bluuebunny/crossref_metadata_2025 huggingface dataset:
Transmission Electron Microscopy of Corrosion of Stainless Steel-Zirconium Metal Waste Forms
Authors: Luo JS , Abraham DP
Published: July 2020

Stainless steel-zirconium (SS-Zr) alloys have been developed as waste forms to immobilize and retain fission products generated during the electrometallurgical treatment of spent nuclear fuel. The baseline waste form is a stainless steel-15 wt.% zirconium (SS-15Zr) alloy, which is prepared by melting appropriate amount of Type 316 stainless steel (SS316) and high purity zirconium. As zirconium has very low solubility in iron, the addition of zirconium to SS316 results in the formation of ZrFe2 -type Laves intermetallic phases. The corrosion behavior of stainless steel has been widely studied; however, the corrosion behavior of the Zr-based-intermetallic has not been previously investigated. In this paper, we present a microstructural characterization of the corrosion layer formed on the Zr-intermetallic phase using energy-filtering transmission electron microscopy (TEM) and energy dispersive x-ray spectroscopy (EDS).

Specimens of SS-15Zr alloy, crushed to 75 to 150 μm sizes, were immersed in 90°C deionized water for a period of two years.
Microstructure of off-Eutectic Nanoscale Pb-Cd Inclusions in Al
Authors: Peters DP , Johnson E , Touboltsev VS , Johansen A , Sarholt L
Published: July 2020

Much work has been devoted to TEM studies of nanoscale precipitates consisting of non-soluble elements in Al, as the Al matrix acts as a unique crucible enabling the study of melting/ solidification and crystal phase transformations at a nanometer scale. Recently, this work has been extended to include the ternary system of Al-Pb-Cd. This system is of particular interest, as both Pb and Cd display a large liquid miscibility gap with Al, but together they form a eutectic system. Bimetallic inclusions consist of an fcc Pb and an hcp Cd segment, joined along an internal interface parallel to the {111}Al planes. In this work, the microstructure and composition of off-eutectic Pb-Cd inclusions are studied. Polycrystalline Al samples were implanted with 1 x 1020 ions/m2 (∼3 at.%) with a Cd:Pb ratio of either 8:2 or 2:8, at temperatures to form inclusions in the 10 nm size range.
SIMS/AEM Characterization of Banded Microstructures in an Ni-Cr-Fe Alloy
Authors: Burke MG , Hyatt BZ , McMahon G
Published: July 2020

Ni-Cr-Fe alloys such as Alloy 600 and Alloy 690 are single phase fee alloys with excellent resistance to corrosion. Alloy 600 is generally used in a thermally-treated condition which is characterized by the intergranular precipitation of Cr-rich M7C3 and, depending upon the aging temperature, Cr-rich M23C6. These alloys are usually cast and processed by rolling or forging. These alloys can exhibit microstructures in which there are nonuniform distributions of intragranular carbides that appear as bands aligned parallel to the working direction (i.e. longitudinal) of the material. It is also possible for these materials to exhibit duplex grain sizes which can be associated with the banded carbide structure. Banded microstructures can become more prominent after further anneals. The resistance of the carbide bands to dissolution during solution annealing at temperatures up to 1150°C has prompted the further evaluation of banding in Alloy 600, specifically in terms of identifying those elements associated with the carbide bands.
Estimation of Interfacial Energies Based on the Shapes of Coherent Intragranular Precipitates
Authors: Chen Xiao , Rabenberg L
Published: July 2020

In a study of the precipitation and aging of Invar particles in Cu, we observed that the particles evolved from spherical to cuboidal as they grew in size. This behavior has been observed in other systems, and predicted by Voorhees et al. ; they showed that the precise shape of the particle is a balance between elastic anisotropy and surface energy. The correlation between our results and their predictions are very good. It is the purpose of this note to point out that simultaneous measurements of size, shape, and mismatch of a coherent particle can be used to estimate the interfacial energy under favorable conditions.

Ternary alloys consisting of Cu - 12.8 wt% Ni -7.2 wt% Fe were arc-melted, held at 1030°C to develop extended regions of the Cu-rich solid solution, quenched, then aged at various times and temperatures to develop FCC precipitates near the Invar composition in the FCC Cu matrix.
Characterization of Interfacial Microstructure in Joints Between Pb-Sn Eutectic Solder and Pd/Ni/Pd-Ni/Ni/Cu Metallization
Authors: Ghosh G
Published: July 2020

In electronic packaging, it is very important to understand and control the interfacial microstructure formed due to the reaction between the metallization scheme and solders. Often, the interfacial reaction is exploited/controlled to obtain an optimum properties of the joints. The strength and interfacial properties of the solder joints are determined by the interfacial microstructure. The evolution of interfacial microstructure in solder joints is governed by the diffusion path during processing and in service. Even though the semiconductor industry uses a wide variety of complex metallization schemes, recently the Pd-Ni metallization scheme on Cu has gained a lot of interest. Furthermore, the design of new metallization scheme(s) to control the interfacial microstructure and to improve the quality of solder joints is the cornerstone of advanced electronic packaging. We have carried out a systematic study of diffusion and interfacial reaction, both in the liquid and solid states, between Pb-Sn eutectic solder and the Pd-Ni metallization scheme on Cu.
Pre-Martensitic State in a Ti50Ni48Fe2 Alloy Studied by Electron Microscopy with Energy-Filtering
Authors: Murakami Y , Ikematsu Y , Shindo D , Oikawa T , Kersker M
Published: July 2020

