Conventional Transmission Electron Microscopy Imaging Research Articles

Comparison of differential phase Contrast, Lorentz and non-Lorentz transmission electron microscopy for microstructural and magnetic imaging of alnico magnet

In this work, non-Lorentz, Lorentz transmission electron microscopy (LTEM) and differential phase contrast (DPC) methods were used to observe the phase and magnetic structure of the alnico magnet. All observations were performed on alnico-5 in transverse orientation. The results showed the magnetic contrast of the domain walls in the conventional TEM images despite the effects of the lens magnetic field, which was verified and explained using DPC. Experimental methodology and sample results are summarized with advice for further research.

DMPFIT: A Tool for Atomic-Scale Metrology via Nonlinear Least-Squares Fitting of Peaks in Atomic-Resolution TEM Images

Despite the wide availability and usage of Gatan’s DigitalMicrograph software in the electron microscopy community for image recording and analysis, nonlinear least-squares fitting in DigitalMicrograph is less straightforward. This work presents a ready-to-use tool, the DMPFIT software package, written in DigitalMicrograph script and C++ language, for nonlinear least-squares fitting of the intensity distribution of atomic columns in atomic-resolution transmission electron microscopy (TEM) images with a general two-dimensional (2D) Gaussian model. Applications of the DMPFIT software are demonstrated both in atomic-resolution conventional coherent TEM (CTEM) images recorded by the negative spherical aberration imaging technique and in high angle annular dark field (HAADF) scanning TEM (STEM) images. The implemented peak-finding algorithm based on the periodicity of 2D lattices enables reliable and convenient atomic-scale metrology as well as intuitive presentation of the resolved atomic structures.

Utilization of TEM with Automated Tile Scan Technique for Length Determination of CNF

Large field of view (FOV) imaging was introduced and evaluated to characterize length and width of cellulose nanofibril (CNF) by transmission electron microscopy (TEM) with automation capability. New imaging method provided an image area as wide as covering one hole on grid. The large FOV image provided 10×10 of tile image which can cover 2500 μm² of sample area. Large FOV imaging has a great advantage to obtain massive information for average length of CNF. The imaging method enable to overcome the limitation of conventional TEM imaging to elicit average length of CNF. The average length and width of CNF was 375 nm and 7 nm, respectively. Length-weighted average length of CNF was 651 nm. Study to select the kind of support film resulted as carbon film was the best among silicon monoxide and carbon. Carbon film was more durable to high electron beam energy exposure and better to disperse CNF separately than silicon monoxide.

Molecular architecture and modifications of full-length myocilin

Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the myocilin olfactomedin domain, the structure and function of full-length myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible. We systematically characterized disulfide-dependent and -independent oligomerization as well as confirmed glycosylation and susceptibility to proteolysis. We identified oligomeric states with glycosylation sites that are inaccessible to enzymatic removal. Low-resolution single particle 2D class averaging from conventional transmission electron microscopy imaging confirms an extended arrangement of tetramers, truncated products consistent with dimers, and a higher-ordered state consistent with octamer. Taken together, our study reveals new myocilin misfolded states and layers of intrinsic heterogeneity, expands our knowledge of olfactomedin-family proteins and lays the foundation for a better molecular understanding of myocilin structure and its still enigmatic biological function.

The measurement of the dislocation density using TEM

The line intercept method is well developed for estimating the dislocation density in metals, but its application is limited by the intersection counting, which is deeply depending on the TEM image quality. Manual interscetion counting is quite time and labor consuming. If most of the dislocations has good contrast and homogenous background in an image, it is possible to perform automatic counting by the computer program. In this work a series of imaging conditions were investigated on a slightly deformed IF steel thin foil. Though the invisibility criterion remains applicable for both the conventional transmission electron microscopy (CTEM) and the scanning transmission electron microscopy (STEM), the STEM images revealed more dislocations and had more homogenous background than the CTEM images. The STEM bright field (BF), annular dark field (ADF) and annular bright field (ABF) images at a low index zone axis contained the most dislocations visible and were suitable for automatic intersection counting of dislocations.

Autocorrected off-axis holography of two-dimensional materials

The reduced dimensionality in two-dimensional materials leads a wealth of unusual properties, which are currently explored for both fundamental and applied sciences. In order to study the crystal structure, edge states, the formation of defects and grain boundaries, or the impact of adsorbates, high resolution microscopy techniques are indispensible. Here we report on the development of an electron holography (EH) transmission electron microscopy (TEM) technique, which facilitates high spatial resolution by an automatic correction of geometric aberrations. Distinguished features of EH beyond conventional TEM imaging are the gap-free spatial information signal transfer and higher dose efficiency for certain spatial frequency bands as well as direct access to the projected electrostatic potential of the 2D material. We demonstrate these features at the example of h-BN, at which we measure the electrostatic potential as a function of layer number down to the monolayer limit and obtain evidence for a systematic increase of the potential at the zig-zag edges.

