bluuebunny/crossref_metadata_2025 huggingface dataset:
MH27 Binds to Several Types of Epithelial Junctions by Em-Immuncytochemistry in the Nematode C. Elegans
Authors: Hall David H.
Published: June 2020

The monoclonal antibody MH27 was used to test a post-embedding method for EM immunocytochemistry in the nematode C. elegans. Primary antibody localization was performed on LR Gold thin sections of lightly fixed him-5 adult animals, followed by a gold-linked anti-mouse IgG secondary antibody (AuroProbe, Amersham Life Science) and viewed on a Philips CM 10 electron microscope. Methods for fixation, embedding, sectioning and antibody procedures followed those of Selkirk et al. (1990) and Hall (1995). Relatively few examples of immunolabelling have been previously demonstrated in C. elegans at the EM level. We have previously found that LR White embedment was unsuitable, as it causes severe shrinkage in nematode tissues. Selkirk and coworkers showed that LR Gold was preferable for EM-immunocytochemistry, and used it to localize several surface antigens. Okamoto and Thomson (1985) previously demonstrated successful results with post-embedding labeling of Epon embedded material, but we anticipate that many antigens will not be well exposed in an Epon thin section.

MH27 was generated during a search for specific antibodies to muscle proteins (Francis and Waterston, 1985). The antibody is now widely used for immunofluorescence studies to demarcate the boundaries of the hypodennal cells, and has proven useful to follow cellular development during embryogenesis and larval stages in C. elegans and other nematode species (Fitch and Emmons, 1995). We were interested to learn the ultrastructural localization of the MH27 antibody, as well as to further develop this technique for C. elegans tissues. We used the him-5 strain so that both male and hermaphrodite animals could be sampled.
An Ultrastructural Comparison of Hepatocytes from ADH+ and ADH−Peromyscus Maniculatus
Authors: Ellzey J. T. , Borunda D. , Stewart B. P.
Published: June 2020

Genetically alcohol deficient deer mice (ADHN/ADHN) (obtained from the Peromyscus Genetic Stock Center, Univ. of South Carolina) lack hepatic cytosolic alcohol dehydrogenase. In order to determine if these deer mice would provide a model system for an ultrastructural study of the effects of ethanol on hepatocyte organelles, 75 micrographs of ADH+ adult male deer mice (n=5) were compared with 75 micrographs of ADH− adult male deer mice (n=5). A morphometric analysis of mitochondrial and peroxisomal parameters was undertaken.

The livers were perfused with 0.1M HEPES buffer followed by 0.25% glutaraldehyde and 2% sucrose in 0.1M HEPES buffer (4C), removed, weighed and fixed by immersion in 2.5% glutaraldehyde in 0.1M HEPES buffer, pH 7.4, followed by a 3,3’ diaminobenzidine (DAB) incubation, postfixation with 2% OsO4, en bloc staining with 1% uranyl acetate in 0.025M maleate-NaOH buffer, dehydrated, embedded in Poly/Bed 812-BDMA epon resin, sectioned and poststained with uranyl acetate and lead citrate. Photographs were taken on a Zeiss EM-10 transmission electron microscope, scanned with a Howtek personal color scanner, analyzed with OPTIMAS 4.02 software on a Gateway2000 4DX2-66V personal computer and stored in Excel 4.0.
An Early Developmental Marker for Radial Glia in Rat Spinal Cord
Authors: Kriho V. , Yang H.-Y. , Lue C.-M. , Lieska N. , Pappas G. D.
Published: June 2020

Radial glia have been classically defined as those early glial cells that radially span their thin processes from the ventricular to the pial surfaces in the developing central nervous system. These radial glia constitute a transient cell population, disappearing, for the most part, by the end of the period of neuronal migration. Traditionally, it has been difficult to definitively identify these cells because the principal criteria available were morphologic only.

Using immunofluorescence microscopy, we have previously defined a phenotype for radial glia in rat spinal cord based upon the sequential expression of vimentin, glial fibrillary acidic protein and an intermediate filament-associated protein, IFAP-70/280kD. We report here the application of another intermediate filament-associated protein, IFAP-300kD, originally identified in BHK-21 cells, to the immunofluorescence study of radial glia in the developing rat spinal cord.

