bluuebunny/crossref_metadata_2025 huggingface dataset:
Bonding in Ion-Implanted Diamond-Like Carbon Films Characterized by TEM Spectrum Lines and Energy-Filtered Imaging
Authors: Bentley J , Walter KC , Evans ND
Published: July 2020

Many of the electrical, mechanical, and electrochemical properties of diamond-like carbon (DLC) coatings are determined by the amount of sp3 bonding. Correlations of bonding and properties are important in efforts to optimize coatings for specific applications. DLC thin films were grown at LANL by plasma deposition and subsequently ion implanted with N or Ar. Cross-sectioned TEM specimens were characterized with a Gatan imaging filter (GIF) interfaced to a LaB6 Philips CM30T at the ORNL SHaRE User Facility. For elemental distribution maps the following conditions were used: incident beam divergence α = 2.9 mrad, collection angle β = 4.8 mrad, slit width ΔE = 30 eV, exposure times of typically 5 s, 2x-binned 512×512 images, and TEM magnification ∼2000. The standard 3-window method was used for producing elemental maps with AE-r background extrapolation; 2-window jump-ratio images were also produced.
Mapping the Subcellular Distribution of Calcium in Depolarized Neurons by Electron Energy Loss Spectrum Imaging
Authors: Andrews SB , Hongpaisan J , Pivovarova NB , Friel DD , Leapman RD
Published: July 2020

In the context of biological specimens, it is in principle desirable to quantitatively map, rather than just point analyze, the distribution of physiologically important elements, and to do so at subcellular resolution. Presently, this can be accomplished by electron energy loss spectrum-imaging (EELSI) in both the scanning transmission electron microscope (STEM) and the energy-filtering transmission electron microscope (EFTEM). Until recently, this approach has been of limited value for mapping the particularly important element Ca, mainly because intracellular total Ca concentrations are normally quite low (<5 mmol/kg dry weight) and because the background in the vicinity of the Ca L23 edge is complex and requires precise background modeling to extract the very weak Ca signals. As a result, the Ca signal is usually not high enough to reach detection threshold during a practical EELSI acquisition time.
New Approaches for Energy Filtered Imaging and EELS Analysis
Authors: Botton GA
Published: July 2020

Electron energy-loss spectroscopy imaging is now becoming an established and indispensable tool in high-resolution materials characterization. Whether the imaging approach is based on post column filters attached to a conventional transmission electron microscope (TEM) or on parallel spectrometers on a scanning (field emission) TEM, energy filtered (EF) microscopy is having a significant impact in materials science (e.g. Ref. 1). Elemental mapping, however, is not the only imaging tool accessible with EF microscopy. EELS images and spectra are often only part of the data that is obtained when samples are characterized in an analytical TEM. Complementary information is also obtained from EDS spectra which are often analyzed independently from the EELS data and from EELS near edge structures. When the EELS fine structure is altered due to changes in chemical state of the probed atoms, additional information is accessible and can potentially be used in novel methods of image acquisition.
Applications of Energy-Filtered Imaging in Mineralogy, I: Pyroxenes
Authors: Elbert DC , Moore KT , Veblen DR
Published: July 2020

Chemical segregation is fundamental to a host of geologically interesting phenomena. These include exsolution, weathering, dynamic recrystallization, and interface formation. Investigation of these mineralogical processes centers on the relationship between crystal structure and chemistry. While there is an abundance of electron microprobe information available at the micron scale, Energy- Filtered Imaging (EFI) offers the first opportunity to investigate the chemistry of these diverse processes at the nearly atomistic level. In particular, we have been using Energy-Filtered Imaging (EFI) in the TEM to acquire quantitative compositional information at the scale of individual unit cells (<1 nm spatial resolution) in exsolved pyroxenes. Along with the two-orders-of-magnitude improvement in spatial resolution, EFI has greater quantum efficiency and the possibility of lighter element detection than X-ray spectroscopic methods.

The complex, subsolidus phase relations of many minerals are routinely exploited by petrologists investigating the thermal and tectonic evolution of the earth, moon and meteorites.
Valence Mapping of Particulate 3D-Transition Metal Oxides Using Energyfiltered Transmission Electron Microscopy
Authors: Stroud RM , Scott JH
Published: July 2020

Particulate, mixed-valence transition metal oxides are frequently used for battery, catalytic and magnetic applications. For example, the Li ion exchange battery exploits charge transfer of mixed Mn+3, Mn+4 materials. Charge localization and phase separation, especially at particle surfaces, are critical issues for determining the materials’ useful properties, be it catalytic activity or saturation magnetization. The ability to image the charge localization and correlate this with crystallographic information would be extremely useful in the study of this class of materials. Using energy-filtered transmission electron microscopy (EFTEM), valence maps of Mn and Co with a ∼ 2 nm scale have been obtained for bulk samples. In principal this technique can de directly extended to the case of particulate samples, however there are some additional experimental challenges, such as thickness and edge effects, that must be addressed. We demonstrate here the feasibility of valence mapping of particulate samples, and discuss the factors that limit quantitative data extraction from the maps.
Future Trends in Modeling Electron Energy Loss Near-Edge Structure (ELNES)
Authors: Scott A J , Brydson R
Published: July 2020

The increasing availability of fast, multiprocessor high performance computing facilities has revolutionized iterative electronic structure calculations of solids, particularly for those systems possessing little or no symmetry. This combined with the semi-commercial dissemination of well-supported software packages for the calculation of both occupied and unoccupied electronic states at high levels of approximation has opened up the possibilities for more routine modeling and understanding of electron energy loss near-edge structure (ELNES) data obtained in the TEM/STEM, and hence the elucidation of chemical bonding in solids at high spatial resolution.

