Nanofabrication of cylindrical STEM specimen of InGaAs/GaAs quantum dots for 3D-STEM observation

We demonstrate the multiazimuth observation (360 degrees in principle) of InGaAs/GaAs quantum dots (QDs) by means of a 300 kV scanning transmission electron microscope (STEM), where both cross-sectional and plan-view observations are performed on a single STEM specimen for the first time. A cylindrical specimen with a diameter of 200-300 nm including the QD layer inside along the rotation axis was fabricated by the focused ion beam (FIB) technique, with the application of a newly developed mesa-cutting method to adjust the position and angle of the QD layer precisely. The 360 degrees STEM observation is realized by mounting the cylindrical specimen on a holder equipped with a specimen-rotation mechanism. High potential of 3D-STEM observation is briefly presented by showing high contrast images of QDs, dark field images, and moire fringes with various incident angles.     

STEM tomography for thick biological specimens

Scanning transmission electron microscopy (STEM) tomography was applied to biological specimens such as yeast cells, HEK293 cells and primary culture neurons. These cells, which were embedded in a resin, were cut into 1-microm-thick sections. STEM tomography offers several important advantages including: (1) it is effective even for thick specimens, (2) 'dynamic focusing', (3) ease of using an annular dark field (ADF) mode and (4) linear contrasts. It has become evident that STEM tomography offers significant advantages for the observation of thick specimens. By employing STEM tomography, even a 1-microm-thick specimen (which is difficult to observe by conventional transmission electron microscopy (TEM)) was successfully analyzed in three dimensions. The specimen was tilted up to 73 degrees during data acquisition. At a large tilt angle, the specimen thicknesses increase dramatically. In order to observe such thick specimens, we introduced a special small condenser aperture that reduces the collection angle of the STEM probe. The specimen damage caused by the convergent electron beam was expected to be the most serious problem; however, the damage in STEM was actually smaller than that in TEM. In this study, the irradiation damage caused by TEM- and STEM-tomography in biological specimens was quantitatively compared.     

Automated three-dimensional X-ray analysis using a dual-beam FIB

We present a fully automated method for three-dimensional (3D) elemental analysis demonstrated using a ceramic sample of chemistry (Ca)MgTiO(x). The specimen is serially sectioned by a focused ion beam (FIB) microscope, and energy-dispersive X-ray spectrometry (EDXS) is used for elemental analysis of each cross-section created. A 3D elemental model is reconstructed from the stack of two-dimensional (2D) data. This work concentrates on issues arising from process automation, the large sample volume of approximately 17 x 17 x 10 microm(3), and the insulating nature of the specimen. A new routine for post-acquisition data correction of different drift effects is demonstrated. Furthermore, it is shown that EDXS data may be erroneous for specimens containing voids, and that back-scattered electron images have to be used to correct for these errors.     

Comparison of intensity distributions in tomograms from BF TEM,ADF STEM,HAADF STEM,and calculated tilt series

The three-dimensional (3D) morphology of a nanometer-sized object can be obtained using electron tomography. Variations in composition or density of the object cause variations in the reconstructed intensity. When imaging homogeneous objects, variations in reconstructed intensity are caused by the imaging technique, imaging conditions, and reconstruction. In this paper, we describe data acquisition, image processing, and 3D reconstruction to obtain and compare tomograms of magnetite crystals from bright field (BF) transmission electron microscopy (TEM), annular dark-field (ADF) scanning transmission electron microscopy (STEM), and high-angle annular dark field (HAADF) STEM tilt series. We use histograms, which plot the number of volume elements (voxels) at a given intensity vs. the intensity, to measure and quantitatively compare intensity distributions among different tomograms. In combination with numerical simulations, we determine the influence of maximum tilt angle, tilt increment, contrast changes with tilt (diffraction contrast), and the signal-to-noise ratio (SNR) as well as the choice of the reconstruction approach (weighted backprojection (WB) and sequential iterative reconstruction technique (SIRT)) on the histogram. We conclude that because ADF and HAADF STEM techniques are less affected by diffraction, and because they have a higher SNR than BF TEM, they are better suited for tomography of nanometer-sized crystals.     

