Assessing Experimental Parameter Space for Achieving Quantitative Electron Tomography for Nanometer-Scale Plastic Deformation

The structures of a series of 3D-SBA-15 materials have been systematically studied by X-ray diffraction (XRD), nitrogen sorption, and electron tomography (ET) techniques. All the 3D-SBA-15 materials possess an ordered 2D hexagonal mesostructure with similar cell parameters, while the pore size measured from nitrogen sorption increases with the hydrothermal treatment temperature, in some cases even larger than the cell parameter calculated from X-ray diffraction. Taking advantage of the ET technique, it is shown that there exist two types of pores in 3D-SBA-15 materials: one is the ordered channels associated with the hexagonal symmetry, and the other is locally disordered and randomly distributed merged pores. During the hydrothermal treatment events at various temperatures, the size of ordered channels changes little, while the volume fraction and the overall size of the latter type of pores increase and eventually become dominant. Thus, the attribution of the pore sizes obtained from nitrogen sorption analysis to which types of pores is synthesis condition dependent and ET is an important tool to answer this question.

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 tomography reveals distinct morphological and optical properties of soot aggregates from coal-fired residential stoves in China

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.

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.

A new methodology to obtain magnetic information on magnetic nanoparticle (MNP) systems via electron tomography techniques is reported in this work. The new methodology is implemented in an under-development software package called Magn3t, written in Python and C++. A novel image-filtering technique that reduces the highly undesired diffraction effects in the tomography tilt-series has been also developed in order to increase the reliability of the correlations between morphology and magnetism. Using the Magn3t software, the magnetic shape anisotropy magnitude and direction of magnetite nanoparticles has been extracted for the first time directly from transmission electron tomography.

Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context. In the present study we used automated electron tomography in conjunction with cryomicroscopy to reveal the internal organization and ultrastructure of mitochondria. The whole isolated cell organelles from Neurospora crassa were quick-frozen and examined in vitrified ice. Single-axis tilt series were recorded with a Philips CM 120 Biotwin under low-dose conditions; the estimated total exposure was 10,000 e−nm−2. The tilt increment was 1° and the tilt series ranged from −65° to +65°. In order to enhance the contrast of the rather thick samples zero-loss energy filtering was employed. The images were recorded with a Ik × Ik CCD camera. The experimental setup has been described by Koster et al2 Three-dimensional reconstructions were performed by weighted backprojection. Prior to the three-dimensional visualization of 3-D data sets a denoising technique based on nonlinear anisotropic diffusion was applied (Figs. 2, 3).

The titanium oxide nanotubes have become a very attractive material with potential applications in biomedicine, photocatalysis, energy etc. Their properties depend mostly on morphology which is relatively easy to change by controlling the conditions of the anodization like the type of electrolyte used, the voltage and the time of anodization. There is a direct linear relation between the anodization voltage and the average diameter of the formed nanotubes, as the voltage increases the diameter of nanotubes also increases [1,2]. The possibility of the preparation of nanotubes with different size, shape and wall thickness leads to the control of their geometric surface area and specific surface area, which is an important parameter in the development of new substrates for example in heterogeneous catalysis. Nanotube surface functionalization with platinum nanoparticles is a way to fabricate active material for catalysis of oxidation reaction of methanol. Such nanostructured complex materials demand advanced methods for characterization and visualization of real structure, therefore application of TEM and electron tomography techniques is desired. In this work titania nanotubes were obtained by electrochemical oxidation of pure titanium at voltage of 10 and 25V, that resulted in creation of TiO 2 nanotubes with 40 and 110 nm in diameter, respectively. After anodization the heat treatment was performed at 450 o C for 1h to change amorphous structure of TiO 2 nanotubes into crystalline anatase structure. A suitable amount of Pt – 0.2 mg/cm 2 on the surface of the nanotubes was deposited using magnetron sputtering. FIB prepared specimens were analyzed in a Hitachi HD‐2700 dedicated STEM (Scanning Transmission Electron Microscopy). Fig. 1 shows cross‐section SEM images of TiO 2 nanotubes with 0.2 mg/cm 2 Pt deposit on the top. The platinum nanoparticles tend to choose the edges of TiO 2 nanotubes and side walls. A high amount of Pt fills the interior of the nanotubes as shown in Fig. 1b. As nanotubes diameter increases from 40 to 110 nm the depth of deposition into nanotubes also increases (Fig. 1c). Morphology of prepared structures was characterized by scanning transmission electron microscopy tomography. This method provides three‐dimensional structural information at nanoscale based on two dimensional projections acquired at different tilt angles. High angle annular dark field (HAADF) imaging was used for 2D projections. The results have shown distribution of platinum nanoparticles inside the nanotubes. The variations in platinum content introduced into different diameter nanotubes were also examined. Segmented volume of nanotubes was analyzed in terms of specific surface area and volume fraction.

The integration of computers and transmission electron microscopes (TEM) in combination with the availability of computer networks evolves in various fields of computer-controlled electron microscopy. Three layers can be discriminated: control of electron-optical elements in the column, automation of specific microscope operation procedures and display of user interfaces. The first layer of development concerns the computer-control of the optical elements of the transmission electron microscope (TEM). Most of the TEM manufacturers have transformed their optical instruments into computer-controlled image capturing devices. Nowadays, the required controls for the currents through lenses and coils of the optical column can be accessed by computer software. The second layer of development is aimed toward further automation of instrument operation. For specific microscope applications, dedicated automated microscope-control procedures are carried out. in this paper, we will discuss our ongoing efforts on this second level towards fully automated electron tomography. The third layer of development concerns virtual- or telemicroscopy. Most telemicroscopy applications duplicate the computer-screen (with accessory controls) at the microscope-site to a computer-screen at another site. This approach allows sharing of equipment, monitoring of instruments by supervisors, as well as collaboration between experts at remote locations.Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments.