The two modeling approaches presented and compared here involve ab-initio band structure calculations based on the full linear augmented plane wave (FLAPW - WIEN97) approach, and full multiple scattering (MS) clusterbased methods. We have performed a systematic study of transition metal carbides and nitrides and compared the modeling results with high resolution EELS data.
Boron K-Edge Electron Energy Loss Fine Structure Study of Gadolinium-Containing Sodium-Alumino-Borosilicate Glasses
Authors: Qian M , Li L , Li H , Strachan D M
Published: July 2020

Up to 47 mass% of gadolinium can be dissolved in a baseline borosilicate glass (mol%) 20 B2O3, 5 A12O3, 60 SiO2, and 20 Na2O. Understanding the dissolution of Gd in borosilicate melts is important in the optimization of glass formulations. However, probing amorphous materials with spectroscopic techniques is challenging research when little knowledge of the local chemical environment of the glass network is available. Electron energy loss fine structure (ELFS) spectroscopy is one available technique that has several advantages for probing the local structure of amorphous materials. This technique gives local atomic structure information equally well for both amorphous and crystalline materials. There is high sensitivity for low Z elements like boron, oxygen, aluminum, and silicon whereas x-ray absorption fine structure (XAFS) technique faces experimental difficulties. Hence, results from ELFS can be complimentary to the results from XAFS. For medium to high Z elements, one can use the L- or M-edges of the elemental spectrum.
First Develoment Step and Test of The SESAM / SATEM
Authors: Kujawa S
Published: July 2020

For the SESAM/SATEM project is for testing and as a first development step a new fully corrected imaging energy filter of the Ω type built. It is incorporated in a 200keV EFTEM equipped with an LaB6 cathode and a objective lens with a spherical and chromatic aberration coefficient of Cs = Cc = 1.2mm. This EFTEM will be used this year by the SESAM project group at the University Tiibungen, Germany and the MPI fur Metallforschung, Stuttgart. At the same time this kind of filter will be also mounted in the final SATEM.

The energy filter is similar to the design of the so-called FHI-filter suggested by Rose and Lanio [1,2] and built at the Fritz-Haber-Institut of the MPG by Krahl [3]. The total length of the proposed filter is half of the FHI-filter and nearly the same as the length of the LEO 912Ω filter.
A New High Performance Detector for Electron Energy Loss Spectroscopy
Authors: Brink HA , Trevor C , Hunt J , Mooney PE
Published: July 2020

The serial detectors traditionally used in Electron Energy Loss Spectroscopy (EELS) have mostly been replaced by parallel detection systems, for reasons of efficiency and ease of use. So far most parallel detection systems have been based on fiber or lens coupled Photo Diode Array (PDA). Although these systems have proven satisfactory they have some limitations: 1) high readout noise, 2) limited correction for gain variations and 3) point spread function or cross talk between channels all of which effects the ultimate sensitivity and resolution. This paper discusses a newly developed parallel detector for EELS based on a CCD and a new type of scintillator and fiber technology. The new detector shows large improvements in all areas of performance.

The sensitivity of a detector is limited by the readout noise at low incident electron dose and the gain correction at high dose. Both these areas have been addressed in the new design. The readout noise is equivalent to approximately 1/2 a primary electron, about 40 times better than a typical PDA.
Chemical Imaging of Polymers Containing Aromatic Groups
Authors: Heckmann W , Mertes J
Published: July 2020

To characterize polymers by transmission electron microscopy it is necessary to use selective staining techniques in order to obtain meaningful results. The combination of a transmission electron microscope with an energy filter system as in the LEO 912 Ω however provides additional analytical information which in particular cases allows imaging without the necessity of staining. Especially a π-π*-resonance at about 6 eV in the low loss electron energy loss spectrum of polymers containing aromatic groups is suitable for imaging. These energy loss electrons are selected by a 2 eV slit in the energy dispersive plane of the spectrometer to form an image. This special type of energy spectroscopic imaging (ESI) can also be referred to as chemical imaging.