A Ti50Ni48Fe2 alloy, which is famous as a typical shape memory alloy, exhibits the cubic (parent phase) to trigonal (R-phase) martensitic transformation upon cooling. It is well known that this structural transformation is accompanied by some precursor phenomena, such as appearance of weak diffuse scattering in electron diffraction patterns of the parent phase. Since the diffuse scattering is visible at around 1/31/3 0 and its equivalent positions in diffraction patterns, as similar to the superlattice reflections in the R-phase, it is believed to be closely related with the formation of the R-phase. However, neither structural nor morphological aspect of this pre-martensitic state is clear, since a precise analysis of the weak diffuse scattering is quite difficult along with a conventional electron microscopy, because of the presence of strong background arising from inelastic scattering. The purpose of the present work is to precisely observe the weak diffuse scattering and to reveal the structural and morphological features of the pre-martensitic state by electron microscopy with the aid of a recently developed omega-type magnet.
Observation of Microstructural Evolution of Aluminum Bonding Wires in Power Electronic Package
Authors: Ron Liu D , Chen K , Jih E
Published: July 2020

Power electronic circuits are used in electric vehicle or hybrid electric vehicle to drive the main electric motor. All the transistors and diodes in a sealed power electronic package will experience repeated high current cycling during their service life. The aluminum bonding wires which connect these devices to the rest of the circuit will experience repeated thermal strain cycling. When design a power electronic package, the thermal fatigue durability of these bonding wires would be a concern. A power electronic module with pure aluminum bonding wires of 0.5mm in diameter from a vendor was power cycled with the following schedule. Each transistor in the circuit was on for 10 seconds with the current at 400 Amperes followed by the transistor being off for 30 seconds. The wires from the diodes to the copper trace lines are about twice as long as those from the transistors to the copper trace lines.
Characterization of Cup Earing in Tin Mill Product by X-Ray Diffraction
Authors: Liao K C , Swartz WC
Published: July 2020

X-ray diffraction (XRD) techniques can readily be utilized for the characterization of textures in crystalline materials. The advantages of XRD techniques are: 1) the test is nondestructive, 2) samples can easily be prepared, and 3) data can be collected and analyzed quickly.

Three low-C steel tin mill coils were manufactured during a mill trial which was conducted to optimize the processing parameters necessary for the production of material with low cup earing behavior. The processing parameters evaluated included hot mill conditions (roughing, finishing and coiling temperatures), cold rolling conditions (during first and second cold reduction), and continuous annealing conditions (line speed and strip temperature). Samples A, B, and C were secured from the coil center-width position of each coil, respectively, and were analyzed via XRD.

Properties such as normal anisotropy (r bar) and planar anisotropy (Δ r), have been correlated with an X-ray Intensity Ratio (XIR) at the Technical Center of National Steel.
Cross-Sectional Sample Preparation and Analysis of Intergranular Stress Corrosion Cracks in FE-NI-CR Alloys.
Authors: Thomas LE , Bruemmer SM
Published: July 2020

Transmission electron microscopy in its modern high-resolution analytical form has great potential for elucidating mechanisms of environmental cracking in structural alloys and other materials. Apart from a few pioneering efforts, the difficulties of preparing suitable samples with the original corrosion structures preserved in narrow crack tips have limited contributions of TEM in this area. Recent work involving cross-sectional crack preparation has focused on intergranular stress-corrosion cracking of Fe- and Ni-base stainless alloys in high-temperature water. This presentation will illustrate preparation methods that have proven successful, and describe limitations of the analysis.

The method for preparing cross-section samples suitable for high-resolution TEM characterization is based on earlier work by Lewis et al. It involves preparing a thin section containing cracks filled with epoxy, dimple grinding selected locations near the crack tip, and ion milling. Figure 1 schematically illustrates this method. The first step is to preserve the crack opening characteristics present under loading and protect the crack surfaces during thinning.
Thickness Measurement of Focused Ion Beam Thinned Silicon Crystals Using Convergent Beam.Electron Diffraction and Electron Energy Loss Spectroscopy.
Authors: Delille D , Pantel R , Auvert G , Van Cappellen E
Published: July 2020

1. Introduction

The FIB (focused ion beam) is now widely accepted as the most site-specific TEM preparation tool and as such proves to be highly valuable when analysing ULSI devices. However, using high-energy Gallium ions for milling induces amorphization of the crystal surfaces. A method able to quantify this surface alteration on silicon using a combination of CBED (convergent beam electron diffraction) and EELS (electron energy loss spectroscopy) is presented. CBED is a powerful tool that also can generate an accurate measure of crystal thickness. EELS can yield the total sample thickness, so from the difference the combined amorphous layers can be assessed. Two sets of application results are presented: the first one is obtained on a FIB thinned sample using an ion energy of 50 keV and the second set of results confirms the validity of the proposed method on a mechanically polished specimen with no subsequent ion milling.
Optimization of FIB-Milling Technique for Preparation of TEM Specimens from Copper/Low-K Materials
Authors: Shaapur F , Brazeau D , Foran B
Published: July 2020

Focused ion beam (FIB) thinning of materials to electron transparency is now a routine procedure for preparation of specimens for transmission electron microscopy (TEM) of microelectronic materials and devices. The nano-scale structural damage, including implantation and amorphization due to this ion milling process has been well investigated and documented. In this paper, we discuss the micro-scale structural damage observed in copper/low-k materials and our efforts to minimize the extent of the damage without compromising the overall specimen preparation time.

