Phase Contrast Nanotomography Research Articles

Unraveling grain growth of metallic tungsten: Investigating the nanoscale realm of hydrogen reduction of tungsten oxides

This paper focuses on the experimental observation of tungsten grain evolution during the process of hydrogen reduction of tungsten trioxide. The objective is to gain insights into the grain size distribution (GSD) evolution and morphology changes of metallic tungsten. The study employs an amalgamation of conventional characterization techniques – such as TGA, SEM-EDS, TEM and XRD – alongside cutting-edge techniques like phase contrast nano-tomography from synchrotron sources. Conducted as a quasi in-situ study, this research offers the opportunity to observe the transient evolution of the formed metallic tungsten phase within its precursor WO2 particles. The paper presents and discusses quantitative results concerning the evolution of GSD and morphological changes, encompassing growth rates of both crystallites and grains of the synthesized metallic phase. Additionally, the quasi in-situ study highlights the dependence of grain size on water concentration during the reduction process. The qualitative and quantitative findings contribute to a more nuanced understanding of the kinetics of grain growth of metallic tungsten and offer insights into the intricate interplay between reduction parameters, GSD evolution and morphological changes. Gaining a deeper understanding of the underlying mechanisms driving the changes in GSD establishes a foundation for predictive theory with immediate academic and industrial impact.

Deep learning to overcome Zernike phase-contrast nanoCT artifacts for automated micro-nano porosity segmentation in bone.

Bone material contains a hierarchical network of micro- and nano-cavities and channels, known as the lacuna-canalicular network (LCN), that is thought to play an important role in mechanobiology and turnover. The LCN comprises micrometer-sized lacunae, voids that house osteocytes, and submicrometer-sized canaliculi that connect bone cells. Characterization of this network in three dimensions is crucial for many bone studies. To quantify X-ray Zernike phase-contrast nanