Specimens For Electron Microscopy Research Articles

How do specimen preparation and crystal perfection affect structure factor measurements by quantitative convergent-beam electron diffraction?

The effectiveness of tripod polishing and crushing as methods of mechanically preparing transmission electron microscopy specimens of hard brittle inorganic crystalline materials is investigatedviathe example of cerium hexaboride (CeB6). It is shown that tripod polishing produces very large electron-transparent regions of very high crystal perfection compared to the more rapid technique of crushing, which produces crystallites with a high density of imperfections and significant mosaicity in the case studied here where the main crystallite facets are not along the natural {001} cleavage planes of CeB6. The role of specimen quality in limiting the accuracy of structure factor measurements by quantitative convergent-beam electron diffraction (QCBED) is investigated. It is found that the bonding component of structure factors refined from CBED patterns obtained from crushed and tripod-polished specimens varies very significantly. It is shown that tripod-polished specimens yield CBED patterns of much greater integrity than crushed specimens and that the mismatch error that remains in QCBED pattern matching of data from tripod-polished specimens is essentially nonsystematic in nature. This stands in contrast to QCBED using crushed specimens and lends much greater confidence to the accuracy and precision of bonding measurements by QCBED from tripod-polished specimens.

New Developments in Cathodoluminescence Spectroscopy for the Study of Luminescent Materials.

Herein, we describe three advanced techniques for cathodoluminescence (CL) spectroscopy that have recently been developed in our laboratories. The first is a new method to accurately determine the CL-efficiency of thin layers of phosphor powders. When a wide band phosphor with a band gap (Eg > 5 eV) is bombarded with electrons, charging of the phosphor particles will occur, which eventually leads to erroneous results in the determination of the luminous efficacy. To overcome this problem of charging, a comparison method has been developed, which enables accurate measurement of the current density of the electron beam. The study of CL from phosphor specimens in a scanning electron microscope (SEM) is the second subject to be treated. A detailed description of a measuring method to determine the overall decay time of single phosphor crystals in a SEM without beam blanking is presented. The third technique is based on the unique combination of microscopy and spectrometry in the transmission electron microscope (TEM) of Brunel University London (UK). This combination enables the recording of CL-spectra of nanometre-sized specimens and determining spatial variations in CL emission across individual particles by superimposing the scanning TEM and CL-images.

X-ray absorption in pillar shaped transmission electron microscopy specimens

The dependence of the X-ray absorption on the position in a pillar shaped transmission electron microscopy specimen is modeled for X-ray analysis with single and multiple detector configurations and for different pillar orientations relative to the detectors. Universal curves, applicable to any pillar diameter, are derived for the relative intensities between weak and medium or strongly absorbed X-ray emission. For the configuration as used in 360° X-ray tomography, the absorption correction for weak and medium absorbed X-rays is shown to be nearly constant along the pillar diameter. Absorption effects in pillars are about a factor 3 less important than in planar specimens with thickness equal to the pillar diameter. A practical approach for the absorption correction in pillar shaped samples is proposed and its limitations discussed. The modeled absorption dependences are verified experimentally for pillars with HfO2 and SiGe stacks.

Focused ion beam sectioning studies of biomimetic hydroxyapatite coatings on Ti-6Al-4V substrates

Biomimetic hydroxyapatite (HA) coating has been used to render titanium implants with excellent osteoconductivity and osseointegration in orthopedic and dental applications. However, to date it has not been possible to reveal the fine details of the coating structure or the coating/substrate interface. In this study, a crack-free biomimetic HA coating has been successfully formed on sand-blasted, acid-etched Ti-6Al-4V discs by simply immersing the discs in a modified simulated body fluid (m-SBF). Dual-beam FIB/SEMs with either gallium ion source (GFIB) or xenon plasma ion source (PFIB) were used to prepare the cross-sections and thin transmission electron microscopy (TEM) specimens of the coating, and the capabilities of GFIB and PFIB were compared. Both the cross-sectional SEM and TEM images confirmed that a well bonded biomimetic HA coating has been formed on the Ti-6Al-4V substrate. The coating exhibits a gradient structure with a dense microstructure adjacent to the titanium substrate to facilitate strong bonding, while there is a porous structure at the surface, which is beneficial to bone cell attachment and subsequent new bone integration.