Figure 1 shows an area-specific cross-sectional specimen prepared from a copper/low-k via-chain test structure using the FIB-milling technique. The procedure involved mechanical thinning of a transverse wafer sliver followed by FIB-milling the area of interest to electron transparency according to conventional steps and conditions' using a liquid Ga+ ion source FIB system. The evidence of structural damage in terms of melting and/or sputtering of the metallization is visible at different areas.
Reactive Ion Beam Etching (RIBE) Technique and Instrumentation for SEM Specimen Preparation of Semiconductors
Authors: Alani R , Mitro RJ , Ogura K
Published: July 2020

Argon ion beam etching has established itself as an alternative technique to “wet chemical” etching for the preparation of cross sectional SEM specimens of semiconductors [1]. Complementing this technique, we are reporting the results of an iodine RIBE method for improved etching/cleaning capabilities with a measurable increase in etching rates as compared to argon ion beam etching. RIBE systems have been used for decades in the semiconductor research/industry for wafer processing, patterning and surface cleaning. This same technique has also been used for high quality TEM specimen preparation of certain semiconductor materials [2,3]. The beneficial aspects of the iodide RIBE technique for surface etching for a variety of semiconductor structures along with the related instrumentation will be discussed. The semiconductor specimens include traditional ICs and more advanced copper technology devices.

The design and construction of the original system used in this work has already been reported [4].
Novel Sample Preparation Methods for Transmission Electron Microscopy Observation of Dopant Profiles in Silicon Devices
Authors: Salvatore Pannitteri
Published: July 2020

I present details of novel sample preparation techniques used for delineating two-dimensional dopant profiles in silicon devices. These techniques are based on selective chemical etch of doped silicon in a mixture of hydrofluoric and nitric acid, or simply in buffered HF. The altered topography of the etched surface is imaged by transmission electron microscopy (TEM). Two different strategies will be presented by focusing on their sensitive, resolution, and field of application.

In the first case the silicon device is subjected to the conventional thinning procedure for TEM observations in cross-sectional configuration. The final thickness is obtained by Ar ion milling and it can vary between 50 to 500 nm. Sample is then immersed in a chemical solution containing HF (40%), HN03 (65%), and CH3COOH (95%) in the ratio 1:10:10. In presence of an intense illumination this mixture preferentially etches those device regions which are doped with boron, while in order to delineate n-type regions, the etching procedure must be performed in the dark.
Microanalytical Techniques for the Determination of Carbonaceous Aerosols in Remote Air and Snow Samples
Authors: Kessler D , Currie A , Newbury E , Windsor ES
Published: July 2020

Remote ice core, snow, and air filter samples can potentially profile past and present atmospheric characteristics, acting as media to preserve populations of particulate matter. Major constituents of these aerosols derive from combustion processes (fossil and biomass) and airborne minerals. Extraction and characterization of these particles with scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDS) provide morphological and elemental data used in determining the presence and sources of atmospheric aerosols. Due to the minute quantity of carbonaceous particles in the snow and ice samples, ca. 1-10 ng g-1, the microscopist faces several principal challenges to prepare and analyze these samples. First, methodology must be established for particle removal in a clean and quantitative fashion. Secondly, since carbon is contained in many of the particles under analysis, techniques must be established to achieve quantitative carbon X-ray data. The SEM requires optimization in order to analyze the greatest number of particles in the least time.
Optimum Window Size for Quantitating Trace Elements Using Linear Least Squares Fit With Eels
Authors: Ho Ruoya , Feng Jiang Lin , Shao Zhifeng , Somlyo Andrew P
Published: July 2020

Trace element quantitation with EELS is a powerful tool for providing information about elemental distribution in biological and materials sciences. Linear least squares fit (LSF) to standards of the signal and the background is a widely used method for retrieving small signals superimposed on a large, slowly varying background. Theoretically, for most accurate background fitting, the fitting window should be as large as possible. However, when using a large window, there may be a mismatch between the fine structure of the standard and the measured background, due to instability of the instrument and the experimental conditions and radiation damage. This mismatch, distributed over the entire window, can severely affect the accuracy of the small signal extracted with LSF. To overcome this problem, a small window, slightly larger than the signal is often used. Fig. 1 shows the results of extracting a small Ca signal by using respectively, a large and a small window for fitting an EELS spectrum collected from a specimen containing 26.3 mmol/kg dry weight Ca.
Electron Holography of Potential Barriers in Zno Varistors
Authors: Elfwing M , Saunders M , Olsson E
Published: July 2020

ZnO varistor materials are polycrystalline materials that exhibit strong non-linear current-voltage characteristics. Their non-ohmic behaviour makes them suitable for overvoltage protection devices. It is the interfaces between ZnO grains that are the key to the non-linear behaviour of the varistor materials. Previous work has shown that the local microstructure determines the breakdown voltage of the individual ZnO/ZnO interface [1]. The pre-breakdown conductivity and the non-linearity in the breakdown region, which are both crucial to the performance of the varistor, are also affected by the height and the width of the potential barriers. In the present work, electron holography has been used to investigate the potential barriers in ZnO varistor materials.

