The type of landmarks used can vary greatly depending on the application. For instance, in the retinal imaging application, the vascular branching and crossover points are a natural choice. Likewise, the locations of cells in Figs. 1 and 2 provide a natural set of landmarks for joining these images.
Association of pituitary adenoma with neuronal christoma: A TEM study
Authors: Horvath Eva , Kovacs Kalman , Scheithauer B. W. , Lloyd R. V. , Smyth H. S.
Published: June 2020

The association of a pituitary adenoma with nervous tissue consisting of neuron-like cells and neuropil is a rare abnormality. In the majority of cases, the pituitary tumor is a chromophobic adenoma, accompanied by acromegaly. Histology reveals widely variable proportions of endocrine and nervous tissue in alternating or intermingled patterns. The lesion is perceived as a composite one consisting of two histogenetically distinct parts. It has been suggested that the neuronal component, morphologically similar to secretory neurons of the hypothalamus, may initiate adenoma formation by releasing stimulatory substances. Immunoreactivity for growth hormone releasing hormone (GRH) in the neuronal component of some cases supported this view, whereas other findings such as consistent lack of growth hormone (GH) cell hyperplasia in the lesions called for alternative explanation.

Fifteen tumors consisting of a pituitary adenoma and a neuronal component have been collected over a 20 yr. period. Acromegaly was present in 11 patients, was equivocal in one, and absent in 3.
An adrenocortical oncocytoma with unusual mitochondrial inclusions and cytoplasmic crystals
Authors: Gunning W.T. , Haselhuhn G.D. , Phillips E.R. , Selman S.H.
Published: June 2020

Within the last few years, adrenal cortical tumors with features concordant with the diagnostic criteria attributed to oncocytomas have been reported. To date, only nine reported cases exist in the literature. This report is the tenth case presentation of a presumptively benign neoplasm of the adrenal gland with a rare differentiation. Oncocytomas are well recognized benign tumors of the thyroid, parathyroid, and salivary glands and of the kidney. Other organs also give rise to these types of tumors, however with less frequency than the former sites. The characteristics generally used to classify a tumor as an oncocytoma include the following criteria: the tumor is 1) usually a solitary circumscribed mass with no gross nor microscopic evidence of metastasis (no tissue nor vascular invasion), 2) fairly bland in terms of mitotic activity and nuclear morphology, and 3) composed of large eosinophillic cells in which the cytoplasm is packed full of mitochondria (Figure 1).
Morphology of Muerto Canyon virus, causative agent of hantavirus pulmonary syndrome
Authors: Goldsmith C. S. , Humphrey C. D. , Elliott L. H. , Zaki S. R.
Published: June 2020

Hantavirus pulmonary syndrome (HPS) is a newly described illness which was first recognized in May, 1993, in the southwestern US. The disease is characterized by fever, myalgias, headache, and cough,with rapid development of fatal respiratory distress. The causative agent, Muerto Canyon virus (MCV), was recently isolated at CDC from a deer mouse, Peromyscus maniculatus, the most common vector for this virus. In order to fully understand this newly discovered virus, we have characterized the isolate by thin section and negative stain TEM, by SEM, and by pre- and post-embedded immunolabeling of thin sections.

MCV is a hantavirus, a member of the family Bunyaviridae. In thin sections of infected Vero E6 culture cells, the extracellular particles are spherical to oval, with fuzzy surface projections along the envelope (figs. 1,2). The interior of the virus contains strands of nucleocapsid. Particles are similar in morphology to Hantann virus, the causative agent of Korean hemorrhagic fever. The negative staining of purified particles (fig. 3) also revealed typical hantavirus morphology of a spherical particle with grid-like surface. Examination by SEM showed scattered particles along the cell surface and among the filopodia.
Identification of the pulmonary syndrome hantavirus by direct and colloidal gold immune Electron Microscopy
Authors: Humphrey C. D. , Goldsmith C.S. , Elliott L. , Zaki S.R.
Published: June 2020

An outbreak of unexplained acute pulmonary syndrome with high fatality was recognized in the spring of 1993 in the southwestern United States. The cause of the illness was quickly identified serologically and genetically as a hantavirus and the disease was named hantavirus pulmonary syndrome (HPS). Recently, the virus was isolated from deer mice which had been trapped near the homes of HPS patients, and cultivated in Vero E6 cells. We identified the cultivated virus by negative-stain direct and colloidal gold immune electron microscopy (EM).

