Electron tomography of dislocation structures

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.

Allvac 718Plus (718Plus) is a newly developed Ni‐based superalloy, high strength, corrosion resistant and has improved higher temperature performance compared to the Inconel 718 superalloy. The 718Plus superalloy is used for applications in power generation, aeronautics and aerospace. The combination of a different chemistry and adequate heat treatments causes precipitation mainly the γ′ and δ or η‐ phases. The 718Plus microstructure consists of a γ matrix with spherical precipitates of ordered face centred cubic γ'‐Ni 3 (Al,Ti) type phase, some orthorhombic δ‐Ni 3 Nb and hexagonal η‐Ni 3 Ti, η*‐Ni 6 AlNb or Ni 6 (Al,Ti)Nb particles precipitated at the grain boundaries. The aim of this study was characterization of phases and 3D visualization of microstructural element morphology using several microscopy techniques, mainly HRSTEM‐HAADF, STEM‐EDX spectrometry. The present work concerns also the application of TEM and FIB‐SEM electron tomography for imaging and evaluation of qualitative and quantitative information about microstructure of materials. Performed analysis in atomic level (HRSTEM‐HAADF) of selected precipitates in the 718Plus superalloy revealed the complex nature of these precipitates, as shown in Fig.1. X‐ray spectroscopic imaging (STEM‐EDX), enables the mapping of local concentrations of selected chemical elements. This technique was used for qualitative and quantitative evaluation of η‐ phase precipitates with resolution about of 0.2 nm. STEM‐EDX maps of selected elements forming η particles in 718Plus superalloy are presented in Fig.2. The electron tomography is currently a relatively new technique in materials science that uses a TEM or FIB‐SEM to 3D imaging of microstructural elements in various engineering materials. The TEM electron tomography technique enables obtaining 3D model of the investigated object(s) from the multiple 2D projection images, acquired over a range of viewing directions (±75°). The TEM investigation was performed on FIB lamella by a Cs‐corrected Titan 3 G2 60‐300 with EDX ChemiSTEM™ technology, which allowed to achieve high X‐ray signal over a large tilt angle of sample and collect a tomographic series of 2D elemental maps at the angular range from ‐40° to +60° (with a step of 4°) of tilting the sample. The STEM‐EDX imaging by ChemiSTEM™ provides new opportunities for 3D visualization of changes in the concentrations of particular chemical elements in nanoparticles or analysis of the microstructure of thin foils. Tomographic reconstruction of a tilt series images was performed using SIRT method, which allowed visualizing the three‐dimensional distribution of selected elements (Al, Cr) in the analysed volume. Application of elemental maps imaging (STEM‐EDX) acquired during tilting the sample was used for 3D imaging of coherent γ′ precipitates in 718Plus superalloy (Fig.3a). FIB‐SEM tomography is based on a serial slicing technique using a FIB‐SEM dual beam workstation. Dual‐beam SEM enables the acquisition of serial images with small (few nanometers) and reproducible spacing between the single imaging planes ‐ because no mechanical stage tilting is necessary between the FIB milling and the electron beam SEM imaging steps. Meso‐scale FIB‐SEM tomography technique, was used for characterization of spatial distribution and metrology of the η‐ phases in 718Plus superalloys with resolution of 12 nm. SEM backscattered electrons (BSE) image of 718Plus superalloy presents different shapes of the η‐ phase precipitated in the γ matrix. Fig. 3b shows three‐dimensional visualization of FIB‐SEM tomographic reconstructed η‐ phase. Fig. 3b shows at different angle of view a morphology of selected η particles precipitated at the γ grain boundary. Platelets, occasionally as a lamellar structure at grain boundaries and in the grains were observed in the microstructure of the 718Plus. The η‐ phase precipitates at the γ grain boundaries had much higher thickness (270 nm) than the thickness of the η‐ phase plates (56 nm) in lamellar precipitates inside γ grains (Fig. 3b). Application of HRSTEM‐HAADF imaging and tomographic techniques (STEM‐EDX, FIB‐SEM) allowed for visualization the precipitates in 718Plus superalloy, their quantitative assessment, spatial distribution and morphology.

Both single-particle analysis and electron tomography are special tools for the structural analysis of biological components by transmission electron microscopy. Single-particle analysis enables us to determine the structures of relatively large proteins and macromolecular complexes without crystallisation. Electron tomography is often used to visualise organelles and subcellular structures at molecular resolution. Recently, the scope of electron tomography has been widened due to the introduction of an automatic data collection system, cryo-electron microscopy, and improved sample preparation techniques. Application of electron tomography to single-particle analysis will be capable of analysing dynamics of large proteins. The techniques described in this review are expected to bridge the gap between the atomic resolution structures of protein molecules and cellular structures obtained by light microscopy.