Results showed that IFAP-300kD appeared very early in rat spinal cord development. In fact by embryonic day 13, IFAP-300kD immunoreactivity was already at its peak and was observed in most of the radial glia which span the spinal cord from the ventricular to the subpial surfaces (Fig. 1). Interestingly, from this time, IFAP-300kD immunoreactivity diminished rapidly in a dorsal to ventral manner, so that by embryonic day 16 it was detectable only in the maturing macroglial cells in the marginal zone of the spinal cord and the dorsal median septum (Fig. 2). By birth, the spinal cord was essentially immuno-negative for this IFAP. Thus, IFAP-300kD appears to be another differentiation marker available for future studies of gliogenesis, especially for the early stages of radial glia differentiation.
Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry
Authors: Kömüves László G.
Published: June 2020

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.

Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.
Enzymatic Digestion is also an Effective Antigenicity-Restoring Method for Immunohistochemistry at the Electron Microscope (EM) Level
Authors: Ahmadi P. , Qu Z. , Kayton R. , Andersern D. , Spangler W. D. , Plank S. R. , Rosenbaum J. T.
Published: June 2020

Immunohistological study using EM technique offers several advantages over light microscopy (LM) such as unequivocal identification of cell type by morphology and accurate subcellular localization of antigenic molecules of interest. These are mainly attributed to fixation with strong cross-linking reagents and embedding in resin-based media. However, they also are associated with a major drawback -- greatly reduced antigenicity or completely abolished accessibility to tissue antigen due to extensive cross-linking and the hydrophobicity of the resin. As a result, the success rate of immunolabeling of routinely processed tissue for EM is far lower than that for LM using paraffin sections. Yet the process is far more laborious. To circumvent this problem, various etching or de-plasticizing methods have been introduced. However, most of them invariably lead to deterioration of the tissue morphology. A trade-off between morphology and antigenicity is generally considered necessary. Here we demonstrate that a brief enzymatic digestion prior to immunolabeling the routinely processed tissue samples for EM greatly improves the success rate of the immunostaining without noticeable effect on morphology. Our results suggest that enzymatic digestion is an effective antigenicity-restoring method for EM.
NMR, crystallography, and electron microscopy of nucleoprotein complexes
Authors: Ottensmeyer F. P.
Published: June 2020

The forte of electron microscopy in biology is its unique ability to elucidate the very diverse structures of biological specimens at a range of sizes, with relationships within tissue sections, between and within cells, subcellular organelles, and macromolecules, for the latter with increasingly finer detail. It is the relationship between molecules that is the essence of biology, and it is here that a major emphasis in effort has been placed in recent years in terms of structure determination, not only in electron microscopy, but also in fields with complementary structural approaches, such as the classical x-ray crystallography and the much more recent NMR spectroscopy. The spatial relationship between nucleic acids and proteins is only one facet of this effort, albeit a most important one.

Conventionally electron microscopy has a spatial resolution of about 20 Å in stained biological specimens, but has succeeded with near-atomic resolutions in several cases. NMR spectroscopy per force has to start at atomic dimensions, since its inter-nuclear signals are only available at short range. It builds outward mathematically to molecular dimensions. In the process the technique has permitted the elucidation of detailed interactions, not only between atoms on the same molecule, but in a number of instances between atoms of interacting macromolecules.
The use of an internal standard in the application of quantitative image-EELS in biology
Authors: Abolhassani-Dadras S. , Vázquez-Nin G.H. , Echeverría O.M. , Fakan S.
Published: June 2020

The energy filtering transmission electron microscope (EFTEM) is employed to examine the possibility of using ribosomes as internal standard for a quantitative in-situ study of phosphorus content of nuclear constituents in a biological section. The problems that can arise from different steps of such an experimental approach are discussed.

Salivary gland cells from fourth instar larvae of Chironomus thummi are selected as test specimens because of their appropriate structure. The nucleus of these cells contains two types of granules, one of which (known as Balbiani ring granule, Brg) has an RNA size known from radioactive phosphorus labelling and subsequent isolation, allowing one to estimate its phosphorus content. The other type of granule (known as small RNP granule) has been recently discovered and its phosphorus content is not known. The EFTEM method is used to verify the phosphorus content of these two nuclear constituents.
Biological crystals formation studied by HTREM and image analysis
Authors: Houlle P. , Cuisinier F.J.G. , Voegel J.C. , Schutz P.
Published: June 2020

High resolution electron microscopy (HREM) allows the determination of the molecular crystal structure by comparing HREM images with simulated images. Direct comparison was not possible for small crystalline areas such as nanometer-sized particles or for thin crystals with weak image contrast. To overcome these problems, we used numerical image analysis to gain access to the structure informations within these minute crystals. We used this approach for the characterization of the initial mineralization steps during human amelogenesis, chicken bone and human dentine crystals growth.