Virus was extracted, clarified, and concentrated from unfixed and 0.25% glutaraldehyde fixed supernatant fluids of infected Vero E6 cells by a procedure described previously. Concentrated virus suspensions tested by direct EM were applied to glow-discharge treated formvar-carbon filmed grids, blotted, and stained with 0.5% uranyl acetate (UA) or with 2% phosphotungstic acid (PTA) pH 6.5. Virus suspensions for immune colloidal gold identification were adsorbed similarly to filmed grids but incubated for 1 hr on drops of 1:50 diluted monoclonal antibody to Prospect Hill virus nucleoprotein or with 1:50 diluted sera from HPS virus infected deer mice.
Acetonitrile is better than ethanol as a dehydrating agent for cells prepared for SEM
Authors: Holshek J. G. , Akins R. E.
Published: June 2020

Acetonitrile has been shown to be a better dehydrating agent than ethanol for whole tissue prepared for TEM studies. In this paper we show that it is also a better choice for dehydration of cell cultures prepared for SEM studies. The procedures used for the isolation and culture of cardiac cells in the present study have been outlined in detail by Schroedl and Hartzell (1983) and Freerksen, et al. (1984). Cells were cultured in suspension using HARV-Bioreactors (Synthecon, Inc., Friendswood, TX) for 24 hours to allow attachment and then fed each subsequent 48 hours. After a total of six days in suspension culture, cell/bead clusters were collected and washed with PBS.

Primary fixation was done at 4°C, all other steps were performed at room temperature. The buffer used in all cases was 0.1M cacodylate, pH 7.3. The cell/bead cultures were processed according to the following protocol. All of the beads were placed in a 1.5 ml microfuge tube and fixed in 2% buffered glutaraldehyde overnight followed by two 5 min washes in buffer, postfixation in 1% buffered OsO4 for 90 min and three washes in buffer. One half of the beads were then placed in another microfuge tube and dehydrated in acetonitrile, while the remaining beads were dehydrated in ethanol.
Air pollution monitoring of the bioindicator lichen,Cetraria cuculata, using microscopy and energy-dispersive x-ray microanalysis
Authors: Crang Richard F. E. , Wu Zheng
Published: June 2020

Lichens, due their sensitivities to variation in environmental conditions have long served as bioindicators of air pollution in arctic and sub-arctic regions for such agents as gaseous air pollutants, acidic rain and misting, and heavy metal deposition. Lichens are an important part of the food chain in arctic regions due to serving as a major food source for animals such as caribou. In this study the fruticose lichen,Cetraria cuculata, was collected from a site 5 km downwind from a major nickel and copper smelting plant near the city of Norilsk in the Siberian region of Russia 330 km north of the Arctic circle. This site is one of the most heavily polluted regions in the world. In contrast, “clean” samples ofC. cuculata, representing controls, were gathered from a remote region 800 km downwind and northeast of Norilsk at the eastern end of Lake Tamyr on the Tamyr Peninsula. Subsequently, different microscopies including scanning electron microscopy (SEM), and energy-dispersive X-ray (EDX) microanalysis were employed in order to determine structural and compositional impacts of the atmospheric deposition. Such techniques have recently been proven to be of considerable value in other studies of contaminated lichen thalli.
Morphological comparison of Pavo cristatus and Pavo cristatus ALB feather barbules
Authors: McKinney M. , Gardner J. S.
Published: June 2020

It was hypothesized that a morphological difference existed between the feather barbules of the colorful, iridescent peafowl and those of the albino peafowl.

Previously molted plume feathers from the Indian (Pavo cristatus) and Albino (Pavo cristatus alb) peafowl were collected from the Tracy Aviary in Salt Lake City, Utah. Segments were selected from the eye region of feathers of both species and they were examined directly with the light microscope (LM). Similar segments were prepared for study with the scanning electron microscope (SEM). Portions of barbs possessing barbules were embedded in Spurr’s resin and sectioned for SEM and transmission electron microscope (TEM) evaluation.

With the light microscope it was observed that only the barbules exhibited brilliant colors. The barbs and rachis did not. By changing the position of the light source, the shade and intensity were altered, but not the color.

SEM images were collected at low magnification (Figs. 1 and 2). Segmentation of the barbules was observed in both Pavo cristatus and Pavo cristatus alb.
A hitchhiker’s guide to microscopy and microanalysis using telecommunications, e-mail, and the Internet
Authors: Zaluzec Nestor J.
Published: June 2020

The Information SuperHighway, Email, The Internet, FTP, BBS, Modems, : all buzz words which are becoming more and more routine in our daily life. Confusing terminology? Hopefully it won't be in a few minutes, all you need is to have a handle on a few basic concepts and terms and you will be on-line with the rest of the "telecommunication experts". These terms all refer to some type or aspect of tools associated with a range of computer-based communication software and hardware. They are in fact far less complex than the instruments we use on a day to day basis as microscopist's and microanalyst's. The key is for each of us to know what each is and how to make use of the wealth of information which they can make available to us for the asking. Basically all of these items relate to mechanisms and protocols by which we as scientists can easily exchange information rapidly and efficiently to colleagues in the office down the hall, or half-way around the world using computers and various communications media. The purpose of this tutorial/paper is to outline and demonstrate the basic ideas of some of the major information systems available to all of us today. For the sake of simplicity we will break this presentation down into two distinct (but as we shall see later connected) areas: telecommunications over conventional phone lines, and telecommunications by computer networks. Live tutorial/demonstrations of both procedures will be presented in the Computer Workshop/Software Exchange during the course of the meeting.
Image-coupling methods in CCD cameras for Electron Microscopy
Authors: Mooney P. E. , Krivanek O. L.
Published: June 2020

It is well established that the charge-coupled device (CCD) is the detector of choice in imaging applications requiring sensitivity, dynamic range, linearity and low geometric distortion. It has also been shown that in the electron microscope, indirect coupling of the image by a scintillator and transfer optic is required to prevent damage to the CCD and to allow for sufficient dynamic range. The question then follows how best to design the coupling to achieve the image quality required for digital imaging in electron microscopy.