Integrating in situ deformation and electron tomography (ET) techniques allows us to visualize the materials’ response to an applied stress with nanometer spatial resolution. The capability of structural, chemical, and morphological characterization in three-dimension real time and at sub-microscopic levels alleviates several persistent problems of two-dimensional imaging such as the projection effect and postmortem appearance. On the other hand, implementing deformation mechanism introduces additional experimental constraints that could influence the accuracy of the reconstructed volumes in a different way. To materialize quantitative and statistically relevant microstructure interpretation by time-resolved ET, we evaluated several key parameters such as angular tilt range, tilt increment, and reconstruction algorithms to characterize their influences on the accuracy of size and morphology reproducibility.

Sampling limits for electron tomography with sparsity-exploiting reconstructions

Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins’ functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000–160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.

The present work focuses on the fundamental plasticity/fatigue mechanisms operating at interfaces in micro/nano scale Ni samples. In‐situ SEM fatigue tests have been performed on FIB prepared single and bi‐crystal micropillars with well‐known orientations as revealed by EBSD. Careful characterizations of the nature and the distribution of deformation dislocations, the character and the local structure of the interface as well as the mechanisms controlling the interaction between these defects under cyclic loads were performed using ex‐situ TEM techniques including diffraction contrast imaging, automated crystallographic orientation and nanostrain mapping in TEM (ACOM‐TEM) as well as electron tomography. The primary TEM results obtained on single crystal micropillars after fatigue tests revealed the presence of dislocation walls structure as shown in Figure 1(a) and (b). ACOM‐TEM revealed local changes of crystal orientation around 1–2 degrees at the position of the dislocation walls. Furthermore, systematic contrast analysis of dislocations in these areas confirmed that only slip systems with the highest Schmid factor have been activated. The analysis of micropillars with GBs subjected to fatigue tests has shown the accumulation of dislocations at the GBs (Figure 1(c) and (d)) without slip transfer or localized plasticity. Electron tomography has been used to investigate the 3D distribution and the interaction of deformation dislocations within the dislocation walls. In order to directly observe the plasticity mechanisms, quantified in‐situ TEM tensile tests were performed on both single and bi‐crystal samples using the Pi 95 picoIndenter instrument and a MEMS device called ‘’Push‐to‐Pull’’ (PTP) from Hysitron.Inc (Fig 2). In order to minimize the effect of FIB on the in‐situ tensile samples, an original sample preparation method combining twin jet electro‐polishing and FIB was used, see figure (2). Nucleation‐controlled‐plasticity has been observed with defects induced by FIB at the edges of the sample acting as preferential sources for the nucleation of deformation dislocations. Furthermore, the in‐situ TEM nanotensile experiments revealed the elementary mechanisms controlling the interaction between dislocations and pre‐selected GBs from the electro‐polished thin foils, as the nucleation of dislocations from GB, see figure (3). These results indirectly shed light on the micropillar's behaviour as the root cause of the deformation is connected to the FIB preparation and the dislocation/GB interaction mechanisms.

Application of electron tomography of dislocations in beam-sensitive quartz to the determination of strain components

ABSTRACTNanoscale tomographic reconstructions from objects with diameters of 100nm or smaller can only be achieved non-destructively with transmission electron tomography. The application of this technique to W tips, which are common probes for scanning tunneling microscopy and nanoindentation, is demonstrated with emphasis on visualizing oxide layers and functionally attached nanoparticles. For the reconstruction of facetted free-standing catalyst nanoparticles, such as CeO2 octahedra, we propose a combination of energy-filtered (EF) and bright field (BF) TEM tomography to achieve high fidelity of the projection relationship via EFTEM, due to its incoherent imaging mode, and high resolution definition of the particle circumference from the edge-enhancement effects of the BF tomogram. Finally, electron tomography applications to CeO2 nanoprecipitates embedded in a multicomponent oxide glass matrix are shown, which comprises the first tomographic 3D reconstruction of a nanoscale dendrite.

Interactions with the physical environment, such as passive haptic feedback, have been previously shown to provide richer and more immersive virtual reality experiences. A strict correspondence between the virtual and real world coordinate systems is a staple requirement for physical interaction. However, many of the commonly employed VR locomotion techniques allow for, or even require, this relationship to change as the experience progresses. The outcome is that experience designers frequently have to choose between flexible locomotion or physical interactivity, as the two are often mutually exclusive. To address this limitation, this paper introduces reactive environmental alignment, a novel framework that leverages redirected walking techniques to achieve a desired configuration of the virtual and real world coordinate systems. This approach can transition the system from a misaligned state to an aligned state, thereby enabling the user to interact with physical proxy objects or passive haptic surfaces. Simulation-based experiments demonstrate the effectiveness of reactive alignment and provide insight into the mechanics and potential applications of the proposed algorithm. In the future, reactive environmental alignment can enhance the interactivity of virtual reality systems and inform new research vectors that combine redirected walking and passive haptics.