Our method consists in HREM associated with both numerical image analysis and image simulation techniques (EMS) Image analysis was performed using the IMAGIC statistical. Nanoparticle subimages, 128 × 128 pixels in size, were extracted from the original micrographs by interactive selection. A circular mask with a radius of 50 pixels was applied. The mean intensity of each subimage was set to zero and its internal variance was normalized to 10. Double direct alignment procedures were used to align the images in rotation and translation against an alignment reference extracted from the data set. An average image was finally calculated to improve the signal to noise ratio.
Scanning electron microscopy of early infection of dyers woad, Isatis tinctoria by germlings of Puccinia thlaspeos
Authors: Binns M. T. , Hooper G. R. , Kropp B. R. , Hansen D. R. , Thomson S. V.
Published: June 2020

Dyers Woad (Isatis tinctoria) is an introduced noxious weed of crop and rangeland in northern Utah. A native rust fungus (tentatively identified as Puccinia thlaspeos) is being investigated as a possible biological control agent of the weed. In order to effectively use the rust in a bio-control program, it is necessary to understand its epidemiology and characteristics.

Weeds were inoculated by suspending previously infected weed leaf fragments over test plants. Teliospores on these fragments produced basidiospores which dropped onto the weed seedling leaf surfaces. Inoculated leaves were prepared for SEM by fixation in 2% cacodylate buffered glutaraldehyde, post fixed in 2% buffered osmium tetroxide and dehydrated through ethyl alcohol and critical point dried. Mounted specimens were sputter-coated with gold. Some tissues were freeze fractured in 100% alcohol and fragments were critical point dried, coated and mounted for SEM.
SEM study of phytoliths produced by grasses indigenous to the desert experimental range in southwestern utah
Authors: Carver Melanie R. , Ball Terry , Gardner John S. , Erickson Kali M.
Published: June 2020

The USDA Desert Experimental Range Station located in southwestern Utah has been conducting an ongoing investigation into the effects of grazing on the ecology of the region, The study reported herein is a preliminary effort to determine if phytolith analysis can be used to gain insight into the ecology of the region prior to the 60 year period over which records have been kept.

Phytoliths are solid deposits of silicon dioxide that form at specific intracellular and extracellular locations in many plant taxa. Grasses produce phytoliths with morphological characteristics diagnostic of their taxa. When a plant dies, its phytoliths resist decomposition thereby becoming micro-fossils of the plant that produced them. Micro-fossil phytoliths provide archaeobotanical and ecological information because they remain in the same soil horizon at which decay occured. In this study phytoliths produced by four grasses indigenous to the Experimental Range were analyzed. Phytoliths extracted from soil samples of different grazing treatments at the Range were then analyzed to determine if any similar to those produced by the four grasses could be identified in the soil. If so, then phytolith analysis could prove to be a viable research tool for those studying the paleoecology of the Range.
Characterization of the effect of heat stress on maternal and embryonic tissues of maize (Zea mays L.) kernels using scanning electron microscopy (SEM)
Authors: Commuri P. D. , Jones R. J.
Published: June 2020

Heat stress during the early formative stages of maize (Zea mays L.) kernel development is detrimental to subsequent growth and grain yield. During this period, even a brief exposure to temperature above the optimum (25°C) can result in significant yield losses. The mechanisms involved in this response are not well understood, but appear to be associated with a thermally induced shift in hormone balance (cytokinin/ABA), which results in a reduction in kernel sink capacity (i.e. number of endosperm cells and starch granules formed) and the disruption of sugar metabolism and starch biosynthesis. However, the effect of heat stress on the morphology and ultra-structure of kernel component tissues (pedicel, pericarp, endosperm, and embryo) has not been studied. Therefore, the objective of our current study was to characterize heat stress induced changes in the pericarp (maternal) and the embryo (embryonic) tissues using SEM.
Cytochemical localization of chloride in the leaves of Ruppia maritima L
Authors: Barnabas A. D.
Published: June 2020

Ruppia maritima L. is a submerged halophyte which occurs frequently in estuaries where sodium chloride is the dominant salt. Unlike terrestrial halophytes, R. maritima does not possess any specialized salt-secreting structures such as salt glands. Knowledge of salt tolerance mechanisms in this plant is important to our understanding of its biology. In the present study, the subcellular distribution of chloride ions in leaf blade epidermal cells of R. maritima was determined.