This paper presents morphological results on polymer systems containing aromatic components. Micrographs of ultra thin sections were acquired with a 2 eV energy slit at 6 eV electron loss in a LEO 912 π
Grain Boundary Characterization in Yttria-Stabilized Zirconia by TEM Spectrum Lines
Authors: Evans N D , Imamura P H , Mecartneyf M L
Published: July 2020

The superplasticity exhibited by some fine-grained ceramics, notably 3-mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP), offers interesting possibilities to lower manufacturing costs by near net shape forming. Both glassy and crystalline materials have been added to pure 3 Y-TZP to limit grain growth and promote grain boundary sliding during sintering and isostatic pressing. EDS spectrum lines have been used to characterize the distribution and extent of additive phases, but were not able to map some light-element components of additives (e.g., O and B in borosilicate glass). Additionally, quantification of the Si Kα peak was compromised by overlap with the Y L and Z L peaks. To both improve light element sensitivity and investigate potential changes in oxygen bonding at the grain boundaries, undoped 3 Y-TZP and 3 Y-TZP powders processed with 1 wt% barium silicate have been examined with TEM spectrum lines. Additionally, some elemental mapping has been performed. TEM specimens were examined in a 300 kV LaB6 Philips CM30T equipped with a Gatan imaging filter (GIF) at the ORNL SHaRE User Facility.
EXEELFS (Extended Electron Energy Loss Fine Structure) Study of Tin Grown by the DC Sputtering Technique.
Authors: A. Duarte-Moller , Espinosa-Maganña F , Martínez-S´anchez R , Contreras O
Published: July 2020

Titanium nitride coatings were grown in a physical vapor deposition system assisted by a direct current reactive magnetron sputtering technique. The vacuum chamber was evacuated with a mechanical pump and cryopump to a base pressure of 10-7 Torr. The Sputtering was performed with a direct current high voltage source (0-1 Kev and 1 A) on a titanium target (99.98% purity). The titanium target was sputtered with a high purity argon -nitrogen mixture. The films were deposited on monocrystalline silicon (mc-Si) (111) substrates at different nitrogen partial pressures from 0.08 mTorr to 1.5 mTorr. Total pressure, power applied to target and substrate temperature were keep constant in all the experiments.

EXEELFS analysis were done using the standard procedure [1,2]. In this case, we are find that the atomic concentration is in good agreement with the respective established stoichioinetry N/T=0.99. Figure 1 shows the window of high energy loss where appears their respective N K-edge and Ti -L23.
Characterization of JEOL 3000f TEM Equipped for Electron Holography
Authors: Schofield M A , Zhu Y
Published: July 2020

Quantitative off-axis electron holography in a transmission electron microscope (TEM) requires careful design of experiment specific to instrumental characteristics. For example, the spatial resolution desired for a particular holography experiment imposes requirements on the spacing of the interference fringes to be recorded. This fringe spacing depends upon the geometric configuration of the TEM/electron biprism system, which is experimentally fixed, but also upon the voltage applied to the biprism wire of the holography unit, which is experimentally adjustable. Hence, knowledge of the holographic interference fringe spacing as a function of applied voltage to the electron biprism is essential to the design of a specific holography experiment. Furthermore, additional instrumental parameters, such as the coherence and virtual size of the electron source, for example, affect the quality of recorded holograms through their effect on the contrast of the holographic fringes.
Structure of Membrane Proteins Revealed by Electron Crystallography
Authors: Mitsuoka K
Published: July 2020

Because membrane proteins are localized in a continuous lipid bilayer in the native environment, the situation of membrane proteins in the two-dimensional (2D) crystals is quite similar to the environment in vivo. Thus, electron crystallography using 2D crystals is one of the suitable techniques for structure determination of membrane proteins at atomic or near-atomic resolution. Here we describe the structures of the two membrane proteins, bacteriorhodopsin and aquaporin-1, which were solved by electron crystallography at 2.5 and 4.0 Å resolution, respectively.

Bacteriorhodopsin (bR) is a light-driven proton pump found in Halobacterium salinarium. The atomic model of the protein was first proposed by electron crystallography and we improved the resolution of the structure determination up to 3.0 Å by collecting 366 electron diffraction patterns and 129 images. The resulted map showed not only a bR molecule but also eight surrounding lipids in the asymmetric unit.
Isolation and Characterization of Connexin26 Gap Junctions from Transfected Hela Cells
Authors: Hand G , Nguyen V , Downing K , Mueller D , Engel A , Nicholson Bruce , Sosinsky G
Published: July 2020

Gap junctions are composed of a multigene family of proteins called the connexins with a similar sequence and folding topology. Connexons, half-channels made from a particular connexin, will dock and form functional channels with some, but not all, connexons made of other isoforms. This is surprising considering that the primary sequences of the docking domains are conserved and in some cases there are limited amino acid changes in the extracellular loops between compatible connexins. Therefore, some tertiary or quaternary interactions between the extracellular loops of the two docking hemichannels must contribute to the compatibility of a connexon assembled from one connexin for a connexon made from a different connexin. The 3D structures of gap junctions composed of only three isoforms have been investigated with only one case of a gap junction structure at better than 1 nm resolution [1]. The rat liver hemichannel structure is the only one that contains an exposed extracellular surface [2].
Cryo-EM 3D Reconstruction of Skeletal Muscle α-Actinin
Authors: Tang Jinghua , Taylor Dianne W , Taylor Kenneth A
Published: July 2020

α-Actinin is a member of the spectrin superfamily of actin crosslinking proteins. The molecule is an antiparallel homodimer with a polypeptide chain weight of 94-103 kDa. Each chain can be divided into three domains: the N-terminal 250 amino acids forms an actin binding domain, the central domain consisting of four spectrin-like, triple-helical repeats and the Cterminal which contains two EF hand motifs (Baron et al, 1987).