Electron holograms were recorded at 200kV using a Hitachi HF-2000 field emission gun transmission electron microscope [2]. The electrostatic bi-prism was located near the second image plane. The bi-prism voltage was about 9 V and the fringe spacing in the holograms was about 1 nm.
Visualization of Pure Phase Objects by Amplitude-Division Three Wave Interference
Authors: Hirayama T , Saito T
Published: July 2020

Pure phase objects having low spatial frequency such as electromagnetic fields in a vacuum can be visualized by electron holography or three-wave interference.1,2) Wavefront-division interference using a biprism requires a field-emission electron gun having high spatial coherency. On the other hand, amplitude-division interference using a thin crystal does not need high spatial coherency of electron source, i.e. an ordinary electron microscope with a LaB6 filament is good enough to form lattice fringes of high contrast. This is an advantage of amplitude-division interference.3,4)

We previously reported three-wave interference achieved by using two electron biprisms. Recently, we have realized amplitude-division three-wave interference by using a Si crystal as a splitter of an electron wave. Figure 1 shows the experimental set-up used to form an amplitude-division threewave interference pattern. A thin crystal prepared by ion-milling is installed at the standard specimen position, and a thin carbon film on which latex particles are sticking is placed at the selected area aperture position.
Interpretation of Holographic and Lorentz Images of an Array of Reverse Biased P-N Junctions in a Semi-Infinite Specimen
Authors: Beleggia M , Capelli R , Pozzi G
Published: July 2020

The problem of observing reverse-biased p-n junctions by means of transmission electron microscopy techniques is very challenging both from the experimental and theoretical points of view. In particular, in addition to standard Lorentz microscopy techniques, whose image contrast is able to give some information on the region of high field, also holographic methods have been successfully applied and an interference map of the twodimensional phase on the specimen plane obtained.

In this work we consider, in particular, the theoretical and interpretative aspects, as we have improved a model for the electric field associated to a periodic array of alternating p and n stripes lying in a half plane (physically corresponding to an array of zero-width step junctions) by taking into account that the junctions are tilted with respect to the specimen edge.
Characterization of Multi-Phase and Multi-Component Polymer Systems Using the Atomic Force Microscope
Authors: VanLandingham M , Gu X , Raghavan D , Nguyen T
Published: July 2020

Recent advances have been made on two fronts regarding the capability of the atomic force microscope (AFM) to characterize the mechanical response of polymers. Phase imaging with the AFM has emerged as a powerful technique, providing contrast enhancement of topographic features in some cases and, in other cases, revealing heterogeneities in the polymer microstructure that are not apparent from the topographic image. The enhanced contrast provided by phase images often allows for identification of different material constituents. However, while the phase changes of the oscillating probe are associated with energy dissipation between the probe tip and the sample surface, the relationship between this energy dissipation and the sample properties is not well understood.

As the popularity of phase imaging has grown, the capability of the AFM to measure nanoscale indentation response of polymers has also been explored. Both techniques are ideal for the evaluation of multi-phase and multi-component polymer systems.
Atomic Force Microscopy and Near-Field Scanning Optical Microscopy Study of Quantum-Dot Assemblies and Fractal Films
Authors: Bragg W D , Banerjee K , Campin M J , Ying Z C , Zhu Jane G , Martin J E , Wilcoxon J P
Published: July 2020

Quantum dots and nanocrystalline films are interesting materials due to their novel properties, not achievable from the bulk materials [1]. New materials fabricated by assembling of quantum dots and nanostructured materials exhibit, for example, high optical nonlinearities. Fractal structures [2] can be formed from quantum-dot aggregates or films. Scanning probe microscopy is the essential technique to characterize these nanometer-scaled materials?

Gold nanocluster colloids are synthesized in the interior of surfactant aggregates known as inverse micelles, without the use of water to solubilize the metal salt [3]. The size of the Au quantum dots is well controlled in the colloid synthesis and selected using chromatography. The Au clusters are then sprayed onto a glass slides to form thin films made of Au quantum dots. We have also used the laser ablation technique [4] to grow Ag nanoparticles, fractal aggregates and thin films. These samples have been studied using atomic force microscopy (AFM) and nearfield scanning optical microscopy.
Novel aspects of micro-thermal analysis of polymer blends
Authors: Conroy M , Pollock H M , Hammiche A , Mills G , Weaver JMR , Reading M , Price D M
Published: July 2020

In “ac” scanning thermal microscopy, an “active” thermal probe is used also as a heater, so as to inject evanescent temperature waves into a sample and to allow sub-surface imaging of polymers and other materials [1]. The sub-surface detail detected corresponds to variations in heat capacity or thermal conductivity. By suitably choosing the temperature modulation frequency, and hence the penetration depth of the wave, we control the depth of material below the sample surface that is contributing to the image.