Chloride ions in the cells were localized by precipitation with silver, resulting in the formation of silver chloride (AgCl2) deposits. Leaf samples were fixed in a mixture of osmium and silver acetate under red safe light conditions to prevent reduction of silver by light. The material was dehydrated and embedded in a chloride-free resin. Leaf samples fixed only in osmium were used as controls. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) microanalysis using a Joel 6100 SEM with a Noran Voyager 2100 EDX microanalyser, were used to study AgCl2 distribution. Ultrathin sections for TEM were examined unstained. EDX microanalysis was performed on thick sections of resinembedded material mounted on glass slides.
Rigid-body translation (RBT) of a NiO-ZrO2(cubic) bicrystal and its implications for interface atomic structure
Authors: Dickey E. C. , Dravid V.P.
Published: June 2020

Any internal interface between two crystalline materials can be described by five macroscopic and three microscopic degrees of freedom. The five macroscopic, or geometric, degrees of freedom describe the relative orientations of the two crystalline lattices and interface plane. For the case of low-energy NiO-ZrO2 boundaries this is defined by the orientation relationship: NiO(111)//ZrO2(100), NiO[110]//ZrO2[010]. The three microscopic degrees of freedom describe the relative in-plane and out-of-plane RBT (Tx,Ty,Tz); these RBTs are geometric mechanisms by which the interface can relax to a low-energy configuration. The combination of these eight degrees of freedom describes completely the bicrystallography. This paper investigates the RBT associated with NiO-ZrO2 interfaces as measured from HREM and Z-contrast images (see fig. 1).
Low-voltage fesem study of TiO2 surface structure and metallization
Authors: Cosandey F.
Published: June 2020

Recent developments of Field Emission Scanning Electron Microscopes (FESEM) are now permitting material science studies of surfaces with nanometer scale resolution. For the study of oxide materials with low conductivity it is particularly important to image surfaces at low voltage in order to minimize both the electron range and charging. The unique electron optic design of the LEO (ex ZEISS) 982 GEMINI microscope combining retarding field and electrostatic lens concepts with Schottky field emission source is particularly well optimize for high resolution imaging of materials at low voltage. In this study, we are presenting results on performance evaluation of the LEO 982 FESEM microscope with a study of TiO2 surface structure and metallization.
Energy-filtered imaging of polymer microstructure
Authors: Siangchaew K. , Bentley J. , Libera M.
Published: June 2020

Energy-filtered electron-spectroscopic TEM imaging provides a new way to study the microstructure of polymers without heavy-element stains. Since spectroscopic imaging exploits the signal generated directly by the electron-specimen interaction, it can produce richer and higher resolution data than possible with most staining methods. There are basically two ways to collect filtered images (fig. 1). Spectrum imaging uses a focused probe that is digitally rastered across a specimen with an entire energy-loss spectrum collected at each x-y pixel to produce a 3-D data set. Alternatively, filtering schemes such as the Zeiss Omega filter and the Gatan Imaging Filter (GIF) acquire individual 2-D images with electrons of a defined range of energy loss (δE) that typically is 5-20 eV.
Digital imaging and quantitative image analysis of polymer blends
Authors: Zhang Xiao
Published: June 2020

Polymer microscopy involves multiple imaging techniques. Speed, simplicity, and productivity are key factors in running an industrial polymer microscopy lab. In polymer science, the morphology of a multi-phase blend is often the link between process and properties. The extent to which the researcher can quantify the morphology determines the strength of the link. To aid the polymer microscopist in these tasks, digital imaging systems are becoming more prevalent. Advances in computers, digital imaging hardware and software, and network technologies have made it possible to implement digital imaging systems in industrial microscopy labs.
Crystal structure and morphology of syndiotactic polypropylene single crystals
Authors: Bu Jenny Z. , Cheng Stephen Z. D.
Published: June 2020

In the past several years there have been an increased interest in the crystal structure and morphology of s-PP due to the new development of homogeneous metallocene catalysts which can produce s-PP having a high stercoregularity. In this research, the crystal structure and morphology of s-PP single crystals grown from the melt were investigated. A series of ten fractions of s-PP was studied with dillcrent molecular weights ranging from 10,300 to 234,000 (g/mol). These fractions all possess narrow molecular weight distributions (around 1.1-1.2) and high syndiotacticitics ([r]˜95%). The main techniques employed including transmission electron microscopy (T1:M), atomic: lorcc microscopy (AFM), wide-angle X-ray diflraction (WAXD), and small-angle X-ray scattering (SAXS).
SEM analysis of in situ polymerization products for chromatographic separations
Authors: Cutler Bruce , Algaier Joseph , Svec Frantisek
Published: June 2020