Two models have been proposed for the alignment of the triple helical repeats in the α-actinin structure, an aligned model (Baron et al., 1987) and a staggered model (Taylor and Taylor, 1993). In order to resolve the controversy, we proceeded with the cryo-EM 3D reconstruction from 2D crystal grown on a positively charged lipid monolayer. From rabbit erector spinae aactinin, we obtain better ordered 2D crystals from which we have calculated a 3D reconstruction to ∼15 Å resolution.
Vive La Différence! Mapping Macromolecular Complexes by Generalized Difference Imaging
Authors: Steven Alasdair C , Conway James F , Cheng Naiqian , Watts Norman R , Wingfield Paul T
Published: July 2020

Overview. Three-dimensional cryo-electron microscopy holds great potential for the investigation of macromolecular complexes, including viruses, the cytoskeleton, and the nanomachines that carry out many vital cellular functions. Such complexes often contain protein subunits of different kinds, with cumulative molecular weights running well into the megadalton range. To rationalize their functional assignments in structural terms, the first order of business is to map the locations of the component subunits of a given complex. A more detailed objective then becomes to understand the organization of their domains and the overall layout of the polypeptide chains. With current methodology, resolutions below 20Å are often achievable in cryo-EM, extending below 10 Å in favorable cases. Although resolutions of this order yield valuable morphological information, they do not usually suffice to identify individual subunits within a complex. Generalized difference imaging offers a solution to this problem of molecular mapping.
Three-Dimensional Structure of Isometric Capsids of Bacteriophage T4
Authors: Baker T S , Olson N H , Gingery M , Eiserling F A
Published: July 2020

Coliphage T4 is a large, double-stranded DNA virus (family Myoviridae) which infects Escherichia coli. It is the classic example of a contractile-tailed phage and a wealth of information on viral morphogenesis processes have been obtained on it. Extensive studies of T4 by negative-stain and metal-shadow electron microscopy have shown that mature virions, which contain over 40 different polypeptides, have a prolate capsid that encapsidates the genome (∼175 × 106Da), and several other structural components, including the collar with whiskers, neck, a contractile sheath, and a baseplate with fibers. A complete understanding of processes such as assembly of the viral capsids will require atomic-scale knowledge of the various protein interactions. Details of the process can be extrapolated from knowledge of the initial, transitional, and final states of a structure.

We have initiated an examination of T4 head morphogenesis by studying mutant, isometric capsids ('isometrics’) using cryo-electron microscopy and three-dimensional (3D) image reconstruction methods as applied to particles with icosahedral symmetry.
Conformational Changes In GROEL Induced by a Protein Substrate
Authors: Falke S , Fisher M , Gogol E
Published: July 2020

The GroEL/GroES chaperonin system of E. coli facilitates nucleotide dependent folding of select proteins. The structure of GroEL has been described by three-dimensional electron microscopy and at higher resolution by X-ray crystallography. The GroEL oligomer is a cylindrical tetradecamer composed of two heptameric rings of 57 kDa protein subunits, stacked back to back. Each subunit is comprised of two large domains, equatorial and apical, connected by a smaller intermediate domain. The intermediate “hinge” domain links the apical and equatorial domain and provides flexibility for en bloc rearrangement associated with nucleotide and GroES binding. Equatorial domains are responsible for interactions between the two heptamers and contain the ATPase activity of GroEL. Each ring of GroEL has a central cavity that is the binding site for denatured protein substrate. GroES and denatured substrate binding sites are located on the apical domains facing the central cavity.
An Integrated System for Transmission Electron Microscopy.
Authors: Carragher B , Kisseberth N , Kriegman D , Milligan RA , Potter CS , Pulokas J , Reilein A
Published: July 2020

Macromolecular microscopy is becoming an increasingly important tool for structural biology. The development of improved capabilities for three-dimensional electron microscopy is critical for optimal progress in emerging integrative research in molecular cell biology. These techniques currently suffer from several severe disadvantages related to the tremendous time and effort required to acquire and analyze the data.

For several years we have been developing software for automated and intelligent acquisition of transmission electron micrographs [1,2,3]. Our overall goal is to develop a system for rapid and routine structure determination of macromolecular assemblies from specimens preserved in vitreous ice. Ultimately we plan to develop an integrated system that can produce an electron density map of a structure within a few hours of inserting a specimen into the electron microscope. With this goal in mind it is essential that the images be acquired using a digital camera rather than film.
Cryo-Em Imaging of a Pseudotyped Adenovhtus with Ocular Cell Tropism
Authors: Stewart PL , Chiu CY , Von Seggern D , Nemerow GR
Published: July 2020

Human adenovirus (Ad) provides unique model system for studying the multistage events in viral entry. We are applying cryo-electron microscopy and single particle reconstruction methods to study the cell entry pathway of Ad as well as to examine the structure of reengineered Ad vectors. Ad cell entry is mediated by interactions with two different cellular receptors: the fiber receptor, CAR for most Ad serotypes or a 50 kDa receptor for a subset of serotypes including Ad37, for attachment; and the αvβ3 and αvβ5 integrins for internalization. We have previously reconstructed both Ad2 and Adl2 virus particles complexed with a soluble form of αvβ5 integrin. We learned during our comparative cryo-EM study of the Ad2/αvβ5 and Adl2/αvβ5 complexes that the length of the variable RGD loop in the penton base affects the relative height of the integrin density over the penton base.
An Interactive User Interface for Automated Acquisition of Transmission Electron Micrographs
Authors: Pulokas J , Kisseberth N , Potter CS , Carragher B
Published: July 2020

For several years we have been developing a software application, called Leginon, for the automated control and acquisition of images from a transmission electron microscope. The system has been developed around a Philips CM200 and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images under low dose conditions from a specimen embedded in vitreous ice. In order to set up and manage this system we have developed a number of simple interactive graphical tools that enable the user to design, oversee and manage protocols for controlling the microscope and collecting the images. Many of these simple tools have also proved generally useful as stand alone applications.
An Automated System for Maintaining Liquid Nitrogen Levels in the Gatan Cryostage.
Authors: Robinson SJ , Fried G , Pulokas J
Published: July 2020

For several years we have been developing a system for the automated control and acquisition of images from a transmission electron microscope [1,2,3]. The system has been developed around a Philips CM200 equipped with a Gatan cryostage and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images over several days of completely unattended operation.

The current implementation of our automated system allows data to be automatically collected from the TEM for more than 36 hours. There is no intervention required from the operator except for one critical exception, refilling the Gatan cryostage. The cryostage, which maintains the temperature of the vitreous ice specimens, must be refilled with liquid nitrogen (LN2) approximately every two hours.
Three-Dimensional Structure Of C3D From Low Temperature Scanning Transmission Electron Microscopy and X-Ray Crystallography
Authors: Martin DJ , Ottensmeyer FP
Published: July 2020

Macromolecular structure can be solved by x-ray crystallography to atomic resolution provided that the molecule can be crystallized, that the crystals diffract x-rays to high resolution, and that the phases of the diffracted x-rays can be determined. Though the resolution of single particle imaging by electron microscopy is lower than that of x-ray diffraction by crystals, electron microscopy can directly image a large molecular weight range of macromolecules in a non-crystalline environment, and provide the basis for the three-dimensional reconstruction of these structures. To investigate combining structural information from x-ray crystallography and electron microscopy for unknown structures, we have imaged a small protein of known structure (1), the 35 kDa human complement protein fragment C3d, in a scanning transmission electron microscope (STEM). The intention is to eventually combine the knowledge of electron densities and molecular boundaries from electron microscopy to assist in phase determination in x-ray crystallography.
Ultrastructural Localization of Antigens in the Mitotic Spindle of Budding Yeast Using High Presure Freezing and Freeze Substitution
Authors: Müller-Reichert Thomas , Ashford Anthony , Antony Claude , Hyman Anthony
Published: July 2020

The preservation of S. cerevisiae ultrastructure is not a trivial task, because the cell wall serves as a diffusion barrier during all steps of preparation. In addition, a high density of proteins in the cytoplasm and in the nucleus renders the visualization of cellular details difficult.

High pressure freezing in combination with freeze substitution has proven to be an advantageous technique for the preservation of the yeast mitotic spindle (1). However, the composition of the freeze substitution “cocktail” is crucial, when immunolabeling of antigens in the sample is intended. A high concentration of crosslinking agents might improve ultrastructural preservation of the sample, but will possibly destroy the antigenicity of the target proteins. In contrast, under conditions of high labeling efficiency, the ultrastructural detail may be lost. We were interested in a method, which gives a good ultrastructural preservation of the mitotic spindle and maintains the antigenicity of proteins associated with the spindle.
Cryosections for Immunocytochemistry
Authors: Webster Paul
Published: July 2020

Immunocytochemistry describes methods for visualization of specific intracellular molecules within cells. These methods use high affinity markers to specifically bind to target molecules in tissues or cells. For electron microscopy, the sites where these markers are bound are visualized using electron opaque probes. Successful immunocytochemistry depends upon immobilization of target molecules, retention of subcellular morphology, and upon total accessibility of affinity marker to the target molecules.

Accessibility to extracellular molecules is not usually a problem, but for intracellular molecules, labeling protocols must be able to preserve subcellular morphology and allow the affinity markers to gain access to their target molecules. Currently, the best way to gain access to the inside of cells for electron microscopy (TEM) is to cut thin sections through them. In this way, morphology is retained and affinity markers can be applied to the cut face of the cells.
The Utilization of Cryopreservation in Small Gold Surface Labeling
Authors: Ackerley CA , Tilups A , Kanazawa T , Becker LE
Published: July 2020

Cryopreservation has many advantages over routine chemical fixation. Two of these are the almost spontaneous arrest of biological activity and the retention of material that normally would be lost with chemical fixation and dehydration. In the current study 2 systems were evaluated. Hyaluronic acid (HA) was covalently bound to 1.4nm nanogold® and applied to astrocytoma cell cultures and the uptake monitored over time using both cryopreparative methods and conventional chemical fixation. The second system evaluated was the detection of liposomes containing CFTR covalently bound to 1.4nm gold in the airways of mice from a phenotype of an inbred congenic strain of CFTR-knockout mice that spontaneously develop lung disease.

