Ocular surface diseases (OSDs) represent a widely investigated field of research given their growing incidence and the negative impact on quality of life. During OSDs, cytokines generated by damaged epithelia trigger and deregulate the lymphoid cells composing the eye-associated lymphoid tissues, inducing an immune-mediated chronic inflammation that amplifies and propagates the disease during time. The conjunctiva-associated lymphoid tissue (CALT), given its particular position that permits immune cells covering the cornea, might play a crucial role in the development of OSDs. Despite the recognized inflammatory role of mucosa-associated lymphoid tissues in other stations taking contact with the external environment (gut or bronchus), CALT did not gain the deserved consideration. In the last years, the diffusion of the in vivo confocal microscopy (IVCM) stimulated the interest to CALT, especially in dry eye, ocular allergy, and glaucoma. Though the initial stimuli were different, IVCM documented similar changes, represented by increased lymphoid cells within the diffuse layer, follicles and interfollicular spaces. These findings, which need to be validated by immunohistology, support the CALT stimulation during OSDs. However, while an involvement of the CALT in OSDs is hypothesizable, the exact role of this structure in their pathogenesis remains unclear and warrants further investigations.

Micro-electro-mechanical systems (MEMS)-based heating holders offer exceptional control of temperature and heating/cooling rates for transmission electron microscopy experiments. The use of such devices is relatively straightforward for nano-particulate samples, but the preparation of specimens from bulk samples by focused ion beam (FIB) milling presents significant challenges. These include: poor mechanical integrity and site selectivity of the specimen, ion beam damage to the specimen and/or MEMS device during thinning, and difficulties in transferring the specimen onto the MEMS device. Here, we describe a novel FIB protocol for the preparation and transfer of specimens from bulk samples, which involves a specimen geometry that provides mechanical support to the electron-transparent region, while maximizing the area of that region and the contact area with the heater plate on the MEMS chip. The method utilizes an inclined stage block that minimizes exposure of the chip to the ion beam during milling. This block also allows for accurate and gentle placement of the FIB-cut specimen onto the chip by using simultaneous electron and ion beam imaging during transfer. Preliminary data from Si and Ag on Si samples are presented to demonstrate the quality of the specimens that can be obtained and their stability during in situ heating experiments.

A detailed knowledge of the atomic structure of magnetic semiconductors is crucial to understanding their electronic and magnetic properties, which could enable spintronic applications. Energy-dispersive X-ray spectrometry (EDX) in the scanning transmission electron microscope and atom probe tomography (APT) experiments reveal the formation of Cr-rich regions in Cd1−x Cr x Te layers grown by molecular beam epitaxy. These Cr-rich regions occur on a length scale of 6–10 nm at a nominal Cr composition of x=0.034 and evolve toward an ellipsoidal shape oriented along <111> directions at a composition of x=0.083. Statistical analysis of the APT reconstructed volume reveals that the Cr aggregation increases with the average Cr composition. The correlation with the magnetic properties of such (Cd,Cr)Te layers is discussed within the framework of strongly inhomogeneous materials. Finally, difficulties in accurately quantifying the Cr distribution in the CdTe matrix on an atomic scale by EDX and APT are discussed.

The morphology, composition, and structure of precipitates in an Al–Si–Mg–Hf alloy after heat treatment at 560°C for 20 h were studied by means of Cs-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), energy dispersive X-ray spectrometry (EDS), high-resolution transmission electron microscopy (HRTEM), and first-principle calculations. Precipitates with three kinds of morphologies were observed. The rectangular and square precipitates were predominantly (Si2−xAlx)Hf phases, while the nanobelt-like precipitate is the Si2Hf phase. First-principle calculations were used to show that the Si6 and Si8 sites were the most favorable sites for Al incorporation in the orthorhombic Si2Hf phase.