Micro-Thermal Analysis [2, 3] builds upon this technique, in order to add spatial resolution to two well-established methods of chemical fingerprinting, DTA and DMA. In both cases, a temperature ramp is used to subject the sample to “events” such as a glass transition or melting. The chief advantages of using the active thermal probe to provide the temperature ramp as well as the modulation, without the use of a heating stage, are: (a) the data are obtained from localised regions chosen from a previously-obtained thermal image, (b) apart from these regions, the rest of the sample is preserved in its original unheated state.
Imaging Mechanisms in Dynamic Force Microscopy of Polymers
Authors: Haugstad Greg D , Hammerschmidt Jon A , Gladfelter Wayne L
Published: July 2020

Applications of scanning force microscopy (SFM) in polymer studies have flourished in this decade, reflecting (a) the power of SFM to image both structure and propertiesdown to the nanometer scale, and (b) the low cost and ease of getting useful results in ambient environments. One difficulty in SFM of polymers has been damage incurred by soft materials during the imaging process. The problem was alleviated by the development of special dynamic modes of operation, in which the probe spends little or no time in contact with the polymer surface. Such modes were dubbed “tapping”, “intermittent-contact”, “non-contact”, “near-contact”, etc. As studies proliferated, it became apparent that different researchers were using different terms to refer to the same apparent imaging mechanism, or the same term to refer to different imaging mechanisms. This quandary derived from a poor understanding of exactly how the SFM probe interacts with the sample surface.1-3,5
Structure, Flexibility and Intramolecular Forces Observed on Individual Proteins Using Afm
Authors: Müller Daniel J , Engel Andreas
Published: July 2020

Atomic force microscopy (AFM) allows the surfaces of native biological macromolecules to be imaged in aqueous solution with submolecular resolution. Short range forces govern the interactions between the AFM tip and the sample and produce the submolecular information of high resolution topographs. In contrast, the long range electrostatic double-layer (EDL) force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area [1]. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteineous substructures. Consequently, the deformation of the native protein is minimized and polypeptide loops of individual proteins were imaged in aqueous solution with a lateral resolution < 5 Åand a vertical resolution < 1 Å (Fig. 1).
Multimodal Atomic Force Imaging of Open Hemichannelinduced Modulation of Cell Volume and Viscoelastic Properties
Authors: Rhee Seung K , Quist Arjan P , Lin Hai , Almqvist Nils , Lai Ratneshx
Published: July 2020

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.

Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.
Atomic Force Microscopy (Afm) of Chromatin Fibers: What Can We Learn?
Authors: Leuba SH , Bash R , Lohr D , Lindsay S M , Zlatanova J
Published: July 2020

DNA in the eukaryotic nucleus is not naked. Instead, it is complexed at frequent intervals with an equal molecular mass of histones to form nucleosomes. In the nucleosome, ∼146 bp of DNA are wrapped around a core histone octamer composed of two copies each of histones H4, H3, H2B, and H2A. The family of linker histones (H1, H5 and H1°) bind to the DNA between successive nucleosomes and help maintain the three-dimensional arrangement of nucleosomes within the chromatin fiber. AFM studies of chromatin fibers with the histones either selectively trypsinized or selectively reconstituted demonstrate a specific role for the H3 N-termini in maintaining fiber structure, in conjunction with the 80 amino acid linker histone globular domain. These AFM results structurally agree with the location of the H3 N-termini in the histone octamer or the nucleosome core particle.
Resolving Spatial Conformations of Immuno-Proteins with Cryo-Atomic Force Microscopy (Cryo-Afm)
Authors: Shaon Zhifeng , Sheng Sitong
Published: July 2020

A major advantage of atomic force microscopy (AFM) is its intrinsic high contrast, owing to its extremely high sensitivity to height variations. For clean, flat specimens, high-resolution images of a variety of bio-molecules have been obtained, demonstrating the usefulness of AFM in structural biology. However, for large molecular complexes, the effectiveness of AFM is seriously limited, partly owing to the excessive deformation induced by the probe force exerted on the specimen. To overcome this difficulty, we have developed a cryo-AFM, operated under liquid nitrogen vapor at a temperature only a few degrees above 77K. In this system, specimen or tip contamination is significantly reduced, and the resolution achieved with isolated macromolecules is generally higher than that obtainable at room temperature with AFM. Therefore, cryo-AFM has been proven to be a worthy alternative for conventional electron microscopy (EM) with the potential of higher resolution without image averaging.