Two inherent problems associated with chromatographic separations are peak broadening and column efficiency. These are directly attributable to the interparticular porosity of the separation media in which limited utilization of column space results in column void volumes. In theory, a minimum of 26% void volume will occur if a column is packed using perfectly arranged monodispersed beads. Typically, with the best packed columns void volumes are 30 to 40%. Svec and Fréchet have introduced an HPLC separation media with no discontinuity that are prepared in a single step by a free-radical polymerization within a column acting as a mold. The resulting rods consist of a continuous molded monolith of rigid, highly porous polymer. These columns proved to be very efficient and extremely fast for reverse-phase HPLC separation of proteins and for the separation of small and mid-size molecules. In this study, we continue to explore various reaction conditions with the resulting polymerization products examined by SEM, for internal morphology.
The study of multiphase polymer-blend morphologies by HVEM
Authors: Cavanaugh T.J. , Buttle K. , Turner J.N. , Nauman E.B.
Published: June 2020

Multiphase polymer blends are important in the polymer industry. Most commercial blends consist of two main polymers combined with a third, compatibilizing polymer, typically a graft or block copolymer. The most common examples are those involving the impact modification of a brittle thermoplastic by the microdispersion of a rubber into the matrix. Recently, a model of ternary polymer blends has provided a wealth of morphologies for examination. Even though this model can give an excellent basis for the design of a polymer blend, experimental verification is necessary. A correlation of blend properties such as impact strength with blend morphology must also be made. The focus is to confirm the predicted morphologies in binary and ternary blends using HVEM.

The polymer blends were produced by compositional quenching. In this process, the polymers were dissolved in a solvent. The solution was pumped through a heat exchanger and then flashed across a needle valve to remove the solvent.
Length-Scale-Dependent surface roughness measurements of bioactive polymer thin films using scanning probe microscopy
Authors: Buchko Christopher J. , Kozloff Kenneth M. , Martin David C.
Published: June 2020

The development of implantable biomedical devices requires active biological interfaces that minimize the body’s immune response. We have identified candidate protein polymer coatings for biomedical applications and methods of processing these coatings that promote a favorable in vitro biological performance. However, the chronic implantation of biomedical devices, specifically the neural prosthetic devices developed at the University of Michigan, places additional requirements on the polymer coatings. Among these constraints are the efficient transport of neuronal signals from tissue to the device and the prevention of physical displacement of the device through tissue. To meet these potentially conflicting goals, we are developing discontinuous, porous polymer coatings that are rough at biologically-relevant length scales.

In order to quantify the surface morphology of these thin polymer films, which are typically 0.02 μm to 5.0 μm thick, we employ a number of complementary techniques, including scanning probe microscopy (SPM), scanning electron microscopy (SEM), light optical microscopy (LOM), and transmission electron microscopy (TEM).
Scanning thermal microscopy (SThM) of polymer blends: Phase separation, localised calorimetric analysis
Authors: Hammiche A. , Pollock H. M. , Leckenby J. N. , Song M. , Reading M. , Hourston D. J.
Published: June 2020

We are combining thermal analysis with the ability of thermal waves to give sub-surface imageing at sub-micron resolution. Our aim is that the image contrast will be determined by the “modulated-temperature differential scanning calorimetry” peak (M-T DSC) or feature chosen according to the particular component of the specimen that is of interest. As described elsewhere, this will allow us to distinguish in the image between reversible processes such as amorphous-to-glass transitions, from recrystallisations, cure reactions and other irreversible processes within the polymer.

The SThM probe gives micron-resolution images whose contrast is determined by spatial variations in thermal properties. Either the temperature at any point is held constant, or we generate and detect evanescent thermal waves whose penetration depth depends on the frequency chosen.The evidence is that a 1 μm-radius probe in air will give a lateral resolution of ca. 200 nm, with a depth detection of a few μm.
Microprobe study of diode corrosion
Authors: Hlava Paul , Braithwaite Jeff , Sorensen Rob
Published: June 2020

A few diodes from a production lot were discovered to have unacceptable current leak rates after about 5 years of storage. Inspection revealed the presence of copper sulfide deposits that bridged the external body of the diode and presumably provided a leakage path. Figure 1 shows the physical configuration of a diode. The function of this device is performed by a silicon-based semiconductor that is bonded between two cylinders of tungsten with copper headpins (Cu wire with a flat “nail-head” formed on one end) brazed to the opposite ends of the cylinders. A toroidal ring of glass protects the Si chip. All exposed metal parts are covered by an immersion plating of Ag. Then the entire assembly is coated with black epoxy and a band of green ink is applied to the cathode end. During storage, each diode was placed in a cardboard holder and secured in stacks of about ten with rubber bands.