We demonstrate that the surface topography of a sample can be reconstructed from electron backscatter diffraction (EBSD) patterns collected with a commercial EBSD system. This technique combines the location of the maximum background intensity with a correction from Monte Carlo simulations to determine the local surface normals at each point in an EBSD scan. A surface height map is then reconstructed from the local surface normals. In this study, a Ni sample was machined with a femtosecond laser, which causes the formation of a laser-induced periodic surface structure (LIPSS). The topography of the LIPSS was analyzed using atomic force microscopy (AFM) and reconstructions from EBSD patterns collected at 5 and 20 kV. The LIPSS consisted of a combination of low frequency waviness due to curtaining and high frequency ridges. The morphology of the reconstructed low frequency waviness and high frequency ridges matched the AFM data. The reconstruction technique does not require any modification to existing EBSD systems and so can be particularly useful for measuring topography and its evolution during in situ experiments.

The capability to perform liquid in situ transmission electron microscopy (TEM) experiments provides an unprecedented opportunity to examine the real-time processes of physical and chemical/electrochemical reactions during the interaction between metal surfaces and liquid environments. This work describes the requisite steps to make the technique fully analytical, from sample preparation, through modifications of the electrodes, characterization of electrolytes, and finally to electrochemical corrosion experiments comparing in situ TEM to conventional bulk cell and microcell configurations.

In this study we compare two thermal annealing series of III/V semiconductor heterostructures on Si, where during the first series nitrogen is present in the in situ holder. The second, comparative, measurement is done in a tertiarybutylphosphine (TBP) environment. The sample annealed in a TBP environment shows favorable thermal stability up to 500°C compared to the unstabilized sample, which begins to degrade at less than 300°C. Evaporation of P from the material is tracked qualitatively by measuring the thickness of the sample during thermal annealing with and without stabilization. Finally, we investigate the in situ thermal annealing processes at atomic resolution. Here it is possible to study phase separation as well as the diffusion of As from a Ga(NAsP) quantum well in the surrounding GaP material during thermal annealing. To make these investigations possible we developed an extension for our in situ transmission electron microscopy setup for the safe usage of toxic and pyrophoric III/V semiconductor precursors. A commercially available gas cell and gas supply system were expanded with a gas mixing system, an appropriate toxic gas monitoring system and a gas scrubbing system. These components allow in situ studies of semiconductor growth and annealing under the purity conditions required for these materials.

The recent technological advance of the gas field ion source (GFIS) and its successful integration into systems has renewed the interest in the focused ion beam (FIB) technology. Due to the atomically small source size and the use of light ions, the limitations of the liquid metal ion source are solved as device dimensions are pushed further towards the single-digit nanometer size. Helium and neon ions are the most widely used, but a large portfolio of available ion species is desirable, to allow a wide range of applications. Among argon and hydrogen, $${\rm N}_{2}^{{\plus}} $$ ions offer unique characteristics due to their covalent bond and their use as dopant for various carbon-based materials including diamond. Here, we provide a first look at the $${\rm N}_{2}^{{\plus}} $$ GFIS-FIB enabled imaging of a large selection of microscopic structures, including gold on carbon test specimen, thin metal films on insulator and nanostructured carbon-based devices, which are among the most actively researched materials in the field of nanoelectronics. The results are compared with images acquired by He+ ions, and we show that $${\rm N}_{2}^{{\plus}} $$ GFIS-FIB can offer improved material contrast even at very low imaging dose and is more sensitive to the surface roughness.

The xenon plasma focused ion beam instrument (PFIB), holds significant promise in expanding the applications of focused ion beams in new technology thrust areas. In this paper, we have explored the operational characteristics of a Tescan FERA3 XMH PFIB instrument with the aim of meeting current and future challenges in the semiconductor industry. A two part approach, with the first part aimed at optimizing the ion column and the second optimizing specimen preparation, has been undertaken. Detailed studies characterizing the ion column, optimizing for high-current/high mill rate activities, have been described to support a better understanding of the PFIB. In addition, a novel single-crystal sacrificial mask method has been developed and implemented for use in the PFIB. Using this combined approach, we have achieved high-quality images with minimal artifacts, while retaining the shorter throughput times of the PFIB. Although the work presented in this paper has been performed on a specific instrument, the authors hope that these studies will provide general insight to direct further improvement of PFIB design and applications.

This paper reports on the substantial improvement of specimen quality by use of a low voltage (0.05 to ~1 keV), small diameter (~1 μm), argon ion beam following initial preparation using conventional broad-beam ion milling or focused ion beam. The specimens show significant reductions in the amorphous layer thickness and implanted artifacts. The targeted ion milling controls the specimen thickness according to the needs of advanced aberration-corrected and/or analytical transmission electron microscopy applications.

Sub-angstrom scanning transmission electron microscopy (STEM) allows quantitative column-by-column analysis of crystalline specimens via annular dark-field images. The intensity of electrons scattered from a particular location in an atomic column depends on the intensity of the electron probe at that location. Electron beam channeling causes oscillations in the STEM probe intensity during specimen propagation, which leads to differences in the beam intensity incident at different depths. Understanding the parameters that control this complex behavior is critical for interpreting experimental STEM results. In this work, theoretical analysis of the STEM probe intensity reveals that intensity oscillations during specimen propagation are regulated by changes in the beam’s angular distribution. Three distinct regimes of channeling behavior are observed: the high-atomic-number (Z) regime, in which atomic scattering leads to significant angular redistribution of the beam; the low-Z regime, in which the probe’s initial angular distribution controls intensity oscillations; and the intermediate-Z regime, in which the behavior is mixed. These contrasting regimes are shown to exist for a wide range of probe parameters. These results provide a new understanding of the occurrence and consequences of channeling phenomena and conditions under which their influence is strengthened or weakened by characteristics of the electron probe and sample.

Beam damage is a major limitation in electron microscopy that becomes increasingly severe at higher resolution. One possible route to circumvent radiation damage, which forms the basis for single-particle electron microscopy and related techniques, is to distribute the dose over many identical copies of an object. For the acquisition of low-dose data, ideally no dose should be applied to the region of interest before the acquisition of data. We present an automated approach that can collect large amounts of data efficiently by acquiring images in a user-defined area-of-interest with atomic resolution. We demonstrate that the stage mechanics of the Nion UltraSTEM, combined with an intelligent algorithm to move the sample, allow the automated acquisition of atomically resolved images from micron-sized areas of a graphene substrate. Moving the sample stage automatically in a regular pattern over the area-of-interest enables the collection of data from pristine sample regions without exposing them to the electron beam before recording an image. Therefore, it is possible to obtain data with minimal dose (no prior exposure during focusing), which is only limited by the minimum signal needed for data processing. This enables us to minimize beam-induced damage in the sample and to acquire large data sets within a reasonable amount of time.

Trefoil factor family 3 (Tff3) peptide is present during intrauterine endochondral ossification in mice, and its deficiency affects cancellous bone quality in secondary ossification centers of mouse tibiae. The aim of this study was to quantitatively analyze parameters describing the growth plate and primary ossification centers in tibiae of 1-month-old wild-type and Tff3 knock-out mice (n=5 per genotype) by using free and open-source software. Digital photographs of the growth plates and trabecular bone were processed by open-source computer programs GIMP and FIJI. Histomorphometric parameters were calculated using measurements made with FIJI. Tff3 knock-out mice had significantly smaller trabecular number and significantly larger trabecular separation. Trabecular bone volume, trabecular bone surface, and trabecular thickness showed no significant difference between the two groups. Although such histomorphological differences were found in the cancellous bone structure, no significant differences were found in the epiphyseal plate histomorphology. Tff3 peptide probably has an effect on the formation and quality of the cancellous bone in the primary ossification centers, but not through disrupting the epiphyseal plate morphology. This work emphasizes the benefits of using free and open-source programs for morphological studies in life sciences.

Biological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter, κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

We present the first demonstration of a general method for the chemical characterization of small surface features at high magnification via simultaneous collection of mass spectrometry (MS) imaging and tandem MS imaging data. High lateral resolution tandem secondary ion MS imaging is employed to determine the composition of surface features on poly(ethylene terephthalate) (PET) that precipitate during heat treatment. The surface features, probed at a lateral resolving power of<200 nm using a surface-sensitive ion beam, are found to be comprised of ethylene terephthalate trimer at a greater abundance than is observed in the surrounding polymer matrix. This is the first chemical identification of PET surface precipitates made without either an extraction step or the use of a reference material. The new capability employed for this study achieves the highest practical lateral resolution ever reported for tandem MS imaging.

Coenzyme Q10 (Q10) plays an important role in the cellular antioxidant system by protecting the cells from free-radical oxidative damage and apoptosis. In the present study, we have investigated the effect of Q10 on the preimplantation development of porcine parthenogenetic embryos, as well as the underlying mechanism. The results showed that 100 μM was the optimal concentration of Q10, which resulted in significantly increased cleavage and blastocyst formation rates and improvement of blastocyst quality. Q10 improved the blastocyst hatching rate and cellular proliferation rate in hatching blastocysts and increased the expression of hatching-related genes. Furthermore, Q10 not only decreased reactive oxygen species production, DNA damage levels, and apoptosis in the blastocysts from H2O2-induced oxidative injury, but also maintained mitochondrial function. Taken together, these results indicate that Q10 has beneficial effects on the development of porcine parthenogenetic embryos by preventing oxidative damage and apoptosis.

Vascular stenosis, the abnormal narrowing of blood vessels, arises from defective developmental processes or atherosclerosis-related adult pathologies. Stenosis triggers a series of adaptive cellular responses that induces adverse remodeling, which can progress to partial or complete vessel occlusion with numerous fatal outcomes. Despite its severity, the cellular interactions and biophysical cues that regulate this pathological progression are poorly understood. Here, we report the design and fabrication of a three-dimensional (3D) in vitro system to model vascular stenosis so that specific cellular interactions and responses to hemodynamic stimuli can be investigated. Tubular cellularized constructs (cytotubes) were produced, using a collagen casting system, to generate a stenotic arterial model. Fabrication methods were developed to create cytotubes containing co-cultured vascular cells, where cell viability, distribution, morphology, and contraction were examined. Fibroblasts, bone marrow primary cells, smooth muscle cells (SMCs), and endothelial cells (ECs) remained viable during culture and developed location- and time-dependent morphologies. We found cytotube contraction to depend on cellular composition, where SMC-EC co-cultures adopted intermediate contractile phenotypes between SMC- and EC-only cytotubes. Our fabrication approach and the resulting artery model can serve as an in vitro 3D culture system to investigate vascular pathogenesis and promote the tissue engineering field.

This letter describes an innovative spin-coating system, built from off-the-shelf components, that can easily and inexpensively be integrated into any laboratory environment. Combined with a liquid suspension of conductive polymer, such a “rotary coater” enables simple coating of planar samples to create a physical protective barrier on the sample surface. This barrier aids in charge dissipation during scanning electron microscope and focused ion beam (FIB) imaging and provides wide-scale protection of the sample surface from ion bombardment during FIB imaging and gas-assisted deposition. This polymer layer replaces the localized and time-consuming electron beam deposition step typically performed during transmission electron microscopy lamella preparation. After observation, the coating can be easily removed, if desired. The described spin-coating procedure has minimal cost while providing repeatable positive results, without the need for expensive commercial coating instrumentation.

The history and iconography of Swiss stained glass dating between the 16th and 18th centuries are well studied. However, the chemical and morphological characteristics of the glass and glass paints, particularly the nature of the raw materials, the provenance of the glass, and the technology used to produce it are less well understood. In this paper, we studied two sets of samples from stained-glass panels attributed to Switzerland, which date from the 16th to 17th centuries: the first set comes from Pena National Palace collection, the second from Vitrocentre Romont. The aims were to identify the materials used in the production of the glass, to find out more about their production origin and to characterize the glass paints. Both glass and the glass paints were analysed by particle-induced X-ray emission; the paints were additionally analysed by scanning electron microscopy–electron-dispersive X-ray spectroscopy (SEM-EDS). The results show that the glass from both sets was probably produced in the same region and that wood ash was used as a fluxing agent. Different recipes have been used to make the blue enamels. However, the cobalt ore used as a coloring agent in all of the blue enamels came from the mining district in Schneeberg, Germany.