Measuring Lattice Strain in Three Dimensions through Electron Microscopy.

Introduction: Two-dimensional (2D) strain assessment is an important diagnostic and prognostic tool in various clinical conditions, particularly coronary artery disease (CAD). However, these measurements are limited in that the information is obtained in only a single plane (2D). Three-dimensional (3D) strain tracks the myocardium in all 3D, potentially overcoming the limitation of 2D strain. The objective of this study was to establish normal values for 3D strain in a population of healthy, normal controls and to compare these values with 2D strain values. In addition, we sought to evaluate the utility of 3D strain in patients with known or suspected CAD. Methods: We conducted a prospective study at a single major tertiary care center. Individuals were recruited for the study and divided into two groups: a normal control group and a CAD group. Global longitudinal strain (GLS) and global circumferential and global radial strain were calculated by both 2D and 3D strain methods. In addition, 3D was used to calculate area strain. Results: We enrolled a total of 43 individuals (20 normal control group, mean age 33 ± 2.7 years, and 23 CAD group, mean age 57 ± 2.8 years, 80% male). Values for 3D strain were consistently lower for GLS and global circumferential strain in both groups compared with 2D measurements. In the control group, the mean 2D GLS was −20 ± 1.6% versus −17.5 ± 1.5% for 3D GLS (P < 0.001). Similarly, the mean 2D circumferential strain was −17.7 ± 2.3% versus −15.6 ± 2.1% for 3D circumferential strain (P < 0.001). Combining both groups, the sensitivity of GLS for CAD was 80% for 2D versus 93% for 3D. Similar findings were seen for global circumferential strain (sensitivity 87% for 2D vs. 100% for 3D). However, the sensitivity of 3D global radial strain was lower (93% for 2D vs. 47% for 3D). 3D strain data were acquired in a shorter time span compared with 2D (2.2 ± 1 min vs. 3 ± 1 min). Conclusions: 3D strain assessment of longitudinal and circumferential strain is similar but mildly reduced compared with 2D techniques, with similar sensitivity for CAD. Radial strain measurements by 3D, however, are not accurate and correlate poorly with 2D.

Both light and electron microscopy have experienced exciting advances in the past few years, driven by the development of new imaging technologies and the ready availability of powerful computers and associated algorithms for visualization, analysis, and image restoration. The concomitant development of molecular genetic methods for engineering fluorescent molecules into proteins and for targeting indicator dyes to subcellular compartments has led to unprecedented opportunities for the study of dynamic processes and for correlating physiology and structure. Although the effective resolution of light microscopy has been increased through the use of optical sectioning and computational deblussing of images, the resolution of the light microscope is still not sufficient to address Inany issues of cell and tissue structure and protein localization. Electron microscopy has also undergone a quiet revolution, in particular high voltage electron microscopy, where the technique of electron tomography allows 3D reconstruction of relatively large structures such as neuronal spiny dendrites without the need for serial sectioning. In combination with light microscopy, high voltage EM and electron tomography provide a relatively straightforward method to bridge the resolution gap between light and electron microscopy. In this review, we discuss correlated 3D light and electron microscopy imaging of cells and tissues, including the adaptation of fluorescent dyes for electron microscopy imaging. (The J Histotechnol 23:261, 2000)

Three-dimensional (3D) left ventricular (LV) myocardial strain measurements using transthoracic 3D echocardiography speckle tracking analysis have several advantages over two-dimensional (2D) LV strain measurements, because 3D strain values are derived from the entire LV myocardium, yielding more accurate estimates of global and regional LV function. In this review article, we summarize the current status of 3D LV myocardial strain. Specifically, we describe how 3D LV strain analysis is performed. Next, we compare characteristics of 2D and 3D strain, and we explain validation of 3D strain measurements, feasibility and measurement differences between 2D and 3D strain, reference values of 3D strain, and its applications in several clinical scenarios. In some parts of this review, we used a meta-analysis to draw reliable conclusions. We also describe the added value of 3D over 2D strain in several specific pathologies and prognoses. Finally, we discuss novel techniques using 3D strain and suggest its future directions.

Richard Feynman pointed out in his 1959 speech, “It would be very easy to make an analysis of any complicated chemical substance; all one would have to do would be to look at it and see where the atoms are… I put this out as a challenge: Is there no way to make the electron microscope more powerful?” Over more than 50 years, with significant achievements in electron microscope and the developments of aberration-corrected electron optics, scanning transmission electron microscopy (STEM) has reached spatial resolution of 0.5 Å. Recently, in combination of the state-of-the-art scanning transmission electron microscope (STEM) and a tomographic reconstruction technique known as equally sloped tomography (EST), we have achieved electron tomography at 2.4 angstrom resolution [ 1 ]. This general method has been applied to reveal almost all atoms inside a Pt nanoparticle with unprecedented details [ 2 ]. Most recently, Feynman's 1959 challenge has been tackled by successfully determining 3D coordinates of 3,769 individual atoms at the tip of a tungsten needle with a precision of ~19 picometers [ 3 ]. In this talk, I will present the achievements of this novel technique.

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.