Analytical and environmental exposure studies were performed at Allied Signal and Sandia to determine the cause and potential long-term significance of this corrosion-related degradation problem. The objective of the analytical work was to characterize the corrosion product and help identify the corrosion mechanism. Tools included optical microscopy, scanning electron microscopy, electron microprobe analysis, SIMS, and x-ray diffraction.
HRTEM and chemical analysis of NiAl-5Ti
Authors: Wilson A. W. , Howe J. M. , Garg A. , Noebe R. D.
Published: June 2020

NiAl-based alloys are currently being studied to determine the effects of alloying additions on the mechanical and physical properties of NiAl. Microstructural changes due to varying alloy composition have been determined and related to the mechanical properties, but high-resolution imaging and compositional profiles of the nanophases and interfaces in these alloys have not been performed. The current study involves the use of a JEOL 2010F field-emission analytical TEM operating at 200 kV equipped with an ultra-thin window energy dispersive X-ray (EDX) detector to obtain high resolution images and compositional information from various nanophases. A single crystal of NiAl containing 5 at.% Ti was examined in this study. EDX spectroscopy of the specimen revealed the presence of low levels of impurities such as Si and P, as shown below. Bulk chemical analysis revealed that Si was present at 190 ppm while P was present at levels below detectability limits.
Mullite Phase Separation in Nanocomposite Powders
Authors: Yao Nan , Dabbs Daniel M. , Aksay Ilhan A.
Published: June 2020

Mullite/alumina composite materials are of considerable interest in many applications because of their high-temperature properties, such as creep resistance, and thermal shock resistance. Although mullite has relatively low fracture toughness and strength at low temperature (2.2 MPa.m1/2 and 200-500 MPa, respectively), it retains the greater part of its physical strength up to 1500°C. We seek to improve the mechanical properties of mullite monoliths by using nanosized (<100nm diameter) inclusions in the matrix, where the nanosized inclusions would act to strengthen the matrix in a manner akin to precipitation hardening in metals. Our approach is to make nanocomposite powders and monoliths with controlled chemical composition and phase distribution on the nanometer length scale (1-10 nm).

We have recently formed nanocomposite powder using spray pyrolyzed precursors. The precursor was formed from a solution of TEOS and aluminum nitrate nonahydrate in ethanol, mixed in proportions equivalent to the stoichiometry of a mullite/alumina composite composed of 20 wt% alumina in a mullite matrix.
Protein Crystallography Using Capillary-Focused X-Rays
Authors: Balaic D. X. , Barnea Z. , Nugent K. A. , Garrett R. F. , Varghese J. N. , Wilkins S. W.
Published: June 2020

Tapered glass capillaries for X-ray beam concentration have been a topic of much interest for the synchrotron X-ray community in recent years. These optics have long held the promise of high-intensity microbeam generation for the “hard” X-ray energies used in crystallography and fluorescence analysis.

X-ray concentration is achieved by exploiting the total external reflection property of glass surfaces for glancing angles of incidence. X-rays directed into the entrance aperture of the capillary are reflected toward an exit aperture of smaller dimensions, resulting in an increased X-ray flux per unit area at the exit aperture. Capillary designs with a true geometrical focus beyond the capillary exit are also possible.

Our group has recently demonstrated a paraboloidally-tapered glass capillary optic which produced a focused X-ray beam using a monochromatised synchrotron source. The optic was designed to produce a focal region for singly-reflected X-rays at a point 40 mm from the end of the capillary. Such a focal region was observed, with a FWHM intensity gain of two orders of magnitude over the incident X-ray intensity from the channel-cut monochromator. Moreover, this gain was achieved for X-ray energies from 5 to 20 keV. We subsequently used a similar optic to obtain X-ray diffraction patterns from a crystal of hen egg-white lysozyme on image plates. The use of the capillary-focused beam yielded diffraction patterns 70 to 100 times faster than using an unfocused beam from the channel-cut monochromator alone. Placement of the crystal at different positions in the capillary-focused beam demonstrated the focusing of Bragg reflections and diffraction from a small volume of crystal.
Spectral Imaging through the light microscope: Application to the image analysis of colored structures in histology
Authors: Omberg R. L.
Published: June 2020