The majority of engineering steels are ferromagnetic and structually inhomogeneous on special scales ranging from nanometers to micrometers, and physical properties of engineering steels arise from three-dimensional (3D) features of the microstructure. Thus, obtaining 3D representation with a large field of view is desired for transmission electron microscopy (TEM) based microstructure characterization to establish microstructure - physical properties relationships with reasonable statistical relevancy. Here, we venture to use a conventional sample preparation process,i.e., mechanical polishing followed by electro-polishing, and experimental protocols optimization for electron tomography (ET) for ferromagnetic materials, especially engineering steels’ microstructural characterization are carried out. We found that the sample thickness after the mechanical polishing step is a critical experimental parameter affecting the success rate of tilt-series image acquisition. For example, for a ferritic heat-resistant 9Cr steel with lath martensite structure, mechanically thinning down to 30 μm or thinner was necessary to acquire an adequate tilt-series image of carbide precipitates in the high-angle annular dark-field scanning TEM (HAADF-STEM) mode. On the other hand, tilt-series image acquisition from dislocation structures remains challenging because the electron beam deflection during specimen-tilt was unavoidable and significant in the HAADF-STEM mode. To overcome the electron beam deflection problem, we evaluate several relatively accessible approaches including the “Low-Mag and Lorentz” TEM/STEM modes; although they are rarely used for ET, both the modes reduce or even zero the objective lens current and likely weaken the magnetic interference between the ferromagnetic specimen and the objective lens magnetic field. The advantages and disadvantages of those experimental components are discussed.

Asymmetric Small Protein Structure Determination by Individual Particle Electron Tomography

The mineralization of collagen in vitro has been extensively investigated for hydroxyapatite, silica, calcium carbonate and lepidocrocite (γ-FeOOH). Henceforth, it is interesting to investigate whether collagen also could serve as a generic mineralization template for other minerals, like magnetite. To this end, and inspired by the partial oxidation approach, first a ferrous hydroxide (Fe(OH)2) intermediate is synthesized via the titration of base to a solution of Fe2+. Subsequently, the Fe(OH)2 is mixed with collagen fibrils and poly(aspartic acid) is added to promote the formation of intrafibrillar crystals. Platelet-shaped lepidocrocite crystals being present throughout the entire thickness of the collagen fibrils can be realized, as was confirmed with electron tomography. The formation of lepidocrocite, which is an Fe3+ compound, is hypothesized to be induced via oxidation of the Fe2+ species and, therefore, the oxygen concentration during titration, TEM sample preparation and drying of TEM samples are investigated. Although the reaction is sensitive to small changes in experimental conditions, highly mineralized collagen fibers can be realized.

Rotordynamic analysis in complex coordinates has many advantages. Especially, the Lee diagram, with the concept of the infinity norm in directional frequency response matrix, can an effective design tool for rotating machinery, because the severity of the mode is clearly identified, and it also includes the concept of the worst case response prediction. However, it is not widely used in the field because most of the previous studies and commercial software are based on the real coordinate system. Our objective was to apply the Lee diagram to analyze rotordyanmic characteristics of the power turbine, which is a typical example of turbomachinery. We show that this could be a useful design tool for rotating machinery. The power turbine in this study was used for the super critical carbon dioxide power generation system and the rotor was supported by tilting pad bearings. The equation of motion of the rotor and bearing system was derived in the complex coordinate system. The Lee diagram was calculated with the concept of the infinity norm and unbalance response was predicted. The operation test of the power turbine was done with compressed air, and the rotor vibration was measured with the proximity probes. The results of the analysis show that the resonance speed can be effectively estimated with the Lee diagram and the degree of the anisotropy of the system can be identified. The operation test was done successfully and the measured maximum rotor vibration was about 5 μm. In addition, the measured rotor vibration shows that the anisotropy of the power turbine is very small as predicted, and the predicted vibration amplitude shows good agreement with the test data.

A real vector X in n dimensions whose components are sinusoidally varying with time can be represented by an n-dimensional complex factor Ze/sup j omega t/ whose real part is X. In different real coordinate systems, the components of X transform linearly, but the amplitudes of the components of Z transform nonlinearly. The author describes a method to reconstruct the relative phases of the (complex) components of Z (and, therefore, of X) based on the amplitude measurements of its n components in an orthogonal coordinate system and at least 2n-3 additional amplitude measurements in different directions. The author shows the necessary and sufficient condition on these additional directions to ensure uniqueness of the phase and polarization reconstruction for any arbitrary vector X, and presents an algebraic method for the reconstruction which offers substantial reduction in computing time over the method of reconstruction by nonlinear optimization. The result of this phase of reconstruction is the complete characterization of the polarization of X except for chirality. >