Review of electronic speckle pattern interferometry (ESPI) for three dimensional displacement measurement

Electron tomography (ET) is a method for three-dimensional (3-D) reconstruction of single, transparent objects from a series of images (i.e., a tilt series) recorded with a transmission electron microscope (EM) (Fig. 4.1). The method is related to the procedures used in medical tomography. The 3-D reconstructions are usually computed from the digitized tilt series after a radial weighting scheme has been applied to the Fourier-transformed data. The ET method can be used to reconstruct in 3-D any object that is transparent enough for projection imaging with the transmission EM. This means that specimens of biological origin are usually available for ET 3-D reconstruction, whereas, e.g., colloidal gold particles are not. An ET-calculated 3-D map could be based on projections from objects that have been visualized by EM in several different ways, i.e., from stained or unstained objects, objects visualized at different energy loss levels or objects embedded in different media. The combined analysis of the 3-D structure, imaged in different ways, could thus become very informative. The general applicability also means that it is not restricted to symmetrical or regularly arranged objects, nor to objects with preferred orientations on a support grid. In its present state, the ET method allows reproducible 3-D reconstructions of single molecular objects, with a resolution in the range of 5 nm, of complex cellular specimens. For isolated objects, free of interfering cellular substances, a somewhat higher resolution can be achieved. The ET method covers the intermediate resolution range where there is no other physical technique available to analyze single molecular complexes.

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.

Oxygen reduction reaction (ORR) is the bottleneck of metal‐air batteries and fuel cells. Strain regulation can change the geometry and adjust the surface charge distribution of catalysts, which is a powerful strategy to optimize the ORR activity. The introduction of controlled strain to the material is still difficult to achieve. Herein, we present a temperature‐pressure‐induced strategy to achieve the controlled lattice strain for metal coordination polymers. Through the systematic study of the strain effect on ORR performance, the relationship between geometric and electronic effects is further understood and confirmed. The strained Co‐DABDT (DABDT=2,5‐diaminobenzene‐1,4‐dithiol) with 2 % lattice compression exhibits a superior half‐wave potential of 0.81 V. Theoretical analysis reveals that the lattice strain changes spin‐charge densities around S atoms for Co‐DABDT, and then regulates the hydrogen bond interaction with intermediates to promote the ORR catalytic process. This work helps to understand the catalytic mechanism from the atomic level.

In our previous paper, structural changes of selenium powders ground by a planetary ball mill at various rotational speeds were investigated for the nanostructural modification of particles using mechanical grinding process. The experimental results indicated that the amorphisation of Se by grinding accompanies lattice strain, and the lattice strain arises from impact energy which is more than an energy related to intermolecular interaction. In this paper, molecular dynamics simulations of selenium have been carried out under compressing conditions of various pressure strengths for obtaining information of the lattice strain at atomic level. Then, dynamical behaviour of atomic configuration has been discussed in this process. The structural disordering and formation of the structural defects were estimated by deviations of bond length and angle and the number of created defects before and after compressing from simulated results. The disordering took place during compressing at various pressure strengths, and the disordered atoms return to their initial positions at lower pressure. Stable disordered state and defects after the compression can however remain by compression at more than a certain pressure strength mainly associated with binding energy of selenium.

In electron tomography (ET) based on a transmission electron microscope, the effective thickness of a specimen increases with the tilt angle and, therefore, the projection quality may deteriorate because of electron scattering. The information-missing region, however, can be reduced by broadening the specimen tilt range. To clarify the general influence of these effects on ET, the projection quality varying with the tilt angle was quantitatively evaluated for a 5-microm thick specimen observed with a 3 MV ultrahigh-voltage electron microscope. Simulations of three-dimensional reconstruction were then performed for different specimen thicknesses and tilt ranges. As a result, the ET accuracy was shown to decrease as the specimen thickness increased. However, an optimum specimen-tilt range, at which ET could reach its highest accuracy, was found to exist and become small with the increase of the specimen thickness. The presented results are helpful for determining the specimen thickness limitation on the ET resolution and improving the ET fidelity of thick specimens.

Three-dimensional feature-preserving noise reduction for real-time electron tomography

New Approach To Quantitative Subcellular Imaging Of Phosphorus And Calcium Using Energy-filtered Transmission Electron Microscopy And Tomography

ABSTRACTPrimary Ciliary Dyskinesia (PCD) is a heterogeneous genetic condition characterized by dysfunction of motile cilia. Patients suffer from chronic infection and inflammation of the upper and lower respiratory tract. Diagnosis of PCD is confirmed by identification of a hallmark defect of ciliary ultrastructure or by identification of biallelic pathogenic mutations in a known PCD gene. Since the first description of PCD in 1976, assessment of ciliary ultrastructure by transmission electron microscopy (TEM) has been central to diagnosis and research. Electron tomography is a technique whereby a series of transmission electron micrographs are collected at different angles and reconstructed into a single 3D model of a specimen. Electron tomography provides improved spatial information and resolution compared to a single micrograph. Research by electron tomography has revealed new insight into ciliary ultrastructure and consequently ciliary function at a molecular and cellular level. Gene discovery studies in PCD have utilized electron tomography to define the structural consequences of variants in cilia genes. Modern transmission electron microscopes capable of electron tomography are increasingly being installed in clinical laboratories. This presents the possibility for the use of tomography technique in a diagnostic setting. This review describes the electron tomography technique, the contribution tomography has made to the understanding of basic cilia structure and function and finally the potential of the technique for use in PCD diagnosis.

Review: 91 refs.