A particularly fruitful application of cryo-AFM is the study of the spatial structure of several immuno-proteins, where the 3D surface topology of the molecule has been obtained.
Micro & Nano-Scale Structure of Enamel and Dentin-Enamel Junction of Human Teeth
Authors: Fong Hanson , Sarikaya Mehmet , White Shane , Snead Malcolm
Published: July 2020

Enamel, which covers the anatomical crown of the tooth, is the hardest tissue in human body. Supported by a soft but tough dentin structure, the tooth is an advanced nanocomposite that can endure mastication stresses throughout a lifetime. A detail understanding the structure of the tooth, and sepecifically detin-enamel junction (DEJ), not only provides a sound basis for a model for synthetic dental restoration, but also provides lessons from nature on biomimetic regeneration with mechanical integrity. Enamel is a non-growing mineralized tissue and is subjected to most mechanical abuse. Dentin-enamel junction plays a critical role of distributing load between two very dissimilar materials - enamel and dentin. Bulk scale mechanical tests have shown that induced cracks on enamel tend to be arrested DEJ. Furthermore, nanoindentation measurements have also shown that there is a gradual decrease in hardness from enamel to dentin in the DEJ zone suggests a strong mechanical coupling in both deciduous and adult incisors. The objective of this investigation, through microscopical study, is to understand how these two dental hard tissues structurally couple through their junction zone.
Localization Of Camkiiα mrnas to Dendritic Spines Visualized By High Resolution In Situ Hybridization
Authors: Byrd AL , Shenoy SM , Martinez M , Plociniak M , Zhang H L , Singer RH , Bassell G J
Published: July 2020

Observations that polyribosomes are localized near dendritic spines and beneath synapses has led to a hypothesis of synapse-specific gene expression in which local synthesis would provide a mechanism to influence synaptic structure and strength (l).The active transport of specific mRNAs into dendrites and spines may be a regulated mechanism to target synaptic and regulatory proteins to postsynaptic locations and influence synaptic activity. RNA granules labeled with the vital dye, SYT014, were observed to localize into developing neurites in response to the neurotrophin, NT-3 (2). Neurotrophins have been shown to enhance synaptic activity by a process which requires new protein synthesis (3). The identity and source of newly synthesized proteins which are required to enhance synaptic strength in response to NT-3 are unknown. CaMKIIαRNA localization into dendrites has been shown to occur during long term potentiation (4).
Imaging Protein Interactions and Gene Expression in Individual Cells by Fluorescence Resonance Energy Transfer
Authors: Tsien RY , Miyawaki A , Kerr R , Baird G , Griffin BA , Adams SR , Zlokarnik G , Whitney M , Negulescu PA , Li W-H
Published: July 2020

Interactions between proteins or protein domains can be imaged by fusing them to cyan (CFP) and yellow (YFP) mutants of Green Fluorescent Protein and observing fluorescence resonance energy transfer (FRET). For example, fusions of CFP, calmodulin, a calmodulin-binding peptide, and YFP are transfectable emission-ratioing Ca2+ indicators with many uses. They are highly suitable for twophoton excitation at 770-810 nm, even at video rates. Applications not possible with previous indicators include detection of submicroscopic domains of Ca2+ by fusion of the indicators to key proteins, and dynamic imaging of Ca2+ in transgenic animals. YFPs have been improved as FRET acceptors by reducing their sensitivity to pH changes. Many other applications of GFP mutants to detect fluctuating protein-protein interactions are underway.

A synthetic alternative to GFPs for protein tagging arises from the ability of membrane-permeant biarsenical dyes to seek out and light up alpha-helical Cys-Cys-X-X-Cys-Cys motifs placed in recombinant proteins in live cells. The new system is much smaller than GFP (6 residues vs. 238), can label internal domains not just N- and C-terminii, and offers novel readouts (e.g. red emission peaking > 600 nm) and better temporal control of the labeling.
Protein Dynamics During Apoptosis
Authors: Herman Brian
Published: July 2020

Apoptosis is an actively regulated process of cell death necessary for proper control of tissue growth. The phenomenon is characterized by plasma membrane blebbing, cell shrinkage, chromatin condensation, and degradation of DNA. Apoptosis is distinctly different from necrosis. Unlike necrosis, which is a passive form of cell death, apoptosis appears to be an active physiological process using a controlled genetic “program” of gene expression. Furthermore, apoptosis does not involve severe tissue damage or inflammation. Membrane integrity is preserved, and there is no loss of cellular contents before phagocytosis. Apoptosis is also distinct from necrosis in that it can be triggered or suppressed by tissue-specific hormones and growth factors.

Apoptosis plays a critical role in tissue homeostasis by counterbalancing mitosis. It is an essential component of many physiological processes, including embryonic development, clonal selection in thymocytes, and in protection against disease.
Elastic Properties of Erythrocyte's Membrane Measured by Atomic Force Stretching Method
Authors: Rafi Korenstein , Arkady Bitler , Luana Scheffer , Eshel Ben-Jacob
Published: July 2020

We employed force-distance measurements, carried out by Atomic Force Microscopy (AFM), to explore the local elastic properties of the membrane-skeleton of human erythrocytes. To calculate the elastic constant of the membrane-skeleton we chose that part of the withdraw region in the force-distance curve which corresponds to the stretching of the membrane-skeleton complex. In order to achieve efficient stretching of the membrane we increased the adhesion of the tip to the membrane by polysine coating of the tip.

Fig. 1 is presents the method of stretching AFM on which our measurements are based, and in Fig.2 one can see the theoretical model of the deformation of a circular plate with fixed boundaries by an external force (AFM probe ) used for recalculating the membrane's bending modulus from the experimental data.

