This paper presents a new method for creating and monitoring controlled localized negatively charged regions inside insulators with a scanning electron microscope (SEM). A localized buried charged region is created and observed close to the point where a high voltage primary beam (10 kV) strikes a metal–insulator–silicon specimen. The amount of buried charge within the insulator at any given moment can be dynamically monitored by detecting the appearance of a second peak in the secondary electron (SE) energy spectrum. SE energy spectral signals were obtained through the use of a compact high signal-to-noise energy analyzer attachment that was fitted on to the SEM specimen stage. An electrostatic model, together with Monte Carlo simulations, is presented to explain how the SE charge contrast effect functions. This model is then experimentally confirmed by using the SE energy spectral signal induced by a gallium ion beam inside a dual focused ion beam-SEM instrument.

In this paper, three-dimensional (3D) image data of ore particle systems is investigated. By combining X-ray microtomography with scanning electron microscope (SEM)-based image analysis, additional information about the mineralogical composition from certain planar sections can be gained. For the analysis of tomographic images of particle systems the extraction of single particles is essential. This is performed with a marker-based watershed algorithm and a post-processing step utilizing a neural network to reduce oversegmentation. The results are validated by comparing the 3D particle-wise segmentation empirically with 2D SEM images, which have been obtained with a different imaging process and segmentation algorithm. Finally, a stereological application is shown, in which planar SEM images are embedded into the tomographic 3D image. This allows the estimation of local X-ray attenuation coefficients, which are material-specific quantities, in the entire tomographic image.

A new method of image texture analysis is presented, based on the mean and standard deviation of gray levels within domains in an image. The calculations are performed recursively on domains of various sizes within the images. These gray level calculations are used as the input matrix for principal component analysis. The technique analyzes the entire image as a whole and is not for image segmentation. The analysis routine operates across all distances, frequencies and directions in the image, and is not computationally burdensome. The method was applied to scanning electron microscope images of reverse osmosis membranes on domains from 23 nm to 3 µm. The texture analysis technique performed well in identifying the surface morphology and, once calibrated, in predicting the surface roughness as measured by atomic force microscopy.

The magnetism and microstructure of Cr25Ni35Nb and Cr35Ni45Nb alloy tubes after 5 years of service were investigated in this paper. The saturation magnetization of the Cr25Ni35Nb alloy tube in the thickness direction is more than 20 emu/g, and the tube becomes ferromagnetic. The inner and outer walls of Cr35Ni45Nb alloy tubes also become ferromagnetic. But the saturation magnetization of the Cr35Ni45Nb alloy tubes approaches to zero in the center zone. The primary carbides M7C3 and NbC are changed into M23C6 and G phase at the outer region of the furnace tube. However, the M23C6-type carbides were replaced by carbon-rich carbides M7C3 at the carburization zone. Cr-depleted zones are formed at the inner and outer walls of the furnace tubes owing to oxidation. Carburization and oxidation reduce the Cr content of the matrix. Accordingly, the saturation magnetization is very high at the carburization zone and Cr-depleted zone. The magnetism of Cr25Ni35Nb and Cr35Ni45Nb alloy tubes has a high correlation with the Cr content of the matrix. Carburization and oxidation are the main reasons that make the paramagnetic ethylene pyrolysis furnace tube change to ferromagnetic.

Highly luminescent thin films of zinc tungstate (ZT) have been deposited on top of conventional scintillators (Yttrium Aluminum Perovskite, Yttrium Aluminum Garnet) for electron detection in order to replace the need for a top conducting layer, such as indium tin oxide (ITO) or aluminum, which is non-scintillating and electron absorbing. Such conventional conducting layers serve the single purpose of eliminating electrical charge build-up on the scintillator. The ZT film also eliminates charging, which has been verified by measuring the Duane–Hunt limit and electron emission versus accelerating voltage. The luminescent nature of the ZT film ensures effective detection of low energy electrons from the very top surface of the structure ZT/scintillator, which we call “first-surface scintillator”. The cathodoluminescence has been measured directly with a photodetector and spectrally resolved at different accelerating voltages. All results demonstrate the extended range of operation of the first-surface scintillator, while the conventional scintillators with a top ITO layer decline below 5 kV and have practically no output below 2 kV. Scintillators of different types were integrated in a detection system for backscattered electrons (BSE). The quality of the image at high accelerating voltages is comparable with the conventional scintillator and commercial BSE detector, while the image quality at 1 kV from the first-surface scintillator is superior.

We present a deep learning approach to the indexing of electron backscatter diffraction (EBSD) patterns. We design and implement a deep convolutional neural network architecture to predict crystal orientation from the EBSD patterns. We design a differentiable approximation to the disorientation function between the predicted crystal orientation and the ground truth; the deep learning model optimizes for the mean disorientation error between the predicted crystal orientation and the ground truth using stochastic gradient descent. The deep learning model is trained using 374,852 EBSD patterns of polycrystalline nickel from simulation and evaluated using 1,000 experimental EBSD patterns of polycrystalline nickel. The deep learning model results in a mean disorientation error of 0.548° compared to 0.652° using dictionary based indexing.

Microscopic vision systems based on a stereo light microscope (SLM) are used in microscopic measuring fields. Conventional measuring methods output the disparity surface based on stereo matching methods; however, these methods require that stereo images contain sufficient distinguishing features. Moreover, matching results typically contain many mismatched points. This paper presents a novel method for disparity surface reconstruction by combining an SLM and laser measuring techniques. The surfaces of small objects are scanned by a laser fringe, and a stereo image sequence containing laser stripes is obtained. The central contours of the laser stripes are extracted, and central contours are derived for alignment. A disparity coordinate system is then defined and used to analyze the relationship between the motion direction and reference plane. Next, the method of aligning disparity contours is proposed. The results show that our method can achieve a precision of ±0.5 pixels and that the real and measured shapes described by the disparity surface are consistent based on our method. Our method is confirmed to perform much better than the conventional block-matching method. The disparity surface output obtained by our method can be used to measure the surface profiles of microscopic objects accurately.

Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass’ cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 μm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.

Correlative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur; and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples.

Transparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level of light scattering in their cytoplasm. It has been shown that both cell types contain (1) beaded intermediate filaments as well as (2) α-crystallin globulins. It is known that genetic and chemical alterations to these specialized proteins induce cytoplasmic opaqueness and visual complications. Crystallins were described previously in the retinal Müller cells of frogs. In the present work, using immunocytochemistry, fluorescence confocal imaging, and immuno-electron microscopy, we found that αA-crystallins are present in the cytoplasm of retinal Müller cells and in the photoreceptors of rats. Given that Müller glial cells were recently described as “living light guides” as were photoreceptors previously, we suggest that αA-crystallins, as in other highly transparent cells, allow Müller cells and photoreceptors to minimize intraretinal scattering during retinal light transmission.

Emerging evidence from various studies indicates that plasmid DNA (pDNA) is internalized by cells through an endocytosis-like process when it is used for electrotransfection. To provide morphological evidence of the process, we investigated ultrastructures in cells that were associated with the electrotransfected pDNA, using immunoelectron microscopy. The results demonstrate that four endocytic pathways are involved in the uptake of the pDNA, including caveolae- and clathrin-mediated endocytosis, macropinocytosis, and the clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched early endosomal compartment (CLIC/GEEC) pathway. Among them, macropinocytosis is the most common pathway utilized by cells having various pDNA uptake capacities, and the CLIC/GEEC pathway is observed primarily in human umbilical vein endothelial cells. Quantitatively, the endocytic pathways are more active in easy-to-transfect cells than in hard-to-transfect ones. Taken together, our data provide ultrastructural evidence showing that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA.

The production of Amarone wine is governed by a disciplinary guideline to preserve its typical features; however, postharvest infections by the fungus Botrytis cinerea (B. cinerea) not only represent a phytosanitary problem but also cause a significant loss of product. In this study, we tested a treatment with mild ozoniztion on grapes for Amarone wine production during withering in the fruttaio (the environment imposed by the disciplinary guideline) and evaluated the impact on berry features by a multimodal imaging approach. The results indicate that short and repeated treatments with low O3 concentrations speed up the naturally occurring berry withering, probably inducing a reorganization of the epicuticular wax layer, and inhibit the development of B. cinerea, blocking the fungus in an intermediate vegetative stage. This pilot study will pave the way to long-term research on Amarone wine obtained from O3-treated grapes.

Gilding threads collected from Spanish and Portuguese palaces and from the embroideries and adornments of sculptures of the Virgin and Christ that form part of Sevillian Holy Week were analyzed and compared (20 artifacts were evaluated). The study covered a broad time period with examples from the 13th to 14th centuries, 18th to 20th centuries, and also including modern embroideries. A combination of scanning electron microscopy and energy-dispersive X-ray spectroscopy was used. The knowledge of the layered structures of the threads has provided very valuable information regarding the manufacturing techniques. The different metal threads found in the embroidery studied consisted of gold, silver, copper, and alloys of these metals and aluminium. The fabrication procedures often differed in the different workshops and changed with time. In the modern embroideries, a decrease of precious metal concentration was detected. The threads were wound around a core of silk threads.