In situ liquid cell transmission electron microscopy (LC-TEM) is an innovative method for studying the processes involved in the formation of crystals in liquids. However, it is difficult to capture early stages of crystallization because of the small field of view and the unfavorable changes in sample composition resulting from electron-beam radiolysis. Nevertheless, if the radiolysis required to induce the crystallization of a sample could be controlled in LC-TEM, this would be advantageous for observing the crystallization process. Here, we examined this possibility by using a mixture of sodium chlorate (NaClO3) and acetone in the LC-TEM. The electron beam induced the formation of dendritic crystals in a saturated acetone solution of NaClO3; moreover, these crystals consisted of sodium chloride (NaCl), rather than NaClO3, suggesting that chloride ions (Cl−), which were not present in the initial solution, were generated by radiolysis of chlorate ions ${\rm ( ClO}_3^- )$. As a result, the solution then supersaturated with NaCl because its solubility in acetone is much lower than that of NaClO3. The combination of radiolysis and a solvent in which a solute is much less soluble is potentially useful for establishing crystallization conditions for materials that are difficult to crystallize directly in LC-TEM experiments.

Electron-probe microanalysis of uranium and uranium alloys poses several problems, such as rapid oxidation, large poorly constrained correction factors, and a large number of characteristic x-ray lines. We show that U-metal can grow 10 nm of oxide within ~20 s of air exposure, increasing to 15–20 nm within a few minutes, which can produce a 30% quantification error at 5 kV. A 15 nm carbon coating on the UO2 reference material also produces an ~30% quantification error of the uncoated but surface oxidized U sample at 5 kV. Correcting for both the coating and oxide improved the analysis accuracy to better than ±1% down to 7 kV and ~2% at 5 kV, but the error increases strongly below this. The measurement of C in U identified a previously unreported U N6–O4 line interference on the C Kα peak, which can produce over 1% error in the analysis total. Oxide stoichiometry was demonstrated to have only a small impact on quantification. The measurement of the O Kα and U Mα mass absorption coefficients in U as 9,528 and 798 cm2/g, respectively, shows good agreement with recently published values and also produces small differences in a quantification error.

Determining free lime content in steelmaking slag is crucial for its safe reuse in road construction. A simple method has been recently developed to rapidly derive this value via cathodoluminescence (CL) imaging of steelmaking slag, previously quenched from 1,000°C to room temperature, according to the illuminated areas corresponding to free lime (luminescence peak at 600 nm). This quenching is required to obtain intense CL from free lime, but the mechanism of such signal enhancement is still unknown. Therefore, the present study investigated the mechanism by comparing the microstructures, CL images, and CL spectra of free lime in quenched and unquenched steelmaking slag. Large amounts of defects, including dislocations, were observed in the free lime emitting intense luminescence at 600 nm, whereas the samples without clear CL exhibited only a few defects. These results and previous studies suggest that the luminescence at 600 nm from free lime is enhanced by the CL originating from oxygen vacancies (380 nm); therefore, the enhancement of the intensity of the free lime CL peak could be attributed to the increase in the oxygen vacancies via quenching from 1,000°C to room temperature.

High-temperature differential scanning calorimetry was used to understand the thermal properties of Si-rich metal–silicon alloys. Insoluble metals (A and B) were found to produce an alloy with discrete ASi2 and BSi2 dispersed phases. In contrast, metals that form a solid solution result in a dispersed phase that has a composition of AxB1−xSi2, where x varies continuously across each inclusion. This complex composition distribution is putatively caused by differences in the solidification temperatures of ASi2 versus BSi2. Though this behavior was observed for several different combinations of metals, we focus here specifically on the Cr/V/Si system. To better understand the range and most probable element concentrations in the dispersed silicide domains, a method was devised to generate histograms of their Cr and V concentrations from energy-dispersive X-ray spectroscopy hyperspectral images. Varying the Cr/V/Si ratio was found to change the shape of the element histograms, indicating that the distribution of silicide compositions that form is controlled by the input composition. Adding aluminum was found to result in dispersed phases that had a single composition rather than a range of Cr and V concentrations. This demonstrates that aluminum can be an effective additive for altering solidification kinetics in silicon alloys.