Comparison of intensity distributions in tomograms from BF TEM, ADF STEM, HAADF STEM, and calculated tilt series

The three-dimensional (3D) morphology of a nanometer-sized object can be obtained using electron tomography. Variations in composition or density of the object cause variations in the reconstructed intensity. When imaging homogeneous objects, variations in reconstructed intensity are caused by the imaging technique, imaging conditions, and reconstruction. In this paper, we describe data acquisition, image processing, and 3D reconstruction to obtain and compare tomograms of magnetite crystals from bright field (BF) transmission electron microscopy (TEM), annular dark-field (ADF) scanning transmission electron microscopy (STEM), and high-angle annular dark field (HAADF) STEM tilt series. We use histograms, which plot the number of volume elements (voxels) at a given intensity vs. the intensity, to measure and quantitatively compare intensity distributions among different tomograms. In combination with numerical simulations, we determine the influence of maximum tilt angle, tilt increment, contrast changes with tilt (diffraction contrast), and the signal-to-noise ratio (SNR) as well as the choice of the reconstruction approach (weighted backprojection (WB) and sequential iterative reconstruction technique (SIRT)) on the histogram. We conclude that because ADF and HAADF STEM techniques are less affected by diffraction, and because they have a higher SNR than BF TEM, they are better suited for tomography of nanometer-sized crystals.     

Automated electron tomography with scanning transmission electron microscopy

We report the successful implementation of a fully automated tomographic data collection system in scanning transmission electron microscopy (STEM) mode. Autotracking is carried out by combining mechanical and electronic corrections for specimen movement. Autofocusing is based on contrast difference of a focus series of a small sample area. The focus gradient that exists in normal images due to specimen tilt is effectively removed by using dynamic focusing. An advantage of STEM tomography with dynamic focusing over TEM tomography is its ability to reconstruct large objects with a potentially higher resolution.     

Contact pressure distribution in circular tube expansion using a conical plug

The technique of evaluating contact pressure distributions along a plug metal interface by measuring the deformations in terms of plug surface strains using electrical resistance strain gauges has been explored. To relate the unknown pressure distributions to the strain gauge readings, a finite element model of a plug was used assuming that the normal and shear pressures can be represented as a linear combination of generalized distributions, which have the form of polynomials up to the fourth order.This technique has been suitably applied to a thin brass tube expanding process for a single expansion ratio, and a single conical hollow plug with and without lubricant. To ensure good accuracy in the results, particular care was given to technological plug design and the choice of a number of appropriate strain gauged points on the inside surface of the plug.The more adequate polynomial representations of the normal and shear contact pressures were found by analyzing the degree of randomness in the distribution of the residuals. A comparison was made between experimental pressure distributions and those predicted by the slab method. Results obtained were compared with those derived from the use of a polynomial curve fitting method, besides assuming an average coefficient of friction at the interface.     

Elemental mapping with elastically scattered electrons

We describe a technique for efficient, quantitative, standardless elemental mapping using a high-angle annular detector in a scanning transmission electron microscope (STEM) to collect elastically scattered electrons. With a single crystal specimen, contrast due to thickness variations, diffraction, and channelling effects can be avoided, so that the resulting image contrast quantitatively reflects variations in impurity concentration. We compare a number of simple analytical approximations to the elastic scattering cross sections and show that a standardless analysis is possible over a wide range of atomic number and inner detector angle to an absolute accuracy of better than 20%.     

Extraction of structural and chemical information from high angle annular dark‐field image by an improved peaks finding method

With the development of spherical aberration (Cs) corrected scanning transmission electron microscopy (STEM), high angle annular dark filed (HAADF) imaging technique has been widely applied in the microstructure characterization of various advanced materials with atomic resolution. However, current qualitative interpretation of the HAADF image is not enough to extract all the useful information. Here a modified peaks finding method was proposed to quantify the HAADF‐STEM image to extract structural and chemical information. Firstly, an automatic segmentation technique including numerical filters and watershed algorithm was used to define the sub‐areas for each atomic column. Then a 2D Gaussian fitting was carried out to determine the atomic column positions precisely, which provides the geometric information at the unit‐cell scale. Furthermore, a self‐adaptive integration based on the column position and the covariance of statistical Gaussian distribution were performed. The integrated intensities show very high sensitivity on the mean atomic number with improved signal‐to‐noise (S/N) ratio. Consequently, the polarization map and strain distributions were rebuilt from a HAADF‐STEM image of the rhombohedral and tetragonal BiFeO₃ interface and a MnO₂ monolayer in LaAlO₃/SrMnO₃/SrTiO₃ heterostructure was discerned from its neighbor TiO₂ layers. Microsc. Res. Tech. 79:820–826, 2016. © 2016 Wiley Periodicals, Inc.     

Discrimination of heavy and light elements in a specimen by use of scanning transmission electron microscopy

Heavier elements have a larger scattering cross-section for elastically scattered electrons than lighter ones. Furthermore, the maximum number of scattered electrons is at higher scattering angles for heavier atoms. These differences can be used, in principle, to distinguish heavy and light elements from each other in dark field Scanning Transmission Electron Microscopy (STEM). We have achieved such discrimination in practice by collecting the electrons in a STEM experiment at two different angles. The information about the elemental composition that these two images together contain is visualized by forming linear combinations of the images which are specific for light and heavy elementsrrespectively. The results are demonstrated for a specimen consisting of platinum grains on a holey carbon film and for granulocytes stained with osmium tetroxide.     

