TEAM I is the final product of the Transmission Electron Aberration-corrected Microscope (TEAM) Project, a collaborative project funded by the Department of Energy with the goal of designing and building a platform for a next generation aberration-corrected electron microscope capable of image resolution of up to 50 pm. The TEAM instrument incorporates a number of new technologies, including spherical- and chromatic-aberration correction, an all-piezo-electric sample stage and an active-pixel direct electron detector. This article describes the functionality of this advanced instrumentation, its response to changes in environment or operating conditions, and its stability during daily operation within the National Center for Electron Microscopy user facility.

Within the central nervous system (CNS), antigen-presenting cells (APCs) play a critical role in orchestrating inflammatory responses where they present CNS-derived antigens to immune cells that are recruited from the circulation to the cerebrospinal fluid, parenchyma, and perivascular space. Available data indicate that APCs do so indirectly from outside of CNS vessels without direct access to luminal contents. Here, we applied high-resolution, dynamic intravital two-photon laser scanning microscopy to directly visualize extravascular CX3CR1+ APC behavior deep within undisrupted CNS tissues in two distinct anatomical sites under three different inflammatory stimuli. Surprisingly, we observed that CNS-resident APCs dynamically extend their cellular processes across an intact vessel wall into the vascular lumen with preservation of vessel integrity. While only a small number of APCs displayed intravascular extensions in intact, noninflamed vessels in the brain and the spinal cord, the frequency of projections increased over days in an experimental autoimmune encephalomyelitis model, whereas the number of projections remained stable compared to baseline days after tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma. Our observation of this unique behavior by parenchyma CX3CR1+ cells in the CNS argues for further exploration into their functional role in antigen sampling and immune cell recruitment.

Focused ion beam (FIB) milling using high-energy gallium ions is widely used in the preparation of specimens for transmission electron microscopy (TEM). However, the energetic ion beam induces amorphization on the edge of specimens during milling, resulting in a mischievous influence on the clearness of high-quality transmission electron micrographs. In this work, the amorphization induced by the FIB milling was investigated by TEM for three kinds of materials, metallic materials in bulk shape, and semiconductive and electronic ceramic materials as a substrate for the deposition of thin films.

The effect of heavy metal in fish has been the focus of extensive research for many years. However, the combined effect of heavy metals and nanomaterials is still a new subject that needs to be studied. The aim of this study was to examine histopathologic alterations in the gills of Nile tilapia (Oreochromis niloticus) to determine possible effects of lead (Pb), carbon nanotubes, and Pb+carbon nanotubes on their histological integrity, and if this biological system can be used as a tool for evaluating water quality in monitoring programs. For this, tilapia were exposed to Pb, carbon nanotubes and Pb+carbon nanotubes for 4 days. The main alterations observed were epithelial structure, hyperplasia and displacement of epithelial cells, and alterations of the structure and occurrence of aneurysms in the secondary lamella. The most severe alterations were related to the Pb+carbon nanotubes. We conclude that the oxidized multi-walled carbon nanotubes enhanced the acute lead toxicity in Nile tilapias. This work draws attention to the implications of carbon nanomaterials released in the aquatic environment and their interaction with classical pollutants.

This aim of this study was to assess the discriminatory value of fractal and grey level co-occurrence matrix (GLCM) analysis methods in standard microscopy analysis of two histologically similar brain white mass regions that have different nerve fiber orientation. A total of 160 digital micrographs of thionine-stained rat brain white mass were acquired using a Pro-MicroScan DEM-200 instrument. Eighty micrographs from the anterior corpus callosum and eighty from the anterior cingulum areas of the brain were analyzed. The micrographs were evaluated using the National Institutes of Health ImageJ software and its plugins. For each micrograph, seven parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, GLCM variance, fractal dimension, and lacunarity. Using the Receiver operating characteristic analysis, the highest discriminatory value was determined for inverse difference moment (IDM) (area under the receiver operating characteristic (ROC) curve equaled 0.925, and for the criterion IDM≤0.610 the sensitivity and specificity were 82.5 and 87.5%, respectively). Most of the other parameters also showed good sensitivity and specificity. The results indicate that GLCM and fractal analysis methods, when applied together in brain histology analysis, are highly capable of discriminating white mass structures that have different axonal orientation.