In the HA uptake experiment, astrocytoma cell cultures were treated with HA gold for either 5, 10 or 20 minutes. At the appropriate time point, cells were either plunge frozen in liquid ethane or fixed with 2.5% glutaraldehyde in phosphate buffer.
Combining Cryo and Conventional Specimen Preparation Methods to Improve the Preservation of Cell Ultrastructure
Authors: McDonald Kent L
Published: July 2020

Poor specimen preparation techniques affect all subsequent EM imaging operations, whether simple descriptions or sophisticated computer reconstruction and analysis. Therefore, EM researchers continue to put effort into developing methods that will preserve the fine structure of the cell as close to the native state as possible. Much attention has been paid to the primary or initial fixation because this is probably the singlemost important step in EM processing. Evidence from a variety of sources. shows that cryofixation is more likely to give good preservation than conventional chemical fixation for most cell types. Among the choices for cryofixation, high pressure freezing is probably the best for most samples, even those that are small enough to be frozen by other means.

Dehydration is another key point in processing where high resolution structure can be lost or distorted, though it has received relatively little attention compared to primary fixation.
Electron Tomography of Frozen-Hydrated Samples
Authors: Marko M , Hsieh C-E , Rath BK , Mannella CA , McEwen BF
Published: July 2020

Cryo-electron tomography offers a means of obtaining the 3-D ultrastructure of specimens that have not been chemically fixed or stained. The technique is still under development, but it has already been applied to several biological specimens including prokaryotic cells, centrioles, sperm axonemes, mitochondria, spindle-pole bodies, lipid vesicles, isolated skeletal-muscle triad junctions, chromatin fragments, actin bundles, and macromolecules.

Electron tomography itself is a nearly mature technology. It is most often used with conventional plastic sections 100- 1000nm thick. The specimen is imaged in the TEM, using a tilt stage to record a series of projection images over a large angular range. Using image-processing techniques, the tilt images are back-projected to form a reconstructed volume of the specimen. The reconstruction is a 3-D array of pixels or volume elements (voxels), and can be sliced in any direction to form a series of 2-D images.
The Potential for Correlative and Multiple Labeling With Colloidal Particles of Differing Shapes
Authors: Albrecht RM , Meyer DA
Published: July 2020

Labeling specific tissues, cells, cell components, or individual molecular species is often critical to correlative microscopic studies. In the case of larger structures such as tissues or cells it is often possible to use fluoresecent labels or cytologic/cytochemical based stains which are observable via photon based imaging systems. Specific locations on cells and tissues identified by the labeling or staining procedure can be delineated and then can be viewed via electron or force based instrumentation. This is provided preparative methodologies can be designed which: 1. are compatible with observation of the particular sample by the various desired imaging modes and 2. do not compromise structure viewed at the higher levels of resolution provided by electron and force based imaging technologies. This approach is particularly valuable where simultaneous multiple labeling is required since color differences permit discrimination of differing fluorescent and/or chromatic stains.
Gold Cluster Crystals
Authors: Hainfeld JF , Powell RD , Furuya FR , Wall JS
Published: July 2020

Gold clusters are gold compounds with a core of gold atoms and organic groups covalently bound to the surface gold atoms. An example is undecagold, Au11(P(C6H5)3)7, whose structure was solved by x-ray crystallography using 3-dimensional crystals. These differ from colloidal gold, which are suspensions of metal particles, usually formed by metal ion reduction; although the particles may be approximately the same size, they vary due to the statistical process of formation. Gold clusters are compounds with a definite formula, and should all be perfectly identical. However, it is known that there is a family of stable gold cluster compounds, such as Au6, Au11, Au13, AU55, Au67, etc. In a given preparation of gold clusters, there is usually some mixture of these, thus leading to some size variation. Methods such as gel filtration column chromatography and ultrafiltration can be used to separate most of these species, so that relatively pure preparations may be achieved.
Sodium Dodecyl Sulfate Enhances Immunolabeling for Certain Antigens
Authors: Vandré D D , Robinson J M
Published: July 2020

Immunocytochemistry provides unique information concerning the spatial and temporal distribution of biomolecules in cells and tissues. While techniques associated with immunocytochemical labeling are well developed, there are always improvements that can be achieved. In this abstract, a modification to immunocytochemical methodology that increases the labeling efficiency for certain antigens is described. This modification includes incubation of the sample with sodium dodecyl sulfate (SDS) prior to application of the primary antibody.