异种钢水下摩擦柱/锥塞焊接头组织及性能*

应用X65、Q235C、Q345C和316L塞棒与X65母材塞孔配合对异种钢水下摩擦柱/锥塞焊过程进行试验研究,探讨塞焊缝区微观组织、显微硬度及力学性能变化规律。试验表明：在转速7 000 r/min、塞棒消耗量14 mm、焊接压力分别为30~50 kN和20~40 kN范围内,用X65和Q345C塞棒可获得无缺陷异种钢水下摩擦柱/锥塞焊接头。异种钢塞焊缝组织均与原始组织有明显差异,塞焊缝区域主要为贝氏体或马氏体或其混合组织特征;X65和Q345C塞棒均形成有效扩散冶金连接,结合界面处具有带状细小铁素体组织特征;316L塞棒与X65塞孔很难形成无缺陷摩擦柱/锥塞焊接头。异种钢塞焊缝区硬度普遍高于其母材,这种高匹配摩擦柱/锥塞焊缝接头有利于抗拉强度的提高,但塞焊缝附近的高硬化倾向将降低异种钢塞焊接头的塑性。研究结果为开发基于等静压摩擦柱/锥塞焊接技术的海底管线修复技术提供重要试验依据。     

Revealing local variations in nanoparticle size distributions in supported catalysts: a generic TEM specimen preparation method

The specimen preparation method is crucial for how much information can be gained from transmission electron microscopy (TEM) studies of supported nanoparticle catalysts. The aim of this work is to develop a method that allows for observation of size and location of nanoparticles deposited on a porous oxide support material. A bimetallic Pt‐Pd/Al₂O₃ catalyst in powder form was embedded in acrylic resin and lift‐out specimens were extracted using combined focused ion beam/scanning electron microscopy (FIB/SEM). These specimens allow for a cross‐section view across individual oxide support particles, including the unaltered near surface region of these particles. A site‐dependent size distribution of Pt‐Pd nanoparticles was revealed along the radial direction of the support particles by scanning transmission electron microscopy (STEM) imaging. The developed specimen preparation method enables obtaining information about the spatial distribution of nanoparticles in complex support structures which commonly is a challenge in heterogeneous catalysis.     

Atom probe specimen fabrication methods using a dual FIB/SEM

A dual FIB/SEM provides solutions to many challenges in atom probe specimen preparation. When combined with an in situ lift-out capability, the versatility of this tool allows almost any region of interest, in almost any geometry, to be placed at the apex of a specimen tip. Several preparation techniques have been developed in response to specific application requirements; for example, in cases where materials are not suitable for electropolishing, or where site-specific analysis is required. Two general techniques, with wide-ranging potential applications, are described in detail here. The first is a 'cut-out' technique that provides a relatively quick means of micro-tip specimen preparation from bulk material samples. The second method is a 'lift-out' technique that can be used in an in situ or ex situ mode and does not require the preparation of pre-sharpened mounting points.     

Monte Carlo electron-trajectory simulations in bright-field and dark-field STEM: Implications for tomography of thick biological sections

A Monte Carlo electron-trajectory calculation has been implemented to assess the optimal detector configuration for scanning transmission electron microscopy (STEM) tomography of thick biological sections. By modeling specimens containing 2 and 3 at% osmium in a carbon matrix, it was found that for 1-μm-thick samples the bright-field (BF) and annular dark-field (ADF) signals give similar contrast and signal-to-noise ratio provided the ADF inner angle and BF outer angle are chosen optimally. Spatial resolution in STEM imaging of thick sections is compromised by multiple elastic scattering which results in a spread of scattering angles and thus a spread in lateral distances of the electrons leaving the bottom surface. However, the simulations reveal that a large fraction of these multiply scattered electrons are excluded from the BF detector, which results in higher spatial resolution in BF than in high-angle ADF images for objects situated towards the bottom of the sample. The calculations imply that STEM electron tomography of thick sections should be performed using a BF rather than an ADF detector. This advantage was verified by recording simultaneous BF and high-angle ADF STEM tomographic tilt series from a stained 600-nm-thick section of C. elegans. It was found that loss of spatial resolution occurred markedly at the bottom surface of the specimen in the ADF STEM but significantly less in the BF STEM tomographic reconstruction. Our results indicate that it might be feasible to use BF STEM tomography to determine the 3D structure of whole eukaryotic microorganisms prepared by freeze-substitution, embedding, and sectioning.     