Ligament healing of a grade III injury (i.e., a complete tear) involves a multifaceted chain of events that forms a neoligament, which is more scar-like in character than the native tissue. The remodeling process may last months or even years with the injured ligament never fully recovering pre-injury mechanical properties. With tissue engineering and regenerative medicine, understanding the normal healing process in ligament and quantifying it provide a basis to create and assess innovative treatments. Ligament fibroblasts produce a number of extracellular matrix (ECM) components, including collagen types I and III, decorin and fibromodulin. Using a combination of advanced histology, molecular biology, and nonlinear optical imaging approaches, the early ECM events during ligament healing have been better characterized and defined. First, the dynamic changes in ECM factors after injury are shown. Second, the factors associated with creeping substitution are identified. Finally, a method to quantify collagen organization is developed and used. Each ECM factor described herein as well as the temporal quantification of fiber organization helps elucidate the complexity of ligament healing.

Electron microprobe trace element analysis is a significant challenge. Due to the low net intensity of peak measurements, the accuracy and precision of such analyses relies critically on background measurements, and on the accuracy of any pertinent peak interference corrections. A linear regression between two points selected at appropriate background positions is a classical approach for electron probe microanalysis (EPMA). However, this approach neglects the accurate assessment of background curvature (exponential or polynomial), and the presence of background interferences, a hole in the background, or an absorption edge can dramatically affect the results if underestimated or ignored. The acquisition of a quantitative wavelength-dispersive spectrometry (WDS) scan over the spectral region of interest remains a reasonable option to determine the background intensity and curvature from a fitted regression of background portions of the scan, but this technique can be time consuming and retains an element of subjectivity, as the analyst has to select areas in the scan which appear to represent background. This paper presents a new multi-point background (MPB) method whereby the background intensity is determined from up to 24 background measurements from wavelength positions on either side of analytical lines. This method improves the accuracy and precision of trace element analysis in a complex matrix through careful regression of the background shape, and can be used to characterize the background over a large spectral region covering several elements to be analyzed. The overall efficiency improves as systematic WDS scanning is not required to assess background interferences. The method is less subjective compared to methods that rely on WDS scanning, including selection of two interpolation points based on WDS scans, because “true” backgrounds are selected through an exclusion method of possible erroneous backgrounds. The first validation of the MPB method involves blank testing to ensure the method can accurately measure the absence of an element. The second validation involves the analysis of U-Th-Pb in several monazite reference materials of known isotopic age. The impetus for the MPB method came from efforts to refine EPMA monazite U-Th-Pb dating, where it was recognized that background errors resulting from interference or strong background curvature could result in errors of several tens of millions of years on the calculated date. Results obtained on monazite reference materials using two different microprobes, a Cameca SX-100 Ultrachron and a JEOL JXA-8230, yield excellent agreement with ages obtained by isotopic methods (Thermal Ionization Mass Spectrometry [TIMS], Sensitive High-Resolution Ion MicroProbe [SHRIMP], or Secondary Ion Mass Spectrometry [SIMS]). Finally, the MPB method can be used to model the background over a large spectrometer range to improve the accuracy of background measurement of minor and trace elements acquired on a same spectrometer, a method called the shared background measurement. This latter significantly improves the accuracy of minor and trace element analysis in complex matrices, as demonstrated by the analysis of Rare Earth Elements (REE) in REE-silicates and phosphates and of trace elements in scheelite.

This article presents a detailed study of the microstructure of Iberian expanded clay lightweight aggregates (LWA). Other than more commonly used mercury porosimetry (MP) and water absorption methods, the experimental study involves optical microscopy, scanning electron microscopy (SEM), and microtomography (μ-CT). Pore connectivity and how it is deployed are shown to some degree, and the pore size spectrum is estimated. LWA are in general characterized by a dense outer shell up to 200 μm thick, encasing an inner cellular structure of 10–100 times bigger pore size. Aggregate pore sizes may span from some hundreds of nanometers up to over 1 mm, though the range of 1–25 μm is more typical. A noteworthy fraction of these pores is closed, and they are mainly up to 1 μm. It is also shown that macropore spatial arrangement is affected by the manufacturing process. A step forward is given to understanding how the outer shell and the inner pore network influence the mechanical and physical LWA properties, particularly the density and water absorption. The joint consideration of μ-CT and SEM seems to be the most appropriate methodology to study LWA microstructure. MP analysis is likely to distort LWA pore spectrum assessment.