Mitochondria-targeted antioxidant SkQ1 reduces age-related alterations in the ultrastructure of the lacrimal gland.

Dry eye syndrome is an eye disorder affecting many people at an old age. Because dry eye syndrome is accelerated by aging, a useful approach to the prevention of this syndrome may be an intervention into the aging process. Previously, we showed that the mitochondria-targeted antioxidant SkQ1 delays manifestations of aging and inhibits the development of age-related diseases including dry eye syndrome. Nevertheless, the link between SkQ1's effects and its suppression of age-related changes in the lacrimal gland remains unclear. Here we demonstrated that dietary supplementation with SkQ1 (250 nmol/[kg body weight] daily) starting at age 1.5 months significantly alleviated the pathological changes in lacrimal glands of Wistar rats by age 24 months. By this age, lacrimal glands underwent dramatic deterioration of the ultrastructure that was indicative of irreversible disturbances in these glands' functioning. In contrast, in SkQ1-treated rats, the ultrastructure of the lacrimal gland was similar to that in much younger rats. Morphometric analysis of electron-microscopic specimens of lacrimal glands revealed the presence of numerous secretory granules in acinar cells and a significant increase in the number of operating intercalary ducts. Our results confirm that dietary supplementation with SkQ1 is a promising approach to healthy ageing and to prevention of aberrations in the lacrimal gland that underlie dry eye syndrome.

In situ electron microscopy studies of electromechanical behavior in metals at the nanoscale using a novel microdevice-based system.

Electrically assisted deformation (EAD) is an emerging technique to enhance formability of metals by applying an electric current through them. Despite its increasing importance in manufacturing applications, there is still an unresolved debate on the nature of the fundamental deformation mechanisms underlying EAD, mainly between electroplasticity (non-thermal effects) and resistive heating (thermal effects). This status is due to two critical challenges: (1) a lack of experimental techniques to directly observe fundamental mechanisms of material deformation during EAD, and (2) intrinsic coupling between electric current and Joule heating giving rise to unwanted thermally activated mechanisms. To overcome these challenges, we have developed a microdevice-based electromechanical testing system (MEMTS) to characterize nanoscale metal specimens in transmission electron microscopy (TEM). Our studies reveal that MEMTS eliminates the effect of Joule heating on material deformation, a critical advantage over macroscopic experiments, owing to its unique scale. For example, a negligible change in temperature (<0.02 °C) is predicted at ∼3500 A/mm2. Utilizing the attractive features of MEMTS, we have directly investigated potential electron-dislocation interactions in single crystal copper (SCC) specimens that are simultaneously subjected to uniaxial loading and electric current density up to 5000 A/mm2. Our in situ TEM studies indicate that for SCC, electroplasticity does not play a key role as no differences in dislocation activities, such as depinning and movement, are observed.

Dark-field image contrast in transmission scanning electron microscopy: Effects of substrate thickness and detector collection angle

An annular dark field (ADF) detector was placed beneath a specimen in a field emission scanning electron microscope operated at 30kV to calibrate detector response to incident beam current, and to create transmission images of gold nanoparticles on silicon nitride (SiN) substrates of various thicknesses. Based on the linear response of the ADF detector diodes to beam current, we developed a method that allowed for direct determination of the percentage of that beam current forward scattered to the ADF detector from the sample, i.e. the transmitted electron (TE) yield. Collection angles for the ADF detector region were defined using a masking aperture above the detector and were systematically varied by changing the sample to detector distance. We found the contrast of the nanoparticles, relative to the SiN substrate, decreased monotonically with decreasing inner exclusion angle and increasing substrate thickness. We also performed Monte Carlo electron scattering simulations, which showed quantitative agreement with experimental contrast associated with the nanoparticles. Together, the experiments and Monte Carlo simulations revealed that the decrease in contrast with decreasing inner exclusion angle was due to a rapid increase in the TE yield of the low atomic number substrate. Nanoparticles imaged at low inner exclusion angles (<150mrad) and on thick substrates (>50nm) showed low image contrast in their centers surrounded by a bright high-contrast halo on their edges. This complex image contrast was predicted by Monte Carlo simulations, which we interpreted in terms of mixing of the nominally bright field (BF) and ADF electron signals. Our systematic investigation of inner exclusion angle and substrate thickness effects on ADF t-SEM imaging provides fundamental understanding of the contrast mechanisms for image formation, which in turn suggest practical limitations and optimal imaging conditions for different substrate thicknesses.

Application of backscatter electrons for large area imaging of cavities produced by neutron irradiation

It is shown that with proper optimization, backscattered electrons in a scanning electron microscope can produce images of cavity distribution in austenitic steels over a large specimen surface for a depth of ∼500–700 nm, eliminating the need for electropolishing or multiple specimen production. This technique is especially useful for quantifying cavity structures when the specimen is known or suspected to contain very heterogeneous distributions of cavities. Examples are shown for cold-worked EK-164, a very heterogeneously-swelling Russian fast reactor fuel cladding steel and also for AISI 304, a homogeneously-swelling Western steel used for major structural components of light water cooled reactors. This non-destructive overview method of quantifying cavity distribution can be used to direct the location and number of required focused ion beam prepared transmission electron microscopy specimens for examination of either neutron or ion-irradiated specimens. This technique can also be applied in stereo mode to quantify the depth dependence of cavity distributions.

Rocksalt ZnO nanocrystal formation by beam irradiation of wurtzite ZnO in a transmission electron microscope

Under ambient conditions, ZnO crystallizes in a hexagonal wurtzite structure, but undergoes a phase transformation into a rocksalt structure with increasing hydrostatic pressure. However, in the present study, I have successfully demonstrated that intense beam irradiation of a wurtzite ZnO specimen in a transmission electron microscope produces nanoparticles of rocksalt ZnO as well as wurtzite ZnO, suggesting that the application of pressures is not a necessary condition for the formation of rocksalt ZnO.

Transmission Electron Microscopy Specimen Preparation of Delicate Materials Using Tripod Polisher

Transmission electron microscopy (TEM) is a powerful tool for analyzing a broad range of materials and provides localized information about the microstructure. However, the analysis results are strongly influenced by the quality of the thin foil specimen. Sample preparation for TEM analysis requires considerable skill, especially when the area of interest is small or the material of interest is difficult to thin because of its high hardness and its mechanical instability when thinned. This article selectively reviews recent advances in TEM sample preparation techniques using a tripod polisher. In particular, it introduces two typical types (fl at type and wedge type) of TEM sample preparation and the benefits and drawbacks of each method; finally, a method of making better samples for TEM analysis is suggested.

Qualifying Electrically Conductive Cold Embedding-Media for Scanning Electron Microscopy

The choice of embedding-media for metallographic specimen preparation, where subsequent scanning electron microscopy will be performed, is highly important since the quality of the analytical results in the specimen’s near-surface region is limited by the properties of the embedding-material. Due to their high electrical conductivity and only slight tendency to form gaps between near the sample surface, warm curing embedding-media are usually best suited. Hot embedding-presses use relatively high pressures at elevated temperatures that can be detrimental for sensitive materials or coatings. Cold embedding with conventional embedding-media, which does not require high pressure mounting, requires compromising for a material with a lower electric conductivity, a higher amount of pores in the material, and inferior interfacial connectivity to the specimen. For this reason, the development of alternative electrically conductive and cold hardening embedding-media is of great interest for metallographic sample preparation of electron microscopy specimens. In the present study, various embedding-media were evaluated with respect to their suitability for scanning electron microscopy and a novel conductive mounting compound has been identified and qualified.

Complex use of the diffraction techniques in depth profiling of the crystal lattice parameter and composition of InGaAs/GaAs gradient layers

A technique is proposed for testing thick (1 μm and larger) gradient layers with the composition and relaxation degree alternating over the layer depth on the basis of comparative analysis of X-ray scattered intensity maps in the reciprocal space and depth profiles of the crystal lattice parameters obtained by electron microdiffraction. The informativity of the proposed technique is demonstrated using the example of an InxGa1–xAs/GaAs layer with linear depth variation in x. Complex representation of the diffraction data in the form of the depth-profiled reciprocal space map allows taking into account the additional relaxation caused by thinning electron microscopy specimens.

