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.

Multiple experimental configurations for performing nanoscale orientation mapping are compared to determine their fidelity to the true microstructure of a sample. Transmission Kikuchi diffraction (TKD) experiments in a scanning electron microscope (SEM) and nanobeam diffraction (NBD) experiments in a transmission electron microscope (TEM) were performed on thin electrodeposited hard Au films with two different microstructures. The Au samples either had a grain size that is >50 or <20 nm. The same regions of the samples were measured with TKD apparatuses at 30 kV in an SEM with detectors in the horizontal and vertical configurations and in the TEM at 300 kV. Under the proper conditions, we demonstrate that all three configurations can produce data of equivalent quality. Each method has strengths and challenges associated with its application and representation of the true microstructure. The conditions needed to obtain high-quality data for each acquisition method and the challenges associated with each are discussed.

Improved plasticity models require simultaneous experimental local strain and microstructural evolution data. Microscopy tools, such as electron backscatter diffraction (EBSD), that can monitor transformation at the relevant length-scale, are often incompatible with digital image correlation (DIC) techniques required to determine local deformation. In this paper, the viability of forescatter detector (FSD) images as the basis for the DIC study is investigated. Standard FSD and an integrated EBSD/FSD approach (Pattern Region of Interest Analysis System: PRIAS™) are analyzed. Simultaneous strain and microstructure maps are obtained for tensile deformation of Q&P 1180 steel up to ~14% strain. Tests on an undeformed sample that is simply shifted indicate a standard deviation of error in strain of around 0.4% without additional complications from a deformed surface. The method resolves strain bands at ~2 μm spacing but does not provide significant sub-grain strain resolution. Similar resolution was obtained for mechanically polished and electropolished samples, despite electropolished surfaces presenting a smoother, simpler topography. While the resolution of the PRIAS approach depends upon the EBSD step size, the 80 nm step size used provides seemingly similar resolution as 8,000× (22.7 nm) FSD images. Surface feature evolution prevents DIC analysis across large strain steps (>6% strain), but restarting DIC, using an FSD reference image from an interim strain step, allows reasonable DIC across the stress–strain curve. Furthermore, the data are obtained easily and provide complementary information for EBSD analysis.

The use of fast pixelated detectors and direct electron detection technology is revolutionizing many aspects of scanning transmission electron microscopy (STEM). The widespread adoption of these new technologies is impeded by the technical challenges associated with them. These include issues related to hardware control, and the acquisition, real-time processing and visualization, and storage of data from such detectors. We discuss these problems and present software solutions for them, with a view to making the benefits of new detectors in the context of STEM more accessible. Throughout, we provide examples of the application of the technologies presented, using data from a Medipix3 direct electron detector. Most of our software are available under an open source licence, permitting transparency of the implemented algorithms, and allowing the community to freely use and further improve upon them.

Time-resolved imaging of molecules and materials made of light elements is an emerging field of transmission electron microscopy (TEM), and the recent development of direct electron detection cameras, capable of taking as many as 1,600 fps, has potentially broadened the scope of the time-resolved TEM imaging in chemistry and nanotechnology. However, such a high frame rate reduces electron dose per frame, lowers the signal-to-noise ratio (SNR), and renders the molecular images practically invisible. Here, we examined image noise reduction to take the best advantage of fast cameras and concluded that the Chambolle total variation denoising algorithm is the method of choice, as illustrated for imaging of a molecule in the 1D hollow space of a carbon nanotube with ~1 ms time resolution. Through the systematic comparison of the performance of multiple denoising algorithms, we found that the Chambolle algorithm improves the SNR by more than an order of magnitude when applied to TEM images taken at a low electron dose as required for imaging at around 1,000 fps. Open-source code and a standalone application to apply Chambolle denoising to TEM images and video frames are available for download.