Since we obtained different detachment patterns of tip from the membrane, we set selection criteria for the choice of appropriate curves corresponding to membrane stretching.
Imaging the Lateral Distribution of Fluorescently Labeled Membrane Components of Human Erythrocytes Under Deformation
Authors: Knowles DW , Mohandas N , Ortiz de Solorzano C , Lockett SJ
Published: July 2020

The human erythrocyte membrane comprises many different biological macromolecules arranged into a cohesive two dimensional structure. It has long been a model system for studying cell membrane component interactions and their related underlying cell biology. Erythrocyte membrane rigidity emanates from its hexagonal semi-solid cytoskeletal network of spectrin polymers joined at junctional complexes by globular proteins. The network supports a 2D fluid double layer of lipid in which is dissolved a large array of receptor proteins some of which completely span the lipid bilayer and link to the underlying cytoskeletal network.

To study membrane component - component interactions, we have used the technique of fluorescence imaged microdeformation (1). This technique combines fluorescence labeling of specific membrane components, single cell microdeformation and state-of-the-art image collection and analysis to map the distribution of labeled components on the surface of the cell.
Quantitative Phase Contrast Microscopy of Living Astrocytes By Numerical Reconstruction of Digital Holograms
Authors: Marquet P , Cuche E , Chatton J-Y , Depeursinge Ch , Magistretti PJ
Published: July 2020

We have developed a method of numerical holography which allows simultaneous intensity and quantitative phase contrast imaging. Measurement of optical path length provides precise information about the cell morphology. As presented in Fig. 1, the experimental set-up for the recording of digital holograms is basically a Mach-Zender interferometer. At the exit of the interferometer, a CCD camera records a hologram which is made by the interference between a reference wave R and the object wave O that is transmitted by the biological sample. In order to obtain a plane wave as reference, a beam expander (B.E.) including a pinhole is introduced in the reference arm and enlarges the beam diameter to about 2cm. A similar system which produces a smaller beam diameter (≈5mm) is inserted in the objet arm. To perform holography in the off-axis geometry, the mirror in the reference arm is oriented in such a way that the reference wave reaches the CCD camera with a small incidence angle.
All-Solid-State Diode-Pumped Fluorescence Lifetime Imaging System for Biomedicaine and Microscopy
Authors: Cole M J , Dowling K , French P M W , Jones R , Parsons-Karavassilis D , Lever M J , Dymoke-Bradshaw A K L , Hares J D , Neil M A A , Ju_kaitis R , Wilson T
Published: July 2020

The determination of fluorescence lifetime requires only relative measurements of intensity and so is especially useful for biomedical samples in which the heterogeneous nature of tissue and autofluorescence cause significant problems. Since fluorescence lifetime is dependent upon both radiative and non-radiative decay rates, it may be used to distinguish between different fluorophore molecules (with different radiative decay rates) and to monitor local environmental perturbations that affect the non-radiative decay rate. Fluorescence lifetime probes have been demonstrated for many biologically significant analytes including [O2], [Ca2+] and pH. Fluorescence lifetime imaging (FLIM) can be applied to almost any optical imaging modality, including microscopy and potentially to non-invasive optical biopsy. Fluorescence lifetime data may be acquired in the frequency or time domain. The recent development of user-friendly and relatively portable ultrafast laser technology and the availability of ultrafast gated optical image intensifiers (GOI’s) enable the development of potentially inexpensive time domain FLIM instruments that may be deployed outside specialist laser laboratories.
The Effects of Spaceflight Conditions on Calcium-Dependent Secretion and Cytoskeletal Organization During Fertilization and Cell Divisions In the Sea Urchin Lytechinus Pictus
Authors: Schatten Heide , Chakrabarti Amitabha , Levine Howard , Runco Mario , Anderson Ken , Taylor Meghan
Published: July 2020

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. To investigate the effects of spaceflight on calcium-dependent secretion and cytoskeletal formation in a less complex system we utilized sea urchin eggs and embryos which were fertilized and cultured under spaceflight conditions during the STS-77 shuttle mission. Sea urchin eggs were fertilized and cultured in the newly developed aquatic research facility (ARF) which allowed culture of eggs and embryos in microgravity and in a 1g centrifuge in space. This allowed analysis of the comparison of microgravity and 1g spaceflight treatments with samples cultured on ground. Eggs and embryos were maintained in Standard Container Assemblies (SCAs) with identical sets prepared for culture in microgravity, and at 1g in the middeck compartment of the shuttle Endeavor, as well as for ground observations at the Kennedy Space Center.
Soma/Germline Interactions In Caenorhabditis Elegans Gonad
Authors: Hall David H , Winfrey Virginia P , Blaeuer Gareth , Hoffman Loren H , Furuta Tokiko , Rose Kimberly L , Hobert Oliver , Greenstein David
Published: July 2020

A combination of microscopical methods (TEM, SEM, freeze fracture, confocal, immunoEM) are utilized to demonstrate functional interactions between somatic cells and the germline in the model organism, C. elegans.A variety of signaling mechanisms are required to coordinate activity of maturing oocytes and the somatic sheath of the gonad. Communication via gap junctions allows direct coordination of sheath cell motility and oocyte development. Gap junctions link adjoining sheath cells, and link the sheath to the most mature oocyte. Yolk protein produced in the intestine passes from the pseudocoelom through the basal lamina of the proximal gonad and then through fenestrations (sheath pores) in the gonad sheath before being endocytosed into the most mature oocytes. Myoepithelial specializations anchored by hemi-adherens junctions in the proximal sheath act to squeeze the primary oocyte out of the gonad to the spermatheca for fertilization.
Applications of Confocal Microscopy to Study the Roles of the Cytoskeleton During Early Embryogenesis In Amphibians
Authors: Danilchik M V , Brown E E , Larkin K , Ray K
Published: July 2020

Because of their great size, availability, and capacity to develop autonomously in culture, amphibian eggs have become a productive system for a wide variety of experimental studies of animal cells. Their use has advanced our understanding of fundamental molecular and cellular processes such as transcription and translation, regulation of the cell cycle, and the function of the cytoskeleton. The amphibian egg, particularly that of the frog Xenopus laevis,has further served as an excellent system for studying complex developmental phenomena, including the roles of growth factors, signal transduction, and transcription regulation in cell determination, morphogenesis, and tissue differentiation.

Despite these advantages, the frog egg's great size and opacity have made it a challenging subject for microscopy. Thus, many problems in early development, such as how maternal cytoplasmic determinants become localized during cell division, have resisted traditional optical approaches.
Ultrastructural Pathology of Aids: an Overview
Authors: Orenstein Jan M
Published: July 2020

The ultrastructural pathology of HIV infection is a broad subject, owing to the fact that the transmission electron microscope has played an integral part in our understanding of HIV disease. The subject can be divided into three basic areas: HIV pathogenesis, opportunistic infections (OI), and neoplastic disease. Once HIV was determined to be a retrovirus, TEM confirmed its membership in the Lentivirus family and showed that it has a conical nucleoid. TEM has played a key role in our understanding of: 1) the CNS as a target of HIV, 2) the macrophage as a “trojan horse” for HIV, 3) how lymphocytes and macrophages differ in their HIV expression, 4) attachment of virions to the processes of follicular dendritic cells via complement and Fc receptors, and 5) the formation of multinucleated giant cells.
Microsporidia Infection Results In Redistribution of Host Cell Mitochondria
Authors: Shaw Andrew P , Scanlon Mary , Leitch Gordon J
Published: July 2020

Microsporidia are obligate intracellular parasites that lack mitochondria. Until recently, their importance was primarily limited to fish and insects; but with the onset of the AIDS epidemic, understanding their role as human pathogens has become increasingly important. Encephalitozoon microsporidia develop and mature within a membrane bound parasitophorous vacuole (pv). Following initial infection of the host cell, the developing stages (meronts) of the parasite proliferate in close proximity to the pv membrane. Later, they differentiate through a sporogonial stage and finally into environmentally resistant mature spores located more centrally in the vacuole.

Previous work done in our laboratory has shown that the pv membrane does not act as a significant diffusion barrier to the movement of ions between the host cell cytoplasm and the parasite, permitting the free-acid form of calcein, a fluorescent dye, loaded into the host cell to enter the pv.
The Efects of Spaceflight on Mitochondria in Human Lymphocytes (Jurkat).
Authors: Schatten Heide , Lewis Marian
Published: July 2020

Spaceflight induced mitochondrial alterations have been reported for muscle and may be associated with altered physiological functions in space. Mitochondrial alterations are also indicative of preapoptotic events which are seen in greater amounts in cells exposed to spaceflight when compared with cells cultured at 1 g. Preapoptotic mitochondrial changes include alterations of processes at the inner mitochondrial membrane and can result in changes in mitochondrial volume. Higher amounts of oxidative stress during space flight may be one of the causes for changes which lead to apoptosis. Jurkat cells flown on the STS-76 space shuttle mission showed an increase in the number of cells with apoptotic bodies early in the mission and a time-dependent, microgravity-related increase in the Fas/APO-1 cell death factor. Here we investigated the morphology of mitochondria in Jurkat cells exposed to spaceflight during the STS-76 mission.
Localization of Tissue Factor Pathway Inhibitor In Endothelial Cells: Pitfalls and Challenges
Authors: Olsen R , Hansen J-B , Webster P
Published: July 2020

Tissue Factor Pathway Inhibitor (TFPI) is a potent inhibitor of the extrinsic coagulation system, synthesized in endothelial cells. Membrane bound TFPI has recently been shown to play an important role in the degradation of activated coagulation factors in the endothelium. Comparison of subcellular TFPI localization patterns and TFPI production between Human Umbilical Vein Endothelial Cells (HUVEC) and an immortalized cell line (ECV 304) are presented here, as are the essential protocol modifications that we used to obtain labeling results by both light- and electron microscopy. We show that ECV 304 cells are similar to HUVEC in that TFPI is present in the Golgi complex (fig. 1) as well as in the endocytic pathway of ECV cells. Additionally, they secrete higher levels of TFPI into the culture medium than HUVEC. A colocalization of anti-TFPI with endocytosed BSA-gold, as well as with accumulated DAMP, demonstrated the presence of TFPI in the endocytic pathway.