Tubulin in microtubules of human neutrophils has been extremely difficult to analyze with antibodies to α-tubulin or β-tubulin using immunocytochemical methods. Using an immunofluorescence assay, we have analyzed several preparative procedures in order to improve the labeling efficiency of these microtubules using anti-tubulin antibodies. We find that treatment of chemically fixed neutrophils with the chaotropic agent SDS improves labeling efficiency for neutrophil microtubules in the immunofluorescence assay.
Free-Floating Cryostat Sections for Immunoelectron Microscopy: Bridging the Gap from Light to Electron Microscopy
Authors: Kan RK , Pleva CM , Backof D , Hamilton T , Petrali JP
Published: July 2020

Since skin basement membrane protein components are labile to conventional chemical fixation and since skin is not amenable to vibratome sectioning, frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slidemounting, usually limit study to the light microscopic level. In the present study, we introduce the use of unfixed, free-floating cryostat sections to characterize immunolocalizations of selected basement membrane protein components at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy.
SCFV-6HIS Bioengineered for High Fidelity Labeling.
Authors: Malecki Marek , Tiongco Lea , Hsu Annie , Takeuchi Nina
Published: July 2020

In life sciences, the essential part of the functional molecular analysis is the unambiguous identification of biochemical composition of the observed structures. This analysis is expected to create a bridge between functional data pouring from biochemistry and molecular biology laboratories with the molecular architecture data available from ultrastructural images. This goal can be attained by application of various ultrastructural tags (See: Albrecht et al. 1992).

A new promising approach for high fidelity labeling is offered by molecular cloning and expression of molecules containing metal binding sites making them suitable for electron spectroscopic imaging (ESI) (Malecki 1995). A truly enormous potential of ESI relies in its ability for mapping of various elements within the same sample. Interactions of electrons with an atom result in the electrons specific energy loss. Based upon these energy losses distribution of the elements within the sample can be mapped.
The Morphology of Widmanstatten Cementite Laths
Authors: Fonda R W , Kral M V
Published: July 2020

The proeutectoid precipitation of Widmanstatten cementite has recently been shown to occur in two morphologies, either as broad monolithic plates or as aggregates of narrow laths, in a Fe-13 wt% Mn-1.3 wt% C steel, see Figure 1. Each of these morphologies correlates to a specific orientation of the cementite crystal within the austenite matrix; precipitates with a Pitsch orientation relationship adopt the monolithic plate morphology while precipitates with a Farooque-Edmonds orientation relationship develop as laths. In this paper, we examine the morphology and interfacial structure of these Widmanstatten cementite laths.

The typical three-dimensional morphology of Widmanstatten cementite laths can be revealed by etching with a 10% nitric acid solution in methanol. This procedure selectively etches the austenite matrix without affecting the cementite precipitates (see Figure 1). By similarly etching an electropolished thin foil TEM sample, the three-dimensional morphology and internal structure of entire electron transparent cementite precipitates can be examined.
Precipitation Behavior of Melt Spun Mg-1.5wt%Ca-6wt%Zn Alloys
Authors: Jardim PM , Solórzano IG , Vander Sande JB , You BS
Published: July 2020

For over five decades considerable attention has been given to magnesium-based alloys, primarily from the physical metallurgy point of view. The driving force for this effort has been the potential applications of Mg alloys in light weight structural components, namely in the automotive and aircraft industries. However, the processing of these alloys through conventional casting technologies have not succeed in reaching a high level of formability and ductility while preserving high specific strength. The combination of rapid solidification and consolidation techniques has arisen as a promising approach to overcome these limitations[l,2]. On the other hand, rapid solidification technologies are opening a wide field for fundamental studies, since different sets of thermodynamic (stability), kinetic and structural conditions are established to control a variety of solid-state phase transformations. The present work reports the applications of electron microscopy techniques to the characterization of the precipitation behavior in the rapidly solidified Mg-based alloys we are investigating.
Translation Order Domains of Coherent Particles in Ni Alloys During Coarsening.
Authors: Calderon H A , Calzado L , Mori T
Published: July 2020

Coarsening of particles involves a reduction of the total energy of the system. In fluids, the tendency for larger particles to grow at the expense of smaller ones is driven by the reduction of the total surface energy. In solids, the elastic strain energy Eel also needs to be considered. Reduction of Eel gives rise to particle alignment along elastically soft directions and changes of morphology as the particle volume increases. In addition according to several authors, reduction of Eel produces the splitting of particles that reach a critical size. Regular arrays of two, four and eight particles have been observed and explained in terms of splitting. However, splitting is unlikely from a micromechanical standpoint or as a consequence of the elastic interaction between particles. This investigation deals with the determination of translation order domains in coherent γ′ particles.
The Determination of Carbide Types in Thin-Film Specimens of Low Alloy Steels.
Authors: Papworth AJ , Watanabe M , Williams DB
Published: July 2020

The introduction of “clean steels” was thought to have cured the problem of temper embrittlement in low alloy steels. However, even these steels exhibit temper embrittlement at services temperature above 400°C. Initial studies of temper embrittlement of clean steels used Auger electron spectrometry of fracture surfaces. The failure was found to occur along the prior austenite grain boundaries (PAGBs) where P segregation was found. It has been reported that the concentration of segregants changes along individual PAGB facets, as well as between the facets of different PAGBs. It was proposed that different elements segregate to different PAGBs, depending on their orientation. Differences in segregation along individual PAGBs were attributed to precipitation during thermal exposure, as identified by AEM. The cause of temper embrittlement is still unknown, as there are doubts about the role of precipitation. This implies that each PAGB may have a different chemistry.
EFTEM Elemental Mapping of α-α′ Interconnected Isotropic Nanostructures Formed by Spinodal Decomposition of Ferrite in Aged CF8 Cast Duplex Steel
Authors: Bentley J
Published: July 2020