Development of the epicardium is critical to proper heart formation. It provides all of the precursor cells that form the coronary system and supplies signals that stimulate cardiac myocyte proliferation. The epicardium forms from mesothelial cells associated with the septum transversum and is referred to as the proepicardium (PE). Two different methods by which these PE cells colonize the developing heart have been described. In avians, PE cells form a bridge to the heart over which PE cells migrate onto the heart. In fish and mammals, PE cells form vesicles of cells that detach from the mesothelium, float through the pericardial cavity, and attach to the heart. A previous study of rat PE development investigated this process at the histological level. Protein markers have been developed since this study. Thus, we investigated this important developmental process coupled with these new markers using other visualization techniques such as scanning electron microscopy (SEM) and confocal microscopy. Finally, a novel, three-dimensional (3-D) culture system was used to confirm the identity of the PE cells. In this study, we found convincing evidence that the rat PE cells directly attach to the heart in a manner similar to that observed in avians.

This paper intends to analyze the microstructure of concrete with recycled aggregates (RA) from construction and demolition waste from various Portuguese recycling plants. To that effect, several scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analyses were performed. Various concrete mixes were evaluated in order to analyze the influence of the RA’s collection point and consequently of their composition on the mixes’ characteristics. Afterward all the mixes were subjected to the capillary water absorption test in order to quantitatively evaluate their porosity. Results from the SEM/EDS analysis were compared with those from capillary water absorption test. The SEM/EDS analysis showed that the bond capacity of aggregates to the new cement paste is greatly influenced by the RA’s nature. On the other hand, there was an increase in porosity with the incorporation of RA.

Atomic-resolution cryogenic scanning transmission electron microscopy (cryo-STEM) has provided a path to probing the microscopic nature of select low-temperature phases in quantum materials. Expanding cryo-STEM techniques to broadly tunable temperatures will give access to the rich temperature-dependent phase diagrams of these materials. With existing cryo-holders, however, variations in sample temperature significantly disrupt the thermal equilibrium of the system, resulting in large-scale sample drift. The ability to tune the temperature without negative impact on the overall instrument stability is crucial, particularly for high-resolution experiments. Here, we test a new side-entry continuously variable temperature dual-tilt cryo-holder which integrates liquid nitrogen cooling with a 6-pin micro-electromechanical system (MEMS) sample heater to overcome some of these experimental challenges. We measure consistently low drift rates of 0.3–0.4 Å/s and demonstrate atomic-resolution cryo-STEM imaging across a continuously variable temperature range from ~100 K to well above room temperature. We conduct additional drift stability measurements across several commercial sample stages and discuss implications for further developments of ultra-stable, flexible cryo-stages.

Relationships between volume plasmon excitations and mechanical properties of various materials are considered. Based on systematic evaluation of available data, correlations between the volume plasmon energy, Ep, Young's modulus, Ym, bulk modulus, Bm, shear modulus, Gm, and microhardness, Hm, are established. The resulting correlations indicate that plasmon energies potentially can be used to predict and/or determine the local mechanical properties of technologically important materials, such as metal alloys, semiconductors, and ceramics at the nanometer level.

There are many sources of random and systematic error in composition quantification by atom probe microscopy, often, however, only statistical error is reported. Significantly larger errors can occur from the misidentification of ions and overlaps or interferences of peaks in the mass spectrum. These overlaps can be solved using maximum likelihood estimation (MLE), improving the accuracy of the result, but with an unknown effect on the precision. An analytical expression for the uncertainty of the MLE solution is presented and it is demonstrated to be much more accurate than the existing methods. In one example, the commonly used error estimate was five times too small.

Literature results containing overlaps most likely underestimate composition uncertainty because of the complexity of correctly dealing with stochastic effects and error propagation. The uncertainty depends on the amount of overlapped intensity, for example being ten times worse for the CO/Fe overlap than the Cr/Fe overlap. Using the methods described here, accurate estimation of error, and the minimization of this could be achieved, providing a key milestone in quantitative atom probe. Accurate estimation of the composition uncertainty in the presence of overlaps is crucial for planning experiments and scientific interpretation of the measurements.

Abstract: We have developed a method for the examination of atomic force microscopy (scanning force microscopy) tips using a high-resolution transmission electron microscope (TEM). The tips can be imaged in a nondestructive way, enabling one to observe the shape of an atomic force microscope probe in the vicinity of the apex with high resolution. We have obtained images of atomic force microscopy probes with a resolution on the order of 1 nm. The tips can be imaged repeatedly, so one can examine tips before and after use. We have found that the tip can become blunted with use, the rate of wear depending upon the sample and tip materials and the scanning conditions. We have also found that the tips easily accrue contamination. We have studied both commercially produced tips, as well as tips grown by electron beam deposition. Direct imaging in the TEM should prove useful for image deconvolution methods because one does not have to make any assumptions concerning the general shape of the tip profile.

To better understand the formation and evolution of hierarchical crack networks in shales, observations of microscopic damage, and crack growth were conducted using an in situ tensile apparatus inside a scanning electron microscope. An arched specimen with an artificial notch incised into the curved edge was shown to afford effective observation of the damage and crack growth process that occurs during the brittle fracturing of shale. Because this arched specimen design can induce a squeezing effect, reducing the tensile stress concentration at the crack tip, and preventing the brittle shale from unstable fracturing to some extent. Both induced and natural pores and cracks were observed at different scales around the main crack path or on fractured surfaces. Observations indicate that the crack initiation zone develops around the crack tip where tensile stresses are concentrated and micro/nanoscale cracks nucleate. Crack advancement generally occurs by the continuous generation and coalescence of damage zones having intermittent en echelon microscopic cracks located ahead of the crack tips. Mineral anisotropy and pressure build-up around crack tips causes crack kinking, deflection, and branching. Crack growth is often accompanied by the cessation or closure of former branch cracks due to elastic recovery and induced compressive stress. The branching and interactions of cracks form a three-dimensional hierarchical network that includes induced branch cracks having similar paths, as well as natural structures such as nanopores, bedding planes, and microscopic cracks.

A portable X-ray fluorescence/X-ray diffraction (XRF/XRD) system for artwork studies has been designed constructed and tested. It is based on Debye Scherrer XRD in reflection that takes advantage of many recent improvements in the handling of X-rays (polycapillary optics; advanced two-dimensional detection). The apparatus is based on a copper anode air cooled X-ray source, and the XRD analysis is performed on a 5–20 μm thick layer from the object surface. Energy dispersive XRF elemental analysis can be performed at the same point as XRD, giving elemental compositions that support the interpretation of XRD diagrams. XRF and XRD analyses were tested to explore the quality and the limits of the analytical technique. The XRD diagrams are comparable in quality with diagrams obtained with conventional laboratory equipment. The mineral identification of materials in artwork is routinely performed with the portable XRF-XRD system. Examples are given for ceramic glazes containing crystals and for paintings where the determination of pigments is still a challenge for nondestructive analysis. For instance, lead compounds that provide a variety of color pigments can be easily identified as well as a pigment such as lapis lazuli that is difficult to identify by XRF alone. More than 70 works of art have been studied in situ in museums, monuments, etc. In addition to ceramics and paintings, these works include bronzes, manuscripts, etc., which permit improvement in the comprehension of ancient artistic techniques.

Ionoluminescence (IL) is the emission of light from a material due to excitation by an ion beam. In this work, a helium ion microscope (HIM) has been used in conjunction with a luminescence detection system to characterize IL from materials in an analogous way to how cathodoluminescence (CL) is characterized in a scanning electron microscope (SEM). A survey of the helium ion beam induced IL characteristics, including images and spectra, of a variety of materials known to exhibit CL in an SEM is presented. Direct band-gap semiconductors that luminesce strongly in the SEM are found not do so in the HIM, possibly due to defect-related nonradiative pathways created by the ion beam. Other materials do, however, exhibit IL, including a cerium-doped garnet sample, quantum dots, and rare-earth doped LaPO4 nanocrystals. These emissions are a result of transitions between f electron states or transitions across size dependent band gaps. In all these samples, IL is found to decay with exposure to the beam, fitting well to double exponential functions. In an exploration of the potential of this technique for biological tagging applications, imaging with the IL emitted by rare-earth doped LaPO4 nanocrystals, simultaneously with secondary electron imaging, is demonstrated at a range of magnifications.

Micro-focused synchrotron radiation techniques to investigate actinide elements in geological samples are becoming an increasingly used tool in nuclear waste disposal research. In this article, results using μ-focus techniques are presented from a bore core section of a U-rich tertiary sediment collected from Ruprechtov, Czech Republic, a natural analog to nuclear waste repository scenarios in deep geological formations. Different methods are applied to obtain various, complementary information. Elemental and element chemical state distributions are obtained from μ-XRF measurements, oxidation states of As determined from μ-XANES, and the crystalline structure of selected regions are studied by means of μ-XRD. We find that preparation of the thin section created an As oxidation state artifact; it apparently changed the As valence in some regions of the sample. Results support our previously proposed hypothesis of the mechanism for U-enrichment in the sediment. AsFeS coating on framboid Fe nodules in the sediment reduced mobile groundwater-dissolved U(VI) to less-soluble U(IV), thereby immobilizing the uranium in the sediment.