Scanning transmission electron microscopy image simulations of complex dislocation structures generated by discrete dislocation dynamics

Scanning Transmission Electron Microscopy Diffraction Contrast Imaging (STEM-DCI) has been gaining popularity for the identification and analysis of dislocations in crystalline materials due to its ability to supress undesirable image features that are often present in conventional TEM images. However, there does not yet exist a robust body of work demonstrating expected contrast in these imaging conditions. A novel approach for the simulation of STEM-DCI images was developed using a modified form of the scattering matrix formalism. This algorithm was used to simulate a variety of dislocation configurations generated using three-dimensional discrete dislocation dynamics.

Atomic Resolution Imaging of Light Elements in a Crystalline Environment using Dynamic Hollow-Cone Illumination Transmission Electron Microscopy.

Multiple electron scattering and the nonintuitive nature of image formation with coherent radiation complicate the interpretation of conventional transmission electron microscopy images. Precession of the illuminating beam in transmission electron microscopy (TEM) can lead to more robust and interpretable images with some penalty to image contrast, a technique known as dynamic hollow-cone illumination TEM. We demonstrate direct and robust imaging of light and heavy atoms in a crystalline environment with this technique. This method is similar to the annular bright-field technique in scanning transmission electron microscopy, via the principle of reciprocity. Dynamic hollow-cone illumination TEM is challenging in practice due to sensitivity to the misalignment of the precession axis, microscope objective aperture, and crystal zone axis.

Microstructure and fission products in the UCO kernel of an AGR-1 TRISO fuel particle after post irradiation safety testing

Tristructural isotropic (TRISO) coated particle fuel, as a key fuel concept for the high-temperature gas-cooled reactors and a candidate accident-tolerant fuel, has been investigated under the US-DOE Advanced Gas Reactor Fuel Development and Qualification Program. Extensive studies have been conducted to evaluate the fission-product release, diffusion of Ag, Pd, and Cd in the SiC layer and the TRISO coating system, and safety-test performance. However, to date there are limited reported results on the fuel kernels’ response to irradiation with or without post irradiation safety testing. To incrementally fill this knowledge gap, extensive studies using transmission electron microscopy (TEM) and atom probe tomography (APT) were conducted on a TRISO fuel-particle kernel with a 19.74% 235U enrichment, irradiated to 18.63% FIMA and subsequently subjected to safety testing at 1600 °C for 300 h. Microstructural characterizations, elemental analysis, and phase identification were conducted using conventional TEM and scanning TEM imaging, energy-dispersive X-ray spectroscopy, selected-area electron diffraction and APT. The following findings were made: (1) significant reconstructions and phase evolutions occurred in the irradiated and post safety-tested fuel kernel, and its microstructure consists of two primary phases, namely a higher-Z (atomic mass) UC phase and a lower-Z UO phase, (2) no fission-gas bubbles are identified within the fuel kernel, that can be attributed to the high-temperature post safety-testing, (3) fission products Zr, Nb, Mo, Ru, Tc and Rh were found to segregate preferentially into UC phase or to form metallic precipitates while the lanthanide fission products tend to stay in the solution of UO phase, and (4) Pd was detected in the rod-shaped precipitates.

Atmospheric scanning electron microscopy and its applications for biological specimens.

Scanning electron microscopy in ambient conditions (Air-SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air-SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)-SEM. Sharpness was lower than that of FE-SEM, but foot process of podocytes was observed at high magnification. Air-SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air-SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air-SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.

Electron microscopy determination of crystallographic polarity of aluminum nitride thin films

Aluminum nitride (AlN) crystallizes usually in the wurtzite structure (P63mc) and it has a crystallographic polarity. In this work, the polarity in AlN was characterized by using several methods of transmission electron microscopy (TEM) in order to examine their applicability. AlN was deposited by metalorganic vapor phase epitaxy (MOVPE), followed by annealing at 1550 °C. TEM samples were prepared by using a focused ion beam (FIB) mill. Observation was performed with microscopes of JEM-2100, JEM-ARM200 F and FEI Titan Cubed G2 at Kyushu University (Japan), and the following results were obtained. (1) Conventional TEM imaging: Under a diffraction condition with hkil = 0002, inversion domains or an inversion domain boundary (IDB) was observed. (2) Scanning TEM (STEM) High-Angle Annular Dark Field (HAADF) imaging: Even when atomic column images of Al and N are not resolved completely from each other, the polarity was determined from the shape of atomic column images. (3) Scanning moire fringe imaging: The moire fringe pattern indicated the position of IDB and determine the direction of polarity. (4) Convergent beam electron diffraction (CBED): CBED was applicable for determination of the polarity in AlN at the acceleration voltage of 120 kV. Hence the polarity, direction of polarity and inversion domain boundary was determined using advanced TEM methods.