Location specific in situ TEM straining specimens made using FIB

A method has been devised and demonstrated for producing in situ straining specimens for the transmission electron microscope (TEM) from specific locations in a sample using a dual-beam focused ion beam (FIB) instrument. The specimen is removed from a polished surface in the FIB using normal methods and then attached to a pre-fabricated substrate in the form of a modified TEM tensile specimen. In this manner, specific features of the microstructure of a polished optical mount can be selected for in situ tensile straining. With the use of electron backscattered diffraction (EBSD), this technique could be extended to select specific orientations of the specimen as well.     

Evaluation of two-dimensional strain distribution by STEM/NBD

We proposed a strain mapping technique by Nano Beam electron Diffraction (NBD) combined with an energy filter (EF) and a scanning transmission electron microscopy (STEM) function. The STEM function improves the accuracy of a position where a diffraction pattern is acquired. The EF excludes inelastic scattering and enables novel numerical processing for the appropriate measurement of distances between diffraction disks. Using this technique, strain distributions were measured for two different types of p-MOSFETs, i.e., source/drain regions of each MOSFET is composed of different types of silicide, and the difference of their strain distributions in the channel region was confirmed. The proposed method was able to clarify that the strain distributions are quite different depending on the silicide materials even if the exterior of the MOSFETs was almost identical.     

STEM nanodiffraction technique for structural analysis of CoPt nanoparticles

Studying the structure of nanoparticles as a function of their size requires a correlation between the image and the diffraction pattern of single nanoparticles. Nanobeam diffraction technique is generally used but requires long and tedious TEM investigations, particularly when nanoparticles are randomly oriented on an amorphous substrate. We bring a new development to this structural study by controlling the nanoprobe of the Bright and Dark Field STEM (BF/DF STEM) modes of the TEM. The particularity of our experiment is to make the STEM nanoprobe parallel (probe size 1 nm and convergence angle <1 mrad) using a fine tuning of the focal lengths of the microscope illumination lenses. The accurate control of the beam position offered by this technique allowed us to obtain diffraction patterns of many single nanoparticles selected in the digital STEM image. By means of this technique, we demonstrate size effects on the order-disorder transition temperature in CoPt nanoparticles when their size is smaller than 3 nm.     

Three-dimensional distributions of elements in biological samples by energy-filtered electron tomography

By combining electron tomography with energy-filtered electron microscopy, we have shown the feasibility of determining the three-dimensional distributions of phosphorus in biological specimens. Thin sections of the nematode, Caenorhabditis elegans were prepared by high-pressure freezing, freeze-substitution and plastic embedding. Images were recorded at energy losses above and below the phosphorus L2,3 edge using a post-column imaging filter operating at a beam energy of 120 keV. The unstained specimens exhibited minimal contrast in bright-field images. After it was determined that the specimen was sufficiently thin to allow two-window ratio imaging of phosphorus, pairs of pre-edge and post-edge images were acquired in series over a tilt range of +/-55 degrees at 5 degrees increments for two orthogonal tilt axes. The projected phosphorus distributions were aligned using the pre-edge images that contained inelastic contrast from colloidal gold particles deposited on the specimen surface. A reconstruction and surface rendering of the phosphorus distribution clearly revealed features 15-20 nm in diameter, which were identified as ribosomes distributed along the stacked membranes of endoplasmic reticulum and in the cytoplasm. The sensitivity of the technique was estimated at < 35 phosphorus atoms per voxel based on the known total ribosomal phosphorus content of approximately 7000 atoms. Although a high electron dose of approximately 10(7)e/nm2 was required to record two-axis tilt series, specimens were sufficiently stable to allow image alignment and tomographic reconstruction.     

Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM)

We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.     

Quantitative electron holographic tomography for the 3D characterisation of semiconductor device structures

Electron tomography and electron holography experiments have been combined to investigate the 3D electrostatic potential distribution in semiconductor devices. The experimental procedure for the acquisition and data reconstruction of holographic tilt series of silicon p-n junction specimens is described. A quantitative analysis of the experimental results from specimens of two different thicknesses is presented, revealing the 3D electrostatic potential variations arising from the presence of surfaces and damage generated by focused ion beam (FIB) sample preparation. Close to bulk-like properties are measured in the centre of the tomographic reconstruction of the specimen, revealing higher electrically active dopant concentrations compared to the measurements obtained at the specimen surfaces. A comparison of the experimental results from the different thickness specimens has revealed a 'critical' thickness for this specimen preparation method of 350nm that is required for this device structure to retain 'bulk'-like properties in the centre of the membrane.