The three-dimensional characterization of distributed particle properties in the micro- and nanometer range is essential to describe and understand highly specific separation processes in terms of selectivity and yield. Both performance measures play a decisive role in the development and improvement of modern functional materials. In this study, we mixed spherical glass particles (0.4–5.8 μm diameter) with glass fibers (diameter 10 μm, length 18–660 μm) to investigate a borderline case of maximum difference in the aspect ratio and a significant difference in the characteristic length to characterize the system over several size scales. We immobilized the particles within a wax matrix and created sample volumes suitable for computed tomographic (CT) measurements at two different magnification scales (X-ray micro- and nano-CT). Fiber diameter and length could be described well on the basis of the low-resolution micro-CT measurements on the entire sample volume. In contrast, the spherical particle system could only be described with sufficient accuracy by combining micro-CT with high-resolution nano-CT measurements on subvolumes of reduced sample size. We modeled the joint (bivariate) distribution of fiber length and diameter with a parametric copula as a basic example, which is equally suitable for more complex distributions of irregularly shaped particles. This enables us to capture the multidimensional correlation structure of particle systems with statistically representative quantities.

Atom probe tomography (APT) analysis conditions play a major role in the composition measurement accuracy. Preferential evaporation (PE), which significantly biases the apparent composition, more than other well-known phenomena in APT, is strongly connected to those analysis conditions. One way to optimize them, in order to have the most accurate measurement, is therefore to be able to predict and then to estimate their influence on the apparent composition. An analytical model is proposed to quantify the PE. This model is applied to three different alloys such as NiCu, FeCrNi, and FeCu. The model explains not only the analysis temperature dependence, as in an already existing model, but also the dependence to the pulse fraction and the pulse frequency. Moreover, the model can also provide an energetic constant directly linked to the energy barrier required to field evaporate atom from the sample surface.

The aim of the study was to investigate the effects of chronic nandrolone decanoate treatment and/or swimming training on immunohistomorphometric parameters on rat pituitary gonadotropic cells. Male Wistar albino rats, 10 weeks old, were classified into four groups: control (T−N−), nandrolone (T−N+), swimming training (T+N−), and swimming training with nandrolone (T+N+). The T+ groups swam for 4 weeks, 1 h/day, 5 days/week. The N+ groups received nandrolone decanoate (20 mg/kg) once per week for 4 weeks. Pituitary tissue sections were processed and stained for immunohistochemical analysis and immunofluorescence. The volume density of luteinizing hormone (LH) cells was decreased by 48% in T−N+ and for 35% in the T+N+ group. The volume density of follicle-stimulating hormone (FSH) cells was decreased by 39% in T−N+ and for 30% in T+N+ compared to the control. Nandrolone alone, or combined with swimming training, decreased the number of LH/FSH cells compared to the control. The levels of the immunofluorescent signal of LH/FSH cells were increased in all experimental groups. Nandrolone alone decreased the serum level of LH by 17%, whereas swimming training alone increased FSH levels by 11% compared to the control. Serum levels of testosterone were increased in all experimental groups. Nandrolone alone, or combined with swimming training, decreased immunohistomorphometric parameters of gonadotropic cells, whereas the levels of immunofluorescent signal were increased.

Schistosomiasis is a multifactorial disease that includes environmental, behavioral, parasitic, vector, and host factors. This study aimed to assess the protective effect of single and polyvalent antigens from cercarial antigen preparations (CAPs), soluble worm antigen preparations (SWAPs), and soluble egg antigens (SEAs) which were used as candidate vaccines in an experimental model of Schistosoma mansoni-infected mice. The efficiency of the antigens was tested by determining their effects on fecal egg count, egg viability analysis, and tissue egg counts. Histological and morphometric analyses of granulomas in liver and intestine tissues were performed. In the present study, all immunized groups showed a significant reduction in the average fecal egg count and tissue egg load compared with infected mice. The most substantial reduction in fecal egg count was observed in the combined vaccinated group (23.23 ± 3.2). The group vaccinated with CAP before infection showed the highest reduction in tissue egg load (liver and intestine: 85.22 and 91.70%, respectively). Immunized animals showed a highly significant reduction in the numbers of hepatic granulomas compared with the infected non-immunized group. In conclusion, combining these different antigens (CAP, SWAP, and SEA) augments the protective immunity compared with other immunized groups.