Application of scanning electron diffraction in the transmission electron microscope for the characterisation of dislocations in minerals

AbstractWe present an application of scanning electron diffraction for the characterisation of crystal defects in olivine, quartz and phase A (a high pressure hydrated phase). In this mode, which takes advantage of the ASTAR™ module from NanoMEGAS, a slightly convergent probe is scanned over the sample with a short acquisition time (a few tens of ms) and the spot patterns are acquired and stored for further post-processing. Originally, orientation maps were constructed from automatic indexing at each probe location. Here we present another application where images are reconstructed from the intensity of diffraction spots, producing either so-called ‘virtual’ bright- or dark-field images. We show that these images present all the characteristics of contrast (perfect crystal or defects) of conventional transmission electron microscopy images. Data are acquired with a very short time per probe location (a few tens of milliseconds), this technique appears very attractive for the characterisation of beam-sensitive materials. However, as the acquisition is done at a given orientation, fine tuning of the diffraction conditions at a given location for each reflection is not possible. This might present a difficulty for some precise, quantitative contrast analysis.

Towards bend-contour-free dislocation imaging via diffraction contrast STEM

Dislocation imaging using transmission electron microscopy (TEM) has been an invaluable tool for characterizing crystallographic defects in metals. Compared to conventional TEM imaging, diffraction contrast imaging scanning transmission electron microscopy (DCI STEM) with appropriate setting can provide better defect contrast with almost negligible bend contour artifacts, enabling more effective analysis of dislocation structures. Here, we investigated why STEM can suppresses bend contour, and how dislocation contrast behaves along with different STEM imaging parameters. Using a body-centered cubic HT-9 ferritic/martensitic alloy as an example, a simple procedure and operational theory are described at the beginning to help set up DCI STEM experiments. Comparing with conventional TEM and the STEM strictly complying with the principle of reciprocity, we found that a pair of STEM convergence and collection semi-angles, αS and βS, a few milliradians in size is essential for bend-contour-free defect imaging. It works in concert such that the convergence STEM probe opens up the reciprocal space, and then a comparable collection region evens out the rocking-curve oscillation and alleviates bend contours from the reciprocal space. This fundamental advantage is unique in DCI STEM. Practical guidelines regarding STEM parameters and specimen orientation and thickness are then provided for DCI STEM dislocation imaging. Lastly, we show that coupling DCI STEM with spectrum images of electron energy loss spectroscopy and of energy-dispersive X-ray Spectroscopy offers a comprehensive characterization of crystallographic defects and chemical information of complex microstructures.

Scanning Transmission Electron Microscopy for Polymer Blends

Physical properties of the polymer can be altered by mixing one or more polymers together also known as polymer blending. The miscibility of polymers is a key parameter in determining the properties of polymer blend. Conventional transmission electron microscopy (CTEM) plays a critical role in determining the miscibility and morphology of the polymers in blend system. One of the most difficult part in polymer microscopy is the staining by heavy metals to generate contrast in CTEM. RuO4 and OsO4 are commonly used to stain the polymer materials for CTEM imaging. CTEM imaging is difficult to interpret for blends due to lack of clear distinction in contrast. Apart from having difficulty in contrast generation, staining procedures are extremely dangerous as improper handling could severely damage skin, eyes, lungs etc. We have used scanning transmission electron microscopy (STEM) to image polymer blends without any staining processes. In current work, Acrylonitrile Butadiene Styrene (ABS)/Methacrylate Butadiene Styrene (MBS) and Styrene Acrylonitrile (SAN) along with filler additive were dispersed on Polycarbonate (PC) matrix and studied by STEM/HAADF (high angle annular dark field). By using HAADF, contrast was generated through molecular density difference to differentiate components in the blend.

Characterization of nanograined powder samples using the Rietveld method applied to electron diffraction ring patterns

A full-pattern fitting procedure based on the Rietveld method was applied to electron diffraction ring patterns of a two-phase system, exhibiting the co-presence of zinc sulfide (sphalerite) and zinc oxide (Wurtzite). Bright and dark field (DF) images reveal the presence of micrometric aggregates, composed of quasi-spherical nanosized crystallites. These conventional transmission electron microscopy imaging methods provide a general morphological characterization of the specimens although, in the present case, they are not suitable for a detailed characterization of the microstructural features of the analyzed samples. Owing to the overlap and broadening of the diffraction rings of the two phases, DF images cannot provide a satisfactory picture of the individual crystallites of each single phase. To overcome this limit, the mentioned Rietveld approach was applied to model the electron diffraction data. The crystalline domain size and relevant shapes for both phases were successfully evaluated using the proposed methodological approach. The excellent results obtained in the microstructural characterization of the nanostructured multiphase samples demonstrate the capability of this technique, that may represents a fully quantitative method for the routine characterization of crystalline nanomaterials.

Exploring the theoretical basis and limitations of cryo-STEM tomography for thick biological specimens.

Scanning transmission electron microscope (STEM) imaging has recently been applied to the cryo-tomography of thick biological specimens. As previously shown for plastic sections, STEM has a number of advantages for cryo-imaging compared to conventional wide-field TEM imaging. STEM is insensitive to phase coherence and is therefore suitable for much thicker specimens than TEM. Imaging in focus, with a long depth of field, also circumvents the complications of an oscillatory contrast transfer function and missing information at low spatial frequencies. Moreover the image signal represents a quantitative measurement of the electron scattering pixel by pixel, so that absolute intensities can be interpreted in terms of material properties in the specimen. Resolution, however, is undoubtedly compromised for thick samples, especially in the regime of multiple elastic scattering. In this work we address the specific issues that arise in cryo-tomography of thick biological specimens. We formulate an imaging model based on a Boltzmann transport equation, complemented by Monte Carlo simulations. Using these theoretical tools, we identify conditions for image acquisition that will be compatible with the basic presumption of tomographic reconstruction, i.e., that for a given composition the imaging signal varies monotonically with thickness. For optimal resolution, contrast, and signal strength, we propose to generalize the on-axis bright field detector to collect at angles well beyond the illumination cone. Our results justify the generation of 3D images for micron thicknesses and beyond.

Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM.

We have refined methods for biological specimen preparation and low-voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130-70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin-embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals-the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon-coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations.

Imaging Irreversible Transformations with Movie-Mode Dynamic Transmission Electron Microscopy

In situ transmission electron microscopy (TEM) has been utilized for decades to image materials processes at high spatial resolution. Yet the relevant dynamics of many of these processes often remain elusive, as they unfold too rapidly to discern at small spatial scales using conventional TEM imaging conditions. For example, consider a transformation front moving with a relatively low velocity of 0.1 mm/s. In situ TEM imaging conducted with conventional acquisition rates of 30 frames/s corresponds to a temporal resolution of ~33 ms and, limited by motion blur, provides a spatial resolution of only ~3 μm. Given the rapid microstructural evolution of many types of irreversible transformation fronts—on the order of mm/s to m/s—nanosecond temporal resolutions are required to capture these processes.

Synergy between transmission electron microscopy and powder diffraction: application to modulated structures.

The crystal structure solution of modulated compounds is often very challenging, even using the well established methodology of single-crystal X-ray crystallography. This task becomes even more difficult for materials that cannot be prepared in a single-crystal form, so that only polycrystalline powders are available. This paper illustrates that the combined application of transmission electron microscopy (TEM) and powder diffraction is a possible solution to the problem. Using examples of anion-deficient perovskites modulated by periodic crystallographic shear planes, it is demonstrated what kind of local structural information can be obtained using various TEM techniques and how this information can be implemented in the crystal structure refinement against the powder diffraction data. The following TEM methods are discussed: electron diffraction (selected area electron diffraction, precession electron diffraction), imaging (conventional high-resolution TEM imaging, high-angle annular dark-field and annular bright-field scanning transmission electron microscopy) and state-of-the-art spectroscopic techniques (atomic resolution mapping using energy-dispersive X-ray analysis and electron energy loss spectroscopy).

TEM and SP-ICP-MS analysis of the release of silver nanoparticles from decoration of pastry.

Metallic silver is an EU approved food additive referred to as E174. It is generally assumed that silver is only present in bulk form in the food chain. This work demonstrates that a simple treatment with water of "silver pearls", meant for decoration of pastry, results in the release of a subfraction of silver nanoparticles. The number-based size and shape distributions of the single, aggregated, and/or agglomerated particles released from the silver pearls were determined by combining conventional bright-field TEM imaging with semiautomatic particle detection and analysis. In addition, the crystal structure of the particles was studied by electron diffraction and chemical information was obtained by combining HAADF-STEM imaging with EDX spectroscopy and mapping. The TEM results were confirmed by SP-ICP-MS. The representative Ag test nanomaterial NM-300 K was used as a positive control to determine the uncertainty on the measurement of the size and shape of the particles.