The three-dimensional characterization of internal features, via metrics such as orientation, porosity, and connectivity, is important to a wide variety of scientific questions. Many spatial and morphological metrics only can be measured accurately through direct in situ three-dimensional observations of large (i.e., big enough to be statistically representative) volumes. For samples that lack material contrast between phases, serial grinding and imaging—which relies solely on color and textural characteristics to differentiate features—is a viable option for extracting such information. Here, we present the Grinding, Imaging, Reconstruction Instrument (GIRI), which automatically serially grinds and photographs centimeter-scale samples at micron resolution. Although the technique is destructive, GIRI produces an archival digital image stack. This digital image stack is run through a supervised machine-learning-based image processing technique that quickly and accurately segments data into predefined classes. These classified data then can be loaded into three-dimensional visualization software for measurement. We share three case studies to illustrate how GIRI can address questions with a significant morphological component for which two-dimensional or small-volume three-dimensional measurements are inadequate. The analyzed metrics include: the morphologies of objects and pores in a granular material, the bulk mineralogy of polyminerallic solids, and measurements of the internal angles and symmetry of crystals.

Cryo-electron microscopy, widely used for high-resolution protein structure determination, does not require staining. Yet negative staining with heavy metal salts such as uranyl acetate has been in persistent demand since the 1950s due to its image contrasting capabilities at room temperature with a common electron microscope. However, uranium compounds are nuclear fuel materials and are tightly controlled worldwide. Acetates of each lanthanoid series elements except promethium are prepared at the same concentration (2%(w/v)) and used as a model on horse spleen ferritin for electron microscopic analysis to systematically evaluate their effectiveness as electron staining reagents for the protein. Analysis shows that the triacetates of samarium and europium, followed by gadolinium and erbium, and then lanthanum and neodymium could function as electron staining reagents. Thulium-triacetate precipitates thin plate-like crystals and may be used for selecting better imaging fields. Of the 14 lanthanoid-triacetates examined, about half are viable alternatives to uranyl acetate as an electron staining reagent for ferritin, and there appears an optimal range in ionic sizes for promising lanthanoids. This is the first systematic investigation of lanthanoid transition heavy metal triacetates from the viewpoint of lanthanoid contraction, using density distribution histograms of electron micrographs as an indicator for comparison with uranyl acetate.

NeXL is a collection of Julia language packages (libraries) for X-ray microanalysis data processing. NeXLCore provides basic atomic and X-ray physics data and models including support for microanalysis-related data types for materials and k-ratios. NeXLMatrixCorrection provides algorithms for matrix correction and iteration. NeXLSpectrum provides utilities and tools for energy-dispersive X-ray spectrum and hyperspectrum analysis including display, manipulation, and fitting. NeXL is integrated with the Julia language infrastructure. NeXL builds on the Gadfly plotting library and the DataFrames tabular data library. When combined with the DrWatson package, NeXL can provide a highly reproducible environment in which to process microanalysis data. Data availability and reproducible data analysis are two keys to scientific reproducibility. Not only should readers of journal articles have access to the data, they should also be able to reproduce the analysis steps that take the data to final results. This paper will both discuss the NeXL framework and provide examples of how it can used for reproducible data analysis.

Considerable information has been gained over the last few decades on several disease processes afflicting free-ranging cetaceans from a pathologist's point of view. Nonetheless, there is still a dearth of studies on the hearts of these species. For this reason, we aimed to improve our understanding of cardiac histological lesions occurring in free-ranging stranded cetaceans and, more specifically, in deep-diving Cuvier's beaked whales. The primary cardiac lesions that have been described include vascular changes, such as congestion, edema, hemorrhage, leukocytosis, and intravascular coagulation; acute degenerative changes, which consist of contraction band necrosis, wavy fibers, cytoplasmic hypereosinophilia, and perinuclear vacuolization; infiltration of inflammatory cells; and finally, the presence and/or deposition of different substances, such as interstitial myoglobin globules, lipofuscin pigment, polysaccharide complexes, and intra- and/or extravascular gas emboli and vessel dilation. This study advances our current knowledge about the histopathological findings in the cardiac muscle of cetaceans, and more specifically, of Cuvier's beaked whales.