Phase separation by spinodal decomposition of body centered cubic Fe-Cr binary alloys to form isotropic fully interconnected nanostructures of Fe-rich α and Cr-enriched α′ phases typically on a scale of 2 to 10 nm is scientifically intriguing and technologically important. In aged dual-phase steels, it is the decomposition of the ferrite to form such α-α′ composition modulations that is primarily responsible for the increased ferrite hardness that contributes to embrittlement. The α′ structures are isotropic since the lattice constants of the two phases are almost identical and consequently the strain energy is small. In other spinodal systems larger strain energy leads to triaxially modulated structures aligned along <100> in cubic materials. A review of TEM characterization of spinodal alloys revealed that aligned structures had received the most attention, because they are more common and their characterization by TEM is more tractable.
Spinodal Decomposition in Fe(1-X)O-Fe3o4 Sintered Ceramics.
Authors: Huerta A , Calderon H A , Umemoto M , Brito M E
Published: July 2020

Ceramic materials in the system FeO-Fe3o4 have been produced by means of mechanical milling and sintering for application of their physical properties. Starting from powder mixtures of Fe and Fe3o4 or Fe and Fe2o4, as well as pure Fe3o4, a metastable solid solution of Fe and O has been produced. The amount of Fe dissolved varies according to the milling time and its initial content in the mixture. A protective Ar atmosphere is used in all stages of the milling process in order to avoid contamination. X-ray diffraction patterns show a clear displacement of the intensity maxima as a function of the milling time, suggesting the formation of a solid solution from the initial mixture. Use of Mossbauer spectroscopy reveals an increasingly higher amount of metastable wustite (Fe(1-X) as a function of milling time i.e., 500 h and 1000 h of milling produce 67 and 74 mol % Fe(1-X)O, respectively.
A Microstructural Study of the Al-Cr-Ru System
Authors: Compton DN , Cornish LA , Witcomb MJ
Published: July 2020

Work undertaken on the Al-Cr-Ru system has shown excellent oxidation resistance in air up to 1500°C for compositions between Al20:Cr60:Ru20 and Al35:Cr32:Ru33 (at.%). Since no phase diagram data for the Al-Cr-Ru system have been published to date, an investigation was initiated. This part of the investigation is to identify the phases which form on solidification, and to derive the liquidus surface for alloys containing in excess of 50 at. % aluminium.

Samples were prepared as arc-melted buttons from 99.9% pure elements, and were analysed in the as-cast state in the SEM using EDS with elemental standards, and by X-ray diffraction.

The B2 phase, based on AlRu, extends to at least 25 at. % Cr. The Al3Ru2 phase has not been identified, except perhaps as a thin peritectic layer which was too fine to analyse. This is not surprising, since it exhibits very limited extension in other Al- Ru-X ternary systems, and in the Al-Ru binary system reacts peritectically forming AlRU2, and also subsequently decomposes at lower temperatures.
Interdiffusion and Phase Behavior in a Vanadium / Polysynthetically Twinned Tial Diffusion Couple
Authors: Pan Ling , Luzzi David E
Published: July 2020

Polysynthetically twinned (PST) TiAl, which has a two phase lamellar structure composed of multiply oriented lamellae of the TiAl phase (γ, with ordered tetragonal L10 structure) with a low density of interspersed layers of the Ti3Al phase (α2, with ordered D019 hexagonal structure), has attracted a large amount of interest in the past decade for its potential as a high temperature structural material. The strongly crystallographic twins and pseudo-twins between the lamellae of the PST TiAl crystal provide an ideal system to study interface diffusion. In this paper, we describe early results on the interrelationships between phase and interface structure, bulk and interface diffusion and phase equilibrium in the diffusion- bonded system PST TiAl and vanadium.

A PST TiAl “single crystal” was grown in an optical floating zone furnace by zone-melting and directional solidification of a polycrystalline as-cast TiAl rod. Within the “single crystal”, all the lamellar interfaces are parallel.
Microstructural Response of DS Nb-Silicide In-Situ Composites During High-Temperature Creep Testing
Authors: Bewlay BP , Sitzman SD
Published: July 2020

Directionally solidified (DS) in-situ composites based on (Nb) and Nb silicides, such as Nb5Si3 and Nb3Si, are being investigated for high-temperature structural applications. The use of alloying additions, such as Hf, Ti and Mo, to these silicides is required to enhance their properties. The present paper describes the microstructural response of a DS Nb-silicide based composite to creep testing.

The composites investigated were directionally solidified from a molten alloy using the Czochralski method as described previously. Creep tests were conducted at 1200°C to strains of up 50%. Microstructure and microtexture characterizations were performed using scanning electron microscopy, electron microprobe analysis (EMPA), and electron backscatter diffraction pattern analysis (EBSP).

Microstructures of the longitudinal section of a DS composite generated from a Nb-12.5Hf-33Ti- 16Si alloy are shown in Figure 1 in the as-DS (left hand side) and the DS+creep tested conditions (right hand side).