Occasionally, Hashimoto's thyroiditis (HT) and papillary thyroid carcinoma (PTC) share similar nuclear features. The current study aims to quantify the differences between the investigated specimens of HT-associated PTC versus the HT alone, to reduce the subjective experience of an observer, by the use of fractal parameters as well as gray-level co-occurrence matrix (GLCM) textural parameters. We have analyzed 250 segmented nuclei per group (nn = 25 per patient and np = 10 patients per group) using the ImageJ software (NIH, Bethesda, MD, USA) as well as an in-house written code for the GLCM analysis. The mean values of parameters were calculated for each patient. The results demonstrated that the malignant cells from the HT-associated PTC specimens showed lower chromatin fractal dimension (p = 0.0321) and higher lacunarity (p = 0.0038) compared with the corresponding cells from the HT specimens. Also, there was a statistically significant difference between the investigated specimens, in the contrast, correlation, angular second moment, and homogeneity, of the GLCM corresponding to the visual texture of follicular cell chromatin. The differences in chromatin fractal and GLCM parameters could be integrated with other diagnostic methods for the improved evaluation of distinctive features of the HT-associated PTC versus the HT in cytology and surgical pathology specimens.

Concurrent three-dimensional imaging of the renal vascular and tubular systems on the whole-kidney scale with capillary level resolution is labor-intensive and technically difficult. Approaches based on vascular corrosion casting and X-ray micro computed tomography (μCT), for example, suffer from vascular filling artifacts and necessitate imaging with an additional modality to acquire tubules. In this work, we report on a new sample preparation, image acquisition, and quantification protocol for simultaneous vascular and tubular μCT imaging of whole, uncorroded mouse kidneys. The protocol consists of vascular perfusion with the water-soluble, aldehyde-fixable, polymeric X-ray contrast agent XlinCA, followed by laboratory-source μCT imaging and structural analysis using the freely available Fiji/ImageJ software. We achieved consistent filling of the entire capillary bed and staining of the tubules in the cortex and outer medulla. After imaging at isotropic voxel sizes of 3.3 and 4.4 μm, we segmented vascular and tubular systems and quantified luminal volumes, surface areas, diffusion distances, and vessel path lengths. This protocol permits the analysis of vascular and tubular parameters with higher reliability than vascular corrosion casting, less labor than serial sectioning and leaves tissue intact for subsequent histological examination with light and electron microscopy.

Accurate elemental quantification of materials by X-ray detection techniques in electron microscopes or microprobes can only be carried out if the appropriate mass absorption coefficients (MACs) are known. With continuous advancements in experimental techniques, databases of MACs must be expanded in order to account for new detection limits. Soft X-ray emission spectroscopy (SXES) is a characterization technique that can detect emitted X-rays whose energies are in the range of 10 eV to 2 keV by using a varied-line-spaced grating. Transitions producing soft X-rays can be detected and accurate MACs are required for use in quantification. This work uses Monte Carlo modeling coupled with multivoltage SXES measurements in an electron probe micro-analyzer (EPMA) to compute MACs for the L2,3-M and Li Kα transitions in a variety of aluminum alloys. Electron depth distribution curves obtained by the software MC X-ray are used in a parametrized fitting equation. The MACs are calculated using a least-squares regression analysis. It is shown that X-ray distribution cross-sections at such low energies need to take into account additional contributions, such as Coster–Kronig transitions, Auger yields, and wave function effects in order to be accurate.