We have characterized slow-scan CCD cameras with three representative optical couplings (Figure 1):

1:1 fiber-optically coupled camera with a large-pixel CCD (TK1024) and both single-crystal and powder scintillators for 100-400 kV applications requiring good sensitivity,

1:1 tandem lens-coupled camera with a large-pixel CCD (TK1024) and a powder scintillator mounted on an ultra-thin Al foil for high voltage applications, and

3:1 reduction macro lens-coupled camera with a fast, small-pixel CCD (Kodak MegaPlus) and thin scintillator mounted on a glass prism for applications requiring fast read-out, but not high sensitivity.

In this abstract we compare the three coupling methods to each other, and also to a TV-rate fiber-optically
coupled CCD camera.
Cryo-temperature atomic-force microscopy: Instrumentation and application
Authors: Yang Jie , Mou Jianxun , Shao Zhifeng
Published: June 2020

Shortly after its invention by Binnig et al. in 1986, the atomic force microscopy (AFM) had already been used to image biological specimens. Particularly, in the past two years, tremendous progress has been made, such as high-resolution and reliable imaging of nucleic acids and 1-2 nm resolution imaging of both membrane-bound and soluble proteins under physiological conditions with randomly distributed specimens. Methodology development, such as the reliable preparation of supported membranes, has led not only to the imaging of membrane-bound cholera toxins at high resolution, but has also made it possible to use the AFM to study the membrane structure and to solve important biological problems. It was found that a commonly used buffer compound, tris(hydroxymethyl)aminomethane (C4H11NO3), induced the ripple phase in unilamellar PC-bilayers. AFM study also revealed that both ethanol and isopropanol caused the interdigitated state as well as the interdigitated domains in unilamellar PC-bilayers with the alcohol concentrations well below the normal threshold value.
Direct on-line imaging and quantification of 8-12 bit digital image information for microscopy
Authors: Peters Klaus-Ruediger
Published: June 2020

In microscopy a digital pixel-accurate intensity processing (PAIP) technology for differential contrast enhancement became available for precise extraction and display of image information. Utilizing PC-based massively parallel processing at 1,600 million instructions per second (MIPS), the PAIP image enhancement can be performed on/off-line in near-real-time for objective image evaluation of 8-12 bit data.

We used a PC-based workstation (PiXision PiX4: Visionary Systems Inc., New Haven, CT) on-line in combination with a JSM-890 FSEM (JEOL, Peabody, MA) as well as off-line with imported digital micrograph data and analyzed digital image contrast characteristics. The system provided automated intensity processing of any data independent of image content, size and depth. The images were first smoothed through averaging within a defined significant intensity range (IRs). Each pixel was provided with a variable automatically adjusting local averaging mask, generating a processing characteristic of non-linear point processing. Then, since the spacial processing was pixel-accurate (no spacial distortions of maintained intensity structures), all the contrast information eliminated by the smoothing could be recovered through subtraction of the smoothed from the original or previously smoothed data, followed by linear scaling to 8 bit.
pH-dependent polymorphism of assemblies of cytokine hMIP-lβ
Authors: Cheng N. , Gronenborn A. M. , Clore C. M. , Steven A. C.
Published: June 2020

Human macrophage inflammatory protein 1β (hMIP-1β), also known as Act-2, is a small protein that belongs to an extensive class of chemotactic cytokines. The high resolution solution structure of hMIP-1β has recently been determined by multidimensional NMR spectroscopy, revealing a dimeric molecule of 2 x 8 kDa. The fold of the monomer is very similar to that of the related cytokine, interleukin-8, which was solved previously. However, the mode of association whereby two monomers form a dimer is quite different. To maintain a sufficiently high concentration of soluble protein for NMR studies, hMIP-1β was dissolved at a pH of 2.5. During solubility trials, it was noticed that the hMIP-1β-containing solutions became birefringent at higher pHs, suggesting the formation of ordered light-scattering structures. We have investigated this phenomenon by negative staining electron microscopy.

hMIP-1β was obtained from an expression vector, extracted from inclusion bodies, and purified as described. Protein solutions at 0.7 mg/ml, 100 mM NaCl, 20mM Na citrate, pH2.3, were dialyzed against 20mM ionic strength buffers at pH 4.5, 5.0 and 5.5 (sodium citrate), and pH 6.0 and 7.0 (sodium phosphate) at 4°C. Samples were withdrawn at various time-points, applied to glow-discharged carbon films, stained briefly (5s) with uranyl acetate, and examined in a Philips EM400RT microscope.