Electron tomography of dislocation structures

Electron microscope tomography is a useful method for deriving three-dimensional (3-D) structures from tilt series. We used an intermediate, high-voltage electron microscope operated at 400 kV to study the 3-D structure of selectively stained or labeled mitochondria from diverse sources. We have examined the structure of neuronal mitochondria and mitochondria from brown adipose tissue (BAT) employing either single-axis or double-axis techniques over a tilt range of ±60° with 1° or 2° angular increments (Fig. 1). Although BAT mitochondria contains only lamellar cristae with no tubular cristae as we have observed in neuronal mitochondria, we found that the inner membrane of BAT mitochondria invaginates to form cristae only through narrow, tubular openings or crista junctions (Fig. 2). Recent interest in the possibility of structural artifacts in chemically fixed mitochondria imaged in situ motivated us to perform electron tomography on cryofixed brain and brown fat tissue.

Janus nanoparticles are nanoparticles (NPs) displaying two sides of different chemical nature which makes them of high interest. The formation of the Janus shell provides the NPs an amphiphilic character, which can therefore self‐assemble in a tunable fashion by varying different experimental parameters such as size, polymer ratio and temperature. Here, Au NPs coated by polyethylene glycol (PEG) and both polystyrene (PS) and poly(N‐isopropylacrylamide) (PNIPAM) were investigated. [1] The true Janus character of the NPs was determined by electron tomography. Characterization of polymer coated particles by electron microscopy is challenging due to the low contrast of the organic materials. The goal was to distinguish the two different polymers in two types of particles (PEG+PNIPAM and PEG+PS coated Au NPs). Different approaches were used to improve the contrast between the two polymers. The PEG+PNIPAM particles were characterized by specific staining of the hemispheres while the character of the PEG+PS particles was investigated by the growth of silica. First, the PEG+PNIPAM coated NPs were stained with 3 mM CuSO 4 .5H 2 O solution to generate sufficient contrast in the Transmission Electron Microscope (TEM). PNIPAM‐coated NPs showed damage of the polymer upon electron beam irradiation. For PEG‐coated NPs a stable shell was observed. In an attempt to distinguish PNIPAM and PEG, PEG+PNIPAM coated particles were stained and investigated with TEM (Fig. 1). Since conventional TEM images are 2D projections from a 3D object, electron tomography was performed. It must be noted that the conventional technique for tomography in materials science, high angle annular dark field scanning TEM (HAADF‐STEM), could not be applied due to the large difference in atomic number Z for Au and the elements in the polymer shell. Instead, Bright‐Field TEM tomography was applied. Images were acquired over the largest possible range (‐76° to +76°) every 2°, aligned and reconstructed by the SIRT (Simultaneous Iterative Reconstruction Technique) algorithm in the Astra Toolbox. [2] The obtained 3D reconstruction displayed rather low contrast between the shell and the background. Manual segmentation based on the orthoslices (xy‐, yx‐ and zx‐direction) could be carried out. This method provided a 3D reconstruction that clearly confirms the formation of stained half‐shells (Fig. 3). In the second case of PEG+PS coated Au NPs, an attempt of staining with calcium phosphate was carried out. In TEM images hemispheres could be observed, but selective staining could not be confirmed. Due to the high affinity of PEG by silica, PEG is able to act as a primer to promote silica condensation. Since PS is hydrophobic and does not allow silica nucleation, the silica shell was expected to occur only at the parts of the NPs that are coated by PEG (Fig. 2). Either staining or silica shell growth over PEG‐coated areas resulted in the observation of semi‐shells (Janus character) in Au NPs by TEM and electron tomography.

Electron tomography is a powerful tool in materials science to characterize nanostructures in three dimensions (3D). In scanning transmission electron microscopy (STEM), the sample under study is exposed to a focused electron beam and tilted to obtain twodimensional (2D) projections at different angles; many imaging modes are available such as high-angle annular dark-field (HAADF). In tomography, the collection of projections is called a tilt-series, from which we can reconstruct a 3D image that represents the sample. While HAADF tomography can clearly reveal the inner structure of the sample, it cannot directly provide compositional information. To better understand nanomaterials with more types of elements, spectral imaging techniques like energy dispersive X-ray spectroscopy (EDS) must be pursued. EDS tomography, however, is currently hampered by slow data acquisition, resulting in a small number of elemental maps with low signalto- noise ratio (SNR). Electron tomography, especially EDS tomography, is an ill-posed inverse problem whose solution is not stable and unique. Although advanced reconstruction techniques may yield a more accurate result by incorporating prior knowledge, they also involve fine-tuning parameters that highly influence the reconstruction quality. Furthermore, while great efforts have been dedicated to developing tomography techniques for image enhancement, directly combining reconstruction volumes at hand has still not been widely considered to the best of our knowledge.

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.

The three-dimensional (3D) atomic structure of nanomaterials, including strain, is crucial to understand their properties. Here, we investigate lattice strain in Au nanodecahedra using electron tomography. Although different electron tomography techniques enabled 3D characterizations of nanostructures at the atomic level, a reliable determination of lattice strain is not straightforward. We therefore propose a novel model-based approach from which atomic coordinates are measured. Our findings demonstrate the importance of investigating lattice strain in 3D.