Atom probe tomography (APT) is used to quantify atomic-scale elemental and isotopic compositional variations within a very small volume of material (typically <0.01 µm3). The small analytical volume ideally contains specific compositional or microstructural targets that can be placed within the context of the previously characterized surface in order to facilitate a correct interpretation of APT data. In this regard, careful targeting and preparation are paramount to ensure that the desired target, which is often smaller than 100 nm, is optimally located within the APT specimen. Needle-shaped specimens required for atom probe analysis are commonly prepared using a focused ion beam scanning electron microscope (FIB-SEM). Here, we utilize FIB-SEM-based time-of-flight secondary ion mass spectrometry (ToF-SIMS) to illustrate a novel approach to targeting <100 nm compositional and isotopic variations that can be used for targeting regions of interest for subsequent lift-out and APT analysis. We present a new method for high-spatial resolution targeting of small features that involves using FIB-SEM-based electron deposition of platinum “buttons” prior to standard lift-out and sharpening procedures for atom probe specimen manufacture. In combination, FIB-ToF-SIMS analysis and application of the “button” method ensure that even the smallest APT targets can be successfully captured in extracted needles.

Surface-sensitive information on a bulk sample can be obtained by using a low incident electron energy (low accelerating voltage/landing voltage) in a scanning electron microscope (SEM). However, topography and composition contrast obtained at low incident electron energies may not be intuitive and should be analyzed carefully. By combining an Auger electron spectrometer (AES) with a low incident electron energy SEM (LE-SEM), we investigated the SEM contrast carefully by separating the secondary electron (SE) and back-scattered electron (BSE) components with high accuracy. For this, we modified an AES to measure the electron energy in the range of 0–0.6 keV with a sample bias voltage of 0 to −0.3 keV. We could clearly observe reversed brightness of gold and carbon (graphite) in BSE images when the energy of the incident electrons was reduced to 0.2–0.3 keV. In addition, reflected electron energy spectroscopy (REELS) is known to be a tool for chemical state analysis of the sample. We demonstrated that it is possible to study the electron states of graphite, diamond, and graphene by acquiring low incident energy REELS spectra from their surfaces with the newly modified AES. This will be a new method for analyzing the electron states of local areas of a surface.

Alluvial mineral sands rank among the most complex subjects for mineral characterization due to the diverse range of minerals present in the sediments, which may collectively contain a daunting number of elements (>20) in major or minor concentrations (>1 wt%). To comprehensively characterize the phase abundance and chemistry of these complex mineral specimens, a method was developed using hyperspectral x-ray and cathodoluminescence mapping in an electron probe microanalyser (EPMA), coupled with automated cluster analysis and quantitative analysis of clustered x-ray spectra. This method proved successful in identifying and quantifying over 40 phases from mineral sand specimens, including unexpected phases with low modal abundance (<0.1%). The standard-based quantification method measured compositions in agreement with expected stoichiometry, with elemental detection limits in the range of <10–1,000 ppm, depending on phase abundance, and proved reliable even for challenging mineral species, such as the multi-rare earth element (REE) bearing mineral xenotime [(Y,REE)PO4] for which 24 elements were analyzed, including 12 overlapped REEs. The mineral identification procedure was also capable of characterizing mineral groups that exhibit significant compositional variability due to the substitution of multiple elements, such as garnets (Mg, Ca, Fe, Mn, Cr), pyroxenes (Mg, Ca, Fe), and amphiboles (Na, Mg, Ca, Fe, Al).

Samples from the sphalerite-dominated zone of a seafloor massive sulfide chimney, the Satanic Mills Chimney of the PACMANUS hydrothermal field, have been investigated to determine the internal macrostructure and microstructure of this zone, the phases present, and the distribution of metals. A combination of electron probe microanalysis, electron backscattered diffraction, and x-ray diffraction has been used. At the macroscale, this zone of the chimney wall is heavily porous and is comprised primarily of sphalerite, enclosing minor chalcopyrite, pyrite, and wurtzite. A Pb–As sulfosalt layer of possible microbial origins is present at the outer edge of the sphalerite matrix, next to a pore. The sphalerite has grown in globules on the order of 300 μm in diameter. At the microscale, the sphalerite features a colloform texture and a duplex-type grain structure consisting of either fine-grain regions in the center surrounded by coarse-grained regions or radiating coarse grains only. Pb- and As-rich bands have been detected in the colloform sphalerite, and growth twins have been observed in both the sphalerite and chalcopyrite crystals. A qualitative description of the growth of a typical globule is given, including nucleation, crystal growth, and solute redistribution.