The ability to analyze nanoparticles in the atom probe has often been limited by the complexity of the sample preparation. In this work, we present a method to lift–out single nanoparticles in the scanning electron microscope. First, nanoparticles are dispersed on a lacey carbon grid, then positioned on a sharp substrate tip and coated on all sides with a metallic matrix by physical vapor deposition. Compositional and structural insights are provided for spherical gold nanoparticles and a segregation of silver and copper in silver copper oxide nanorods is shown in 3D atom maps. Using the standard atom probe reconstruction algorithm, data quality is limited by typical standard reconstruction artifacts for heterogeneous specimens (trajectory aberrations) and the choice of suitable coatings for the particles. This approach can be applied to various unsupported free-standing nanoparticles, enables preselection of particles via correlative techniques, and reliably produces well-defined structured samples. The only prerequisite is that the nanoparticles must be large enough to be manipulated, which was done for sizes down to ~50 nm.

Collagen plays a key role in the strength of aortic walls, so studying micro-structural changes during disease development is critical to better understand collagen reorganization. Second-harmonic generation microscopy is used to obtain images of human aortic collagen in both healthy and diseased states. Methods are being developed in order to efficiently determine the waviness, that is, tortuosity and amplitude, as well as the diameter, orientation, and dispersion of collagen fibers, and bundles in healthy and aneurysmal tissues. The results show layer-specific differences in the collagen of healthy tissues, which decrease in samples of aneurysmal aortic walls. In healthy tissues, the thick collagen bundles of the adventitia are characterized by greater waviness, both in the tortuosity and in the amplitude, compared to the relatively thin and straighter collagen fibers of the media. In contrast, most aneurysmal tissues tend to have a more uniform structure of the aortic wall with no significant difference in collagen diameter between the luminal and abluminal layers. An increase in collagen tortuosity compared to the healthy media is also observed in the aneurysmal luminal layer. The data set provided can help improve related material and multiscale models of aortic walls and aneurysm formation.

The present study investigated the corneal sub-basal nerve plexus (SNP) modifications in glaucoma. Ninety-five glaucomatous patients were enrolled and divided into Group 1 and 2, preserved and preservative-free mono-therapy (30 and 28 patients), and Group 3, multi-therapy (37). Thirty patients with dry eye disease (DED) and 32 healthy subjects (HC) served as controls. In vivo confocal microscopy evaluated the nerve fibers density (CNFD), length (CNFL), thickness (CNFT), branching density (CNBD), and dendritic cell density (DCD). CNFD, CNFL, and CNBD were reduced in Group 3 and DED compared to HC (p < 0.05). CNFL was reduced in Group 3 compared to Group 2 (p < 0.05), and in Group 1 compared to HC (p < 0.001). CNFD, CNBD, and CNFT did not differ between glaucomatous groups. DCD was higher in Group 3 and DED compared to HC and Group 2 (p < 0.01). Group 3 showed worse ocular surface disease index (OSDI) scores compared to Group 1, 2, and HC (p < 0.05). CNFL and DCD correlated with OSDI score in Group 3 (r = −0.658, p < 0.001; r = 0.699, p = 0.002). Medical therapy for glaucoma harms the corneal nerves, especially in multi-therapy regimens. Given the relations with the OSDI score, SNP changes seem features of glaucoma therapy-related OSD and negatively affects the patient's quality of life.

Analyzing porous (nano)materials via atom probe tomography has been notoriously difficult. Voids and pores act as concentrators of the electrostatic pressure, which results in premature specimen failure, and the electrostatic field distribution near voids leads to aberrations that are difficult to predict. In this study, we propose a new encapsulating method for porous samples using a low melting point Bi–In–Sn alloy, known as Field's metal. As a model material, we used porous iron made by direct-hydrogen reduction of single-crystalline wüstite. The complete encapsulation was performed using in situ heating on the stage of a scanning electron microscope. No visible corrosion nor dissolution of the sample occurred. Subsequently, specimens were shaped by focused ion-beam milling under cryogenic conditions at −190°C. The proposed approach is versatile and can be applied to provide good quality atom probe datasets from micro/nanoporous materials.