Color is an important factor in defining structures in both biological and materials microscopy. Indeed, the field of histology/pathology is founded on the use of special dyes and staining procedures which label features of interest with a defining color. Of particular interest is the use of colorigenic substrates in enzyme linked – immunochemistry on tissue sections and the subsequent measurement of the amount of labeling in a given tissue. Typically, colored image objects such as the red deposit produced by the reaction of AEC (aminoethylcarbazole) substrate with peroxidase enzyme can be detected in RGB (red, green, blue) images by a combination of RGB pixel intensities or by HSI (hue, saturation, intensity) pixel intensities. Using a new color imaging system with spectral image acquisition capabilities, we have develop an imaging algorithm based on the absorption spectra of the colored object and the counter stained surrounding space that provides optimal images of segmentation and morphological measurement.

The basic system consists of a low light level CCD coupled to a liquid crystal tunable filter (Cambridge Research Instruments). The filter is based on the Lyot-type birefringent filter and provides continuous wavelength selection from 400 - 720 nm with wavelength dependent resolution of about 10 nm at 500 nm. The system is controlled by a Silicon Graphic workstation and Isee software written by Inovision Corp. Spectral images, i.e., images at a given wavelength, can be obtained and the intensity of objects with in a given image can be sampled as a function of wavelength for microspectro -photometric or -fluorometric analysis.
Crack analysis of unfilled natural rubber using infrared microspectroscopy
Authors: Neumeister L. A. , Koenig J. L.
Published: June 2020

Several spectroscopic methods are available for characterizing the crack tip region of natural rubber on the molecular scale to develop a material with fatigue resistant properties. Attenuated total reflectance or ATR-IR has been used to characterize the structure of many different polymers, including rubbers. Transmission has also been well established as a viable technique for the molecular characterization of transparent materials including thin films of unfilled natural rubber.

Unfilled natural rubber was stretched to 629% elongation until cracks appeared in the surface. A cross section of the sample containing a crack was then microtomed at -85°C into slices approximately 0.5 µm thick. Similarly, samples were cut with a razor and microtomed under the same conditions. The crack tip region was mapped using the IRμs™/SIRM Molecular Microanalysis System. The map consisted often spectra taken in the x and y directions as shown in Figure 1. The same region was analyzed for orientation of molecular structures. Points were selected along the crack tip, crack edges, and the bulk. Dichroic ratios of all prominent peaks were calculated. ATR was used to verify the results of the mapping experiments for both stressed and unstressed material.
Fourier transform infrared chemical imaging microscopy: Applications in neurotoxicity and pathology
Authors: Lewis E. Neil , Kidder Linda H. , Lester David S. , Kalasinsky Victor F. , Levin Ira W.
Published: June 2020

We have developed a new Fourier transform infrared chemical imaging technique which, when coupled with powerful multivariate data processing techniques, allows the visualization of complex intrinsic chemical distributions in biological materials with the potential for performing rapid histological examinations. Integrating spectroscopy with sample visualization and digital image processing is a potent combination of what have traditionally been two distinct methods for studying the chemistry and morphology of a sample. This synergy has been referred to as chemical imaging or hyperspectral imaging and has wide ranging applications in material characterization. In the infrared spectral region the technique relies on the use of infrared focal-plane array detectors, a newly developed commercial technology. These arrays are used in conjunction with standard Cassegrainian infrared microscopes and commercially available step-scan Michelson FTIR spectrometers to construct infrared imaging systems capable of collecting tens of thousands of spatially resolved infrared spectra (3μm2) with less than 1 minute of data acquisition time. The data sets contain both spatial and spectral information and typically consist of hundreds of images resolved in frequency space (wavenumbers, cm−1), with each image containing many tens of thousands of pixels.
Ion-Beam Milling Materials with Applications to TEM Specimen Preparation
Authors: Anderson Ron
Published: June 2020

For the last thirty years, ion milling has been an indispensable part of preparing TEM specimens in the physical sciences. While great improvements have been made in our ability to thin most materials to the point where ion milling may not be a requirement, there will still be a need to utilize ion milling to clean and polish specimens and to provide small amounts of incremental thinning as needed. Thanks mainly to the work of Bama we now understand a great deal about the physics of ion milling. We also benefit from the works of a number of investigators who have studied the artifacts produced by ion milling (see Barber for a review).

Ion milling is a subset of the topic “dry etching,” which consists of two major categories: glow discharge methods and ion beam methods. Glow discharge methods include plasma etching, reactive ion etching, and glow discharge sputter etching. These techniques have little application in TEM specimen preparation aside from surface cleaning. The reactive ion etching literature is a source for suggesting gas/specimen combinations to perform chemically-assisted ion beam etching (CAIBE), to be discussed below. The other major dry etching category, ion beam methods, includes ion milling, reactive ion beam etching, and CAIBE.
Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications
Authors: Marshall W.F. , Oegema K. , Nunnari J. , Straight A.F. , Agard D.A. , Sedat J.W.
Published: June 2020

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).

Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.
3D Microscopy using Confocal Microscopy
Authors: Stelzer Ernst H. K. , Lindek Steffen
Published: June 2020

The importance of confocal fluorescence microscopy in modem biological research results from its optical sectioning capability, which allows the three-dimensional analysis of thick specimens. This property is due to the combination of a point-like light source and a point-like detector, which restrict the illumination and detection volumes, respectively. Only the volume that is illuminated and detected is relevant to the confocal observation volume. The smaller it is, the better is the resolution. The performance of a confocal microscope is thus primarily specified by the spatial extent of the confocal point spread function (PSF). The extent can be estimated, e.g., by the volume enclosed by the isosurface at half maximum of the PSF (VHM – volume at half maximum).

The relationship of the parameters that determine the lateral resolution of a microscope has been described by Ernst Abbé. The diameter of a light spot in the focal plane Δx is proportional to the wavelength λ of the incident light and inversely proportional to the numerical aperture of the optical
system (N.A. = n, ∙ sin α).
Time-Resolved Stimulated-Emission Fluorescence Microscope
Authors: So P. T. C. , Dong C. Y. , Buhler C. , Gratton E.
Published: June 2020

Time-resolved stimulated-emission fluorescence microscopy is a novel technique for obtaining super-diffraction limited spatial resolution and sub-nanosecond time resolution using a multi-photon process. This technique is inspired by traditional asynchronous stimulated-emission pump-probe spectroscopy. Fluorescence sample is first excited by a pump laser pulse, tuned to the molecular absorption band of the molecule. Within the chromophore lifetime, a second probe pulse, tuned to the emission band, stimulates fluorescence emission.

The spatial resolution enhancement originates from the bilinear dependence of the stimulated emission efficiency on both the pump and probe beam intensities. At the objective focal point, the stimulated emission point spread function is the product of the point spread functions of the pump and probe beams. This situation is mathematically equivalent to both the confocal and the two-photon methods. 3-D depth discrimination and superior spatial resolution is expected.
Going Beyond 3-D Imaging: Automated 3-D Montaged image Analysis of Cytological Specimens
Authors: Roysam Badrinath , Ancin Hakan , Becker Douglas E. , Mackin Robert W. , Chestnut Matthew M. , Ridder Gregg M. , Dufresne Thomas E. , Szarowski Donald H. , Turner James N.
Published: June 2020

This paper summarizes recent advances made by this group in the automated three-dimensional (3-D) image analysis of cytological specimens that are much thicker than the depth of field, and much wider than the field of view of the microscope. The imaging of thick samples is motivated by the need to sample large volumes of tissue rapidly, make more accurate measurements than possible with 2-D sampling, and also to perform analysis in a manner that preserves the relative locations and 3-D structures of the cells. The motivation to study specimens much wider than the field of view arises when measurements and insights at the tissue, rather than the cell level are needed.

The term “analysis” indicates a activities ranging from cell counting, neuron tracing, cell morphometry, measurement of tracers, through characterization of large populations of cells with regard to higher-level tissue organization by detecting patterns such as 3-D spatial clustering, the presence of subpopulations, and their relationships to each other. Of even more interest are changes in these parameters as a function of development, and as a reaction to external stimuli. There is a widespread need to measure structural changes in tissue caused by toxins, physiologic states, biochemicals, aging, development, and electrochemical or physical stimuli. These agents could affect the number of cells per unit volume of tissue, cell volume and shape, and cause structural changes in individual cells, inter-connections, or subtle changes in higher-level tissue architecture. It is important to process large intact volumes of tissue to achieve adequate sampling and sensitivity to subtle changes. It is desirable to perform such studies rapidly, with utmost automation, and at minimal cost. Automated 3-D image analysis methods offer unique advantages and opportunities, without making simplifying assumptions of tissue uniformity, unlike random sampling methods such as stereology.12 Although stereological methods are known to be statistically unbiased, they may not be statistically efficient. Another disadvantage of sampling methods is the lack of full visual confirmation - an attractive feature of image analysis based methods.
Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy
Authors: Martin Greg V. , Hubbard Ann L.
Published: June 2020

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.