Local magnification artifacts in atom probe tomography (APT) caused by multiphase materials with heterogeneous evaporation behavior are a well-known problem. In particular, the analysis of the exact size, shape, and composition of small precipitates is, therefore, not trivial. We performed numerical simulations of APT measurements to predict the reconstructed morphology of precipitates with contrasting evaporation thresholds. Based on a statistical approach that avoids coarse graining, the simulated data are evaluated to develop a model for the calculation of the original size of the precipitates. The model is tested on experimental APT data of precipitates with a higher and lower evaporation field in a ferritic alloy. Accurate sizes, proven by a complementary investigation by transmission electron microscopy, are obtained. We show further, how the size information can be used to obtain compositional information of the smallest precipitates and present a new methodology to determine a correct in-depth scaling of the APT reconstruction in case no complementary geometric information about the specimen exists or if no lattice planes are visible in the reconstruction.

This work introduces NexusLIMS, an electron microscopy laboratory information management system designed and implemented by the Office of Data and Informatics and the Materials Science and Engineering Division at NIST for a multi-user electron microscopy co-op facility. NexusLIMS comprises network infrastructure, shared information technology resources, a custom software package to harvest and extract experimental information and construct experimental metadata records, and an intuitive web-based user-facing interface for searching, browsing, and examining research data. These metadata records conform to the Nexus Experiment schema, which is introduced in this work. The NexusLIMS suite of tools requires minimal input and adjustments to user behavior, instead relying on existing organizational procedures and the collection of information from a multitude of sources to construct a complete picture and record of a research experiment. The underlying infrastructure and design considerations for a multi-user data management system are discussed. The core codebase for NexusLIMS is made publicly available alongside this work, and its modular design encourages the adaptation of the presented methods at other research organizations.

The invention of silicon drift detectors has resulted in an unprecedented improvement in detection efficiency for energy-dispersive X-ray (EDX) spectroscopy in the scanning transmission electron microscope. The result is numerous beautiful atomic-scale maps, which provide insights into the internal structure of a variety of materials. However, the task still remains to understand exactly where the X-ray signal comes from and how accurately it can be quantified. Unfortunately, when crystals are aligned with a low-order zone axis parallel to the incident beam direction, as is necessary for atomic-resolution imaging, the electron beam channels. When the beam becomes localized in this way, the relationship between the concentration of a particular element and its spectroscopic X-ray signal is generally nonlinear. Here, we discuss the combined effect of both spatial integration and sample tilt for ameliorating the effects of channeling and improving the accuracy of EDX quantification. Both simulations and experimental results will be presented for a perovskite-based oxide interface. We examine how the scattering and spreading of the electron beam can lead to erroneous interpretation of interface compositions, and what approaches can be made to improve our understanding of the underlying atomic structure.

The ability to accurately and precisely measure the thickness of biomaterial constructs is critical for characterizing both specific dimensional features and related mechanical properties. However, in the absence of a standardized approach for thickness measurements, a variety of imaging modalities have been employed, which have been associated with varying limits of accuracy, particularly for ultrathin hydrated structures. Electron microscopy (EM), a commonly used modality, yields thickness values for extensively processed and nonhydrated constructs, potentially resulting in overestimated mechanical properties, including elastic modulus and ultimate tensile strength. Confocal laser scanning microscopy (CLSM) has often been used as a nondestructive imaging alternative. However, published CLSM-derived image analysis protocols use arbitrary signal intensity cutoffs and provide minimal information regarding thickness variability across imaged surfaces. To address the aforementioned limitations, we present a standardized, user-independent CLSM image acquisition and analysis approach developed as a custom ImageJ macro and validated with collagen-based scaffolds. In the process, we also quantify thickness discrepancies in collagen-based scaffolds between CLSM and EM techniques, further illustrating the need for improved strategies. Employing the same image acquisition protocol, we also demonstrate that this approach can be used to estimate the surface roughness of the same scaffolds without the use of specialized instrumentation.

In the field of transmission electron microscopy, data interpretation often lags behind acquisition methods, as image processing methods often have to be manually tailored to individual datasets. Machine learning offers a promising approach for fast, accurate analysis of electron microscopy data. Here, we demonstrate a flexible two-step pipeline for the analysis of high-resolution transmission electron microscopy data, which uses a U-Net for segmentation followed by a random forest for the detection of stacking faults. Our trained U-Net is able to segment nanoparticle regions from the amorphous background with a Dice coefficient of 0.8 and significantly outperforms traditional image segmentation methods. Using these segmented regions, we are then able to classify whether nanoparticles contain a visible stacking fault with 86% accuracy. We provide this adaptable pipeline as an open-source tool for the community. The combined output of the segmentation network and classifier offer a way to determine statistical distributions of features of interest, such as size, shape, and defect presence, enabling the detection of correlations between these features.

The quantification of the particle size and the number concentration (PNC) of nanoparticles (NPs) is key for the characterization of nanomaterials. Transmission electron microscopy (TEM) is often considered as the gold standard for assessing the size of NPs; however, the TEM sample preparation suitable for estimating the PNC based on deposited NPs is challenging. Here, we use an ultrasonic nebulizer (USN) to transfer NPs from aqueous suspensions into dried aerosols which are deposited on TEM grids in an electrostatic precipitator of an aerosol monitor. The deposition efficiency of the electrostatic precipitator was ≈2%, and the transport efficiency of the USN was ≈7%. Experiments using SiO2 NPs (50–200 nm) confirmed an even deposition of the nebulized particles in the center of the TEM grids. PNCs of the SiO2 NPs derived from TEM images underestimated the expected PNCs of the suspensions by a factor of up to three, most likely resulting from droplet coagulation and NP aggregation in the USN. Nevertheless, single particles still dominated the PNC. Our approach results in reproducible and even deposition of particles on TEM grids suitable for morphological analysis and allows an estimation of the PNC in the suspensions based on the number of particles detected by TEM.

Renal aging is a progressive, physiological, and anatomical change that naturally occurs in all animal species. To date, no information is available concerning the aging-related structural and functional changes in camel kidneys. A total of 25 healthy male camels (14 aged 4–6 years and 11 aged 18–22 years) were included in this study. After the camels were slaughtered, samples were collected from all the camels’ kidneys and prepared for histopathological, immunohistochemical, and gene expression evaluations. The most striking observation was the significant decline in the immunohistochemical abundance of podocin and the significant upregulation of smoothening in the aging camels’ kidneys. However, the nonsignificant changes have reported for nephrin, calbindin, autophagy 5 (ATG5), aquaporin 1, and toll-like receptor 9. Furthermore, the mRNA expressions of sirtuin 1, superoxide dismutase 1, superoxide dismutase 2, peroxisome proliferator-activated receptor alpha, B-cell lymphoma 2 (Bcl-2), and erythropoietin were significantly decreased in the aging camels’ kidneys. While the significant upregulation of Bcl-2-associated X protein and the nonsignificant increase in ATG5 expression levels were reported in the aging camels’ kidneys. The present findings provide better understanding of the complex events and initiating factors of aging, allowing for the development of a future therapeutic strategy to preserve adequate renal function throughout life.

Smooth muscle cells (SMCs) are dynamic and transition from a contractile to a synthetic phenotype under different circumstances. Plasma factors (fibrin and transforming growth factors, TGFs) are possible components affecting SMCs differentiation and behavior. Thus, the objective of this work was to investigate how the fibrin matrix and TGFs affect SMCs differentiation and motility behavior. SMCs invaded the fibrin gel and adopted a stellate phenotype while reducing the expression of differentiation markers (Acta2, Myh11, and Smtn). At the ultrastructural level, SMCs did not assemble a basal lamina and showed numerous blebs along the entire cell surface. This transition was not associated with changes in focal adhesion kinase (FAK) content and phosphorylation status but reflected a marked change in FAK distribution in the cytoplasm. After 48 h in culture, SMCs caused an active degradation of the fibrin gel. Additionally, we tested the SMCs response to TGFs in a cell layer wound repair assay. TGFα, but not TGFβ1 or TGFβ3, had significantly increased motility. In conclusion, prostatic SMCs present a phenotypical transition when cultured on fibrin, adopting a micro-blebbing based motility behavior and increasing migration in response to TGFα.

Engineered biomaterials provide unique functions to overcome the bottlenecks seen in biomedicine. Hence, a technique for rapid and routine tests of collagen is required, in which the test items commonly include molecular weight, crosslinking degree, purity, and sterilization induced structural change. Among them, the crosslinking degree mainly influences collagen properties. In this study, second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy are used in combination to explore the collagen structure at molecular and macromolecular scales. These measured parameters are applied for the classification and quantification among the different collagen scaffolds, which were verified by other conventional methods. It is demonstrated that the crosslinking status can be analyzed from SHG images and presented as the coherency of collagen organization that is correlated with the mechanical properties. Also, the comparative analyses of SHG signal and relative CARS signal of amide III band at 1,240 cm−1 to δCH2 band at 1,450 cm−1 of these samples provide information regarding the variation of the molecular structure during a crosslinking process, thus serving as nonlinear optical signatures to indicate a successful crosslinking.

Obesity is characterized by excess adipose tissue and chronic inflammation and promotes extensive changes that can compromise skeletal muscles’ structural and functional integrity. Obesity can seriously impact the force transmission region between the muscle and the tendon, the myotendinous junction (MTJ). The present study aimed to investigate the plasticity of muscle fibers and MTJ regions in high-fat diet-induced obesity in rat tibialis anterior (TA) and soleus (SO) muscles. Wistar rats were divided into control and obese groups (induced by a high-fat diet). The samples of TA and SO muscles were prepared for histochemical and ultrastructural analysis (sarcomeres and MTJ projection). In the muscle fiber, similar adaptations were observed between the muscles of the smaller fiber (types I and IIa) in the obesity results. The MTJ region demonstrated different adaptations between the analyzed muscles. The TA–MTJ region has shorter ultrastructures, while in the SO–MTJ region, the ultrastructures were larger. We conclude that obesity induced by a high-fat diet promotes similar adaptation in the muscle fibers; however, in the MTJ region, the sarcoplasmatic projections and adjacent sarcomere demonstrate different adaptations according to distinct muscle phenotypes.

Listeriosis is a disease that is induced by infection with the Gram-positive bacterium Listeria monocytogenes. Much is still unknown about the pathogenesis of encephalitic listeriosis. We aimed to identify the contribution of glial fibrillary acidic protein (GFAP), epithelial cadherin (E-cadherin), S100, and acute-phase proteins (APPs) in pathogenesis, clinical and preclinical diagnosis of natural cases of encephalitic listeriosis. Of 1,325 Ossimi sheep, 64 were suspected of having listeriosis from 2018 to 2020. Prospective cohort evaluation of clinical and postmortem findings was performed, in addition to bacterial isolation, the measurement of APPs in serum and cerebrospinal fluid (CSF), and the histopathological and immunohistochemical evaluation of GFAP, S100, and E-cadherin. Infected sheep showed nervous symptoms ranging from neck stretching to complete paralysis. APPs were significantly increased in the CSF of both clinically and preclinically diseased animals; however, serum APPs were only significantly increased in clinically diseased animals. Histopathological evaluation revealed microabscesses, meningoencephalitis, and perivascular cuffing of the brainstem of infected sheep. Immunohistochemical investigations revealed strong expression of GFAP and S100 in necrotic areas and negative expression of E-cadherin. The measurement of CSF APPs could be useful in the preclinical diagnosis of sheep listeriosis. GFAP and S100 proteins could be involved in the pathogenesis of listeriosis; however, E-cadherin does not appear to be involved.

Tramadol is used worldwide and is listed in many medical guidelines to treat both acute and chronic pains. There is a growing evidence of abuse of tramadol in some African and West Asian countries. Tramadol has some side effects. The present study designed to follow up the treatment of the cellular responses which might be induced in the kidney of tramadol mice. Treated mice received daily injection of tramadol dose (125 μg/100 g b.wt) for 20 and 40 days. Other mice received tramadol for 40 days and then were divided into three groups: the first received distilled water, the second received Lagenaria siceraria, and the third received melatonin daily for 40 days. Both the daily injection of tramadol for 20 and 40 days resulted in radical, extensive, and severe alterations in the normal histological architecture of the kidney. Treatment with Lagenaria siceraria or melatonin after tramadol administration for a long-term, markedly changed the collagen content and other chemical components, that may reach nearly normal levels. Such findings propose that although tramadol has many cytological and histopathological side effects on the kidneys of male mice, the treatments via Lagenaria siceraria and melatonin have effective therapeutic impacts on the tramadol side effects.

The mineral composition of eggshells is assumed to be a conserved phylogenetic feature. Avian eggshells are composed of calcite, whereas those of taxa within Chelonia are aragonitic. Yet, the eggshells of a passerine bird were reported to be made of aragonite. Here, we report a new study of the same bird eggshells using a combination of in situ microscopy and chemical techniques. A microstructural analysis finds a similar arrangement to other avian eggshells, despite their very thin and fragile nature. Fourier transform infrared spectrometry (FTIR) and electron backscatter diffraction (EBSD) results also confirm that the eggshells are entirely composed of calcite. Our findings demonstrate that passerine eggshells are not an exception and similar to other birds and reinforce the phylogenetic placement of this bird species.

The palate is a fundamental region in food swallowing and presents different adaptations in species. This research aimed to describe structural and ultrastructural characteristics of the palatine epithelium and the connective tissue cores (CTCs) of ten red-rumped agoutis (Dasyprocta leporina—Linnaeus, 1758) using macroscopic, light microscopy, scanning electron microscopy, and transmission electron microscopy. We found nine palatine ridges in the diastema and hard palate, and a smooth surface in the soft palate. Stratified squamous keratinized epithelium with projections of lamina propria and soft palate had gland clusters. Epithelial removal revealed CTCs with a conical shape with high density in the hard palate and the sides of the soft palate. Near the CTCs were nerve fibers in the hard palate, and the soft palate had muscular tissue below the gland clusters. The structural and ultrastructural characteristics enable stability of the hard palate and fixation to the soft palate sides, while the soft palate center has greater mobility thus assisting in food swallowing. We concluded that structural characteristics are similar to other mammals, although the morphology of agouti's palate differs in the amount and disposition of palatine ridges, and the conical CTC's morphology.

This study presents the oocyte development of Poecilimon ataturki Ünal, 1999 (Orthoptera, Tettigoniidae) with histology, morphology, and histochemistry by using a stereomicroscope, a light microscope, a scanning electron microscope, and a transmission electron microscope. The ovary in this species is a panoistic type which contains many ovarioles which consist of terminal filament, germarium, and vitellarium. Germarium is the region that has undifferentiated cells which generate the oocytes and follicular cells. In the vitellarium region, yolk granules start to cover the whole oocyte. In histochemical studies, to determine the content of the yolk granules, proteins, and carbohydrates in oocytes were treated with a bromophenol blue (BPB) method, a mercury bromophenol blue (mBPB) method, and a periodic acid Schiff (PAS) method, respectively. As a result of these methods, the yolk granules gave positive results in ovariole sections treated with the PAS and the BPB, while the mBPB staining was negative.