Olfactory sensory neurons (OSNs) of fish belong to three main types: ciliated olfactory sensory neurons (cOSNs), microvillous olfactory sensory neurons (mOSNs), and crypt cells. Mercury is a toxic metal harmful for olfaction. We exposed the olfactory epithelium of zebrafish to three sublethal Hg2+ concentrations. Molecular markers specific for the different types of OSNs were immunohistochemically detected. Image analysis of treated sections enabled counting of marked cells and measurement of staining optical density indicative of the response of OSNs to Hg2+ exposure. The three types of OSNs reacted to mercury in a different way. Image analysis revealed that mOSNs are more susceptible to Hg2+ exposure than cOSNs and crypt cell density decreases. Moreover, while the ratio between sensory/nonsensory epithelium areas is unchanged, epithelium thickness drops, and dividing cells increase in the basal layer of the olfactory epithelium. Cell death but also reduction of apical processes and marker expression could account for changes in OSN immunostaining. Also, the differential results between dorsal and ventral halves of the olfactory rosette could derive from different water flows inside the olfactory chamber or different subpopulations in OSNs.

This work investigates how knock-on displacements influence fluctuation electron microscopy (FEM) experiments. FEM experiments were conducted on amorphous silicon, formed by self-ion implantation, in a transmission electron microscope at 300 kV and 60 kV at various electron doses, two different binnings and with two different cameras, a CCD and a CMOS one. Furthermore, energy filtering has been utilized in one case. Energy filtering greatly enhances the FEM data by removing the inelastic background intensity, leading to an improved speckle contrast. The CMOS camera yields a slightly larger normalized variance than the CCD at an identical electron dose and appears more prone to noise at low electron counts. Beam-induced atomic displacements affect the 300 kV FEM data, leading to a continuous suppression of the normalized variance with increasing electron dose. Such displacements are considerably reduced for 60 kV experiments since the primary electron's maximum energy transfer to an atom is less than the displacement threshold energy of amorphous silicon. The results show that the variance suppression due to knock-on displacements can be controlled in two ways: Either by minimizing the electron dose to the sample or by conducting the experiment at a lower acceleration voltage.

Hibernation is a biological status during which hibernating animals acclimatize themselves to reduced energy consumption through extreme but governed decline in self-metabolism. The role of mitochondria (Mt) in metabolic suppression during hibernation has already been elaborated in different organs and species. Nonetheless, the concretely changing process of mitochondrial architecture and the mechanism underlying this transformation during hibernation remains unclear. Herein, the present study was aimed at clarifying the detailed alteration of mitochondrial morphology and its potential role in the Chinese soft-shelled turtle (Pelodiscus sinensis) during different stages of hibernation. Compared with the nonhibernation period, the mitochondrial architecture was changing from round to crescent, and lipid droplet (LD)/Mt interaction was enhanced during hibernation, as observed by transmission electron microscopy (TEM). Further ultrastructural analysis uncovered that mitochondrial fusion was promptly accelerated in the early stage of hibernation, followed by mitochondrial fission in the middle stage, and mitophagy was boosted in the late stage. Moreover, gene and protein expression related to mitochondrial fusion, fission, and mitophagy accorded closely with the mitochondrial ultrastructural changes in different stages of hibernation. Taken together, our results clarified that the transformation of mitochondrial architecture and mitochondrial dynamics are of vital importance in maintaining internal environment homeostasis of Pelodiscus sinensis.

Inorganic ions are essential factors stabilizing nucleosome structure; however, many aspects of their effects on DNA transactions in chromatin remain unknown. Here, differential effects of K+ and Na+ on the nucleosome structure, stability, and interactions with protein complex FACT (FAcilitates Chromatin Transcription), poly(ADP-ribose) polymerase 1, and RNA polymerase II were studied using primarily single-particle Förster resonance energy transfer microscopy. The maximal stabilizing effect of K+ on a nucleosome structure was observed at ca. 80–150 mM, and it decreased slightly at 40 mM and considerably at >300 mM. The stabilizing effect of Na+ is noticeably lower than that of K+ and progressively decreases at ion concentrations higher than 40 mM. At 150 mM, Na+ ions support more efficient reorganization of nucleosome structure by poly(ADP-ribose) polymerase 1 and ATP-independent uncoiling of nucleosomal DNA by FACT as compared with K+ ions. In contrast, transcription through a nucleosome is nearly insensitive to K+ or Na+ environment. Taken together, the data indicate that K+ environment is more preserving for chromatin structure during various nucleosome transactions than Na+ environment.