Crystalline Grain Interior Configuration Affects Lithium Migration Kinetics in Li-Rich Layered Oxide.

The electrode kinetics of Li-ion batteries, which are important for battery utilization in electric vehicles, are affected by the grain size, crystal orientation, and surface structure of electrode materials. However, the kinetic influences of the grain interior structure and element segregation are poorly understood, especially for Li-rich layered oxides with complex crystalline structures and unclear electrochemical phenomena. In this work, cross-sectional thin transmission electron microscopy specimens are "anatomized" from pristine Li1.2Mn0.567Ni0.167Co0.067O2 powders using a new argon ion slicer technique. Utilizing advanced microscopy techniques, the interior configuration of a single grain, multiple monocrystal-like domains, and nickel-segregated domain boundaries are clearly revealed; furthermore, a randomly distributed atomic-resolution Li2MnO3-like with an intergrown LiTMO2 (TM = transitional metals) "twin domain" is demonstrated to exist in each domain. Further theoretical calculations based on the Li2MnO3-like crystal domain boundary model reveal that Li(+) migration in the Li2MnO3-like structure with domain boundaries is sluggish, especially when the nickel is segregated in domain boundaries. Our work uncovers the complex configuration of the crystalline grain interior and provides a conceptual advance in our understanding of the electrochemical performance of several compounds for Li-ion batteries.

Parallel preparation of plan-view transmission electron microscopy specimens by vapor-phase etching with integrated etch stops

Specimen preparation remains a practical challenge in transmission electron microscopy and frequently limits the quality of structural and chemical characterization data obtained. Prevailing methods for thinning of specimens to electron transparency are serial in nature, time consuming, and prone to producing artifacts and specimen failure. This work presents an alternative method for the preparation of plan-view specimens using isotropic vapor-phase etching with integrated etch stops. An ultrathin amorphous etch-stop layer simultaneously serves as an electron transparent support membrane whose thickness is defined by a controlled growth process such as atomic layer deposition with sub-nanometer precision. This approach eliminates the need for mechanical polishing or ion milling to achieve electron transparency, and reduces the occurrence of preparation induced artifacts. Furthermore, multiple specimens from a plurality of samples can be thinned in parallel due to high selectivity of the vapor-phase etching process. These features enable dramatic reductions in preparation time and cost without sacrificing specimen quality and provide advantages over wet etching techniques. Finally, we demonstrate a platform for high-throughput transmission electron microscopy of plan-view specimens by combining the parallel preparation capabilities of vapor-phase etching with wafer-scale micro- and nanofabrication.

Analysis on the laboratory examination characteristics in 22 patients with acute megakaryoblastic leukemia

目的分析急性巨核细胞白血病（AMKL）患者实验室检查特点。方法采集22例AMKL患者骨髓，分离有核细胞，一部分用于制备普通透射电镜标本，观察AMKL患者巨核细胞的形态结构；另一部分用于制备血小板过氧化物酶（PPO）细胞组化电镜标本，分析AMKL患者巨核细胞PPO阳性反应。同时结合分析患者骨髓细胞形态学、细胞化学和染色体核型等检查结果。结果22例患者中，17例患者骨髓巨核细胞以Ⅰ期巨核细胞为主，核圆，胞质含少量微管、膜性囊腔及微小致密颗粒，无分界膜和表面连接管道系统等特征性结构，致密颗粒和α颗粒少；5例患者骨髓巨核细胞大小不等，除Ⅰ期巨核细胞外，同时含有大量Ⅱ、Ⅲ期巨核细胞。22例患者巨核细胞PPO阳性率0~80%。22例患者骨髓涂片中均可见原始和幼稚巨核细胞，CD41免疫组化染色呈阳性反应。复杂染色体核型异常多见。结论大部分AMKL患者骨髓巨核细胞以Ⅰ期巨核细胞为主，少数患者除Ⅰ期巨核细胞外还伴有大量Ⅱ、Ⅲ期巨核细胞；PPO阳性反应差别明显；巨核细胞CD41免疫组化染色具有特异性；染色体核型复杂。