The excitability of local surface plasmon modes in radial trimers composed of gold nanorods was mapped using hyperspectral cathodoluminescence (CL) in the scanning electron microscope. In symmetric trimers, the local plasmon resonances could be excited most effectively at the ends of individual rods. Introducing asymmetry into the structure breaks the degeneracy of the dipole modes and changes the excitability of transverse dipole modes in different directions. CL in the scanning electron microscope has great potential to interrogate individual nanophotonic structures and is a complement to electron energy loss spectroscopy and optical microscopy.

Hyperspectral soft X-ray emission (SXE) and cathodoluminescence (CL) spectrometry have been used to investigate a carbonaceous-rich geological deposit to understand the crystallinity and morphology of the carbon and the associated quartz. Panchromatic CL maps show both the growth of the quartz and the evidence of recrystallization. A fitted CL map reveals the distribution of Ti4+ within the grains and shows subtle growth zoning, together with radiation halos from 238U decay. The sensitivity of the SXE spectrometer to carbon, together with the anisotropic X-ray emission from highly orientated pyrolytic graphite, has enabled the C Kα peak shape to be used to measure the crystal orientation of individual graphite regions. Mapping has revealed that most grains are predominantly of a single orientation, and a number of graphite grains have been investigated to demonstrate the application of this new SXE technique. A peak fitting approach to analyzing the SXE spectra was developed to project the C Kα 2pz and 2p(x+y) orbital components of the graphite. The shape of these two end-member components is comparable to those produced by electron density of states calculations. The angular sensitivity of the SXE spectrometer has been shown to be comparable to that of electron backscatter diffraction.

An Al–Cu–Li aerospace alloy has been investigated to determine the order in which corrosion at different types of sites occurs in AA2099-T83. Specifically, the sequence of galvanic attack on intermetallic (IM) particles and other sites of AA2099-T83 was determined as a function of time, in 0.1 M NaCl, through the use of scanning electron microscopy and electron backscatter diffraction characterization techniques. The earliest attack occurred at isolated grains and grain boundaries and on Li-containing dispersoids. Similarly, some constituent IM particles showed evidence of trenching in the surrounding alloy matrix. These IM particles included Al7Cu2Fe and another group of unidentified particles which displayed complete trenching within the first 10 min of exposure. Al13(Fe, Mn)4 were next most active followed by Al37Fe12Cu2 with Al6(Fe,Mn) and large TiB2 particles being the least active.

Tomography using a focused ion beam (FIB) combined with a scanning electron microscope (SEM) is well-established for a wide range of conducting materials. However, performing FIB–SEM tomography on ion- and electron-beam-sensitive materials as well as poorly conducting soft materials remains challenging. Some common challenges include cross-sectioning artifacts, shadowing effects, and charging. Fully dense materials provide a planar cross section, whereas pores also expose subsurface areas of the planar cross-section surface. The image intensity of the subsurface areas gives rise to overlap between the grayscale intensity levels of the solid and pore areas, which complicates image processing and segmentation for three-dimensional (3D) reconstruction. To avoid the introduction of artifacts, the goal is to examine porous and poorly conducting soft materials as close as possible to their original state. This work presents a protocol for the optimization of FIB–SEM tomography parameters for porous and poorly conducting soft materials. The protocol reduces cross-sectioning artifacts, charging, and eliminates shadowing effects. In addition, it handles the subsurface and grayscale intensity overlap problems in image segmentation. The protocol was evaluated on porous polymer films which have both poor conductivity and pores. 3D reconstructions, with automated data segmentation, from three films with different porosities were successfully obtained.