The purpose of this study is to elucidate the impact of bleomycin on the degree of lung injury and development of mediastinal fat-associated lymphoid clusters (MFALCs) in the lymphoproliferative mouse model (MRL/MpJ-Faslpr/lpr “Lpr”) and its control strain (MRL/MpJ “MpJ”). We analyzed immune cells, the degree of proliferation, lymphatic vessels (LVs), and high endothelial venules (HEVs) in lungs and MFALCs in Lpr and MpJ mice on the 7th and 21st days following intranasal instillation of either bleomycin (BLM group) or PBS (PBS group). The BLM group showed a significant increase in the size of MFALCs, lung injury score, and positive area ratios of LVs, HEVs, and immune cells (especially macrophages, B- and T-lymphocytes) on both days 7 and 21. Interestingly, the lungs in the BLM group on day 21 showed higher collagen deposition and cellular infiltration in MpJ and Lpr, respectively. Moreover, significant positive correlations were observed between the size of MFALCs and lung injury. In conclusion, BLM could exert lung fibrosis or lymphoproliferative infiltration in chronic stages in MpJ and Lpr, respectively, and this varied effect could be due to the variations in the degree of immune cell proliferation and the development of LVs and HEVs among the studied strains.

Laser-assisted atom probe tomography (APT) is a relatively new, powerful technique for sub-nanometric mineral and biomineral analysis. However, the laser-assisted APT analysis of highly anisotropic and chemically diverse minerals, such as phyllosilicates, may prove especially challenging due to the complex interaction between the crystal structure and the laser pulse upon applying a high electric field. Micas are a representative group of nonswelling clay minerals of relevance to a number of scientific and technological fields. In this study, a Mg-rich biotite was analyzed by APT to generate preliminary data on nonisotropic minerals and to investigate the effect of the crystallographic orientation on mica chemical composition and structure estimation. The difference in results obtained for specimens extracted from the (001) and (hk0) mica surfaces indicate the importance of both experimental parameters and the crystallography. Anisotropy of mica has a strong influence on the physicochemical properties of the mineral during field evaporation and the interpretation of APT data. The promising results obtained in the present study open the way to future innovative APT applications on mica and clay minerals and contribute to the general discussion on the challenges for the analysis of geomaterials by atom probe tomography.

In vivo transparent vessel segmentation is important to life science research. However, this task remains very challenging because of the fuzzy edges and the barely noticeable tubular characteristics of vessels under a light microscope. In this paper, we present a new machine learning method based on blood flow characteristics to segment the global vascular structure in vivo. Specifically, the videos of blood flow in transparent vessels are used as input. We use the machine learning classifier to classify the vessel pixels through the motion features extracted from moving red blood cells and achieve vessel segmentation based on a region-growing algorithm. Moreover, we utilize the moving characteristics of blood flow to distinguish between the types of vessels, including arteries, veins, and capillaries. In the experiments, we evaluate the performance of our method on videos of zebrafish embryos. The experimental results indicate the high accuracy of vessel segmentation, with an average accuracy of 97.98%, which is much more superior than other segmentation or motion-detection algorithms. Our method has good robustness when applied to input videos with various time resolutions, with a minimum of 3.125 fps.

Accurately determining local polarization at atomic resolution can unveil the mechanisms by which static and dynamical behaviors of the polarization occur, including domain wall motion, defect interaction, and switching mechanisms, advancing us toward the better control of polarized states in materials. In this work, we explore the potential of atomic-resolution scanning transmission electron microscopy to measure the projected local polarization at the unit cell length scale. ZnO and PbMg1/3Nb2/3O3 are selected as case studies, to identify microscope parameters that can significantly affect the accuracy of the measured projected polarization vector. Different STEM imaging modalities are used to determine the location of the atomic columns, which, when combined with the Born effective charges, allows for the calculation of local polarization. Our results indicate that differentiated differential phase contrast (dDPC) imaging enhances the accuracy of measuring local polarization relative to other imaging modalities, such as annular bright-field or integrated-DPC imaging. For instance, under certain experimental conditions, the projected spontaneous polarization for ZnO can be calculated with 1.4% error from the theoretical value. Furthermore, we quantify the influence of sample thickness, probe defocus, and crystal mis-tilt on the relative errors of the calculated polarization.