Surface Preserving Targeted Preparation using Focused Ion Beam Demonstrated by the Example of Oxide layers on Ni-Ti Alloys

Abstract The use of focused ion beams, whilst permitting the targetted preparation of thin specimens for Transmission Electron Microscopy, also results in modification of the material to be investigated as a result of energy being transferred into the material. This undesirable effect is normally limited to the surface of the material, which is particularly unfavourably orientated towards the impinging ion beam. If the crystal structure and composition of areas close to the surface of such specimens need to be characterised, protective layers may be used. However, those layers, depending on the applied deposition technique, may interact with the sample surface as well thus affecting the results of the analysis. In the work presented here, the possible interactions which might occur between the various protective coatings of ion-beam deposited Platinum, electron beam deposited Platinum, Silicon Oxide or adhesively bonded Gold foil and the subsequent FIB-preparation of the oxide layers on Ni-Ti alloys are investigated, with respect to and how these might affect the TEM-images obtained of areas close to the surface of such specimens. It is shown that the use of adhesively bonded Gold foil as a protective coating, in particular, permits comprehensive characterisation of the surface, including the use of high-resolution TEM, to be carried out, up to the surface of the Oxide layer itself.

膜迷路障害動物モデルにおける前庭各器官の変化と可塑性について

This study was aimed at investigating the morphological change and plasticity of the cupula and other vestibular cells in a model of mechanical rupture of the membranous labyrinth. Ten bullfrogs were used as study subjects. As in our previous reports, mechanical rupturing was accomplished by inserting a fine needle into the center of the saccule. The morphological change of the vestibular organs originated in an inflammatory change, and/or mixture of endolymph and perilymph which might be caused by rupturing. The vestibular membranous labyrinths were sectioned on 1-28 days after insult, and the degrees of cupula change were observed with the stereoscopic microscope. After the fixation and preparation of specimens for transmission electron microscopy (TEM), the ampullary and utricular sensory epithelium and ampullary dark cells were evaluated and compared with cupular changes. As controls, the vestibular organs on the opposite side were observed. Sensory epithelia and dark cells were impaired in the early stage, and those tended to recover thereafter. Morphologically, the epithelia recovered in 14 days after the insult, whereas dark cells needed 28 days for recovery. The cupulae did not change significantly within 3 days after insults. In the lateral semicircular canals, the cupulae were highly affected, and those of the posterior semicircular canals were less affected. As for the dark cells, there were no obvious differences between each canal. Morphological variations and possibilities of plasticity were seen in the vestibular organs with TEM observations, however, no obvious relationship between changes in the cupulae and vestibular cells was found. It is considered that the time lag of the damage development and recovery phase in each cell and cupula are responsible for this phenomenon. The change and plasticity of the cupula might be influenced by not only the anatomical features of the canals but also the degree of ampullary epithelial change.

Preparation of Drosophila Follicles for Transmission Electron Microscopy.

Transmission Electron Microscopy (TEM) provides high resolution and accuracy at the subcellular level for observing and investigating cellular structures. This is essential for understanding a large variety of cellular processes. In this chapter, we describe a detailed protocol for preparing Drosophila follicles in order to be used as a specimen for transmission electron microscopy.

Cross-Sectional Transmission Electron Microscopy Specimen Preparation Technique by Backside Ar Ion Milling

Cross-Sectional Transmission Electron Microscopy Specimen Preparation Technique by Backside Ar Ion Milling

Preparation Method of Plan-View Transmission Electron Microscopy Specimen of the Cu Thin-Film Layer on Silicon Substrate Using the Focused Ion Beam with Gas-Assisted Etch

Preparation Method of Plan-View Transmission Electron Microscopy Specimen of the Cu Thin-Film Layer on Silicon Substrate Using the Focused Ion Beam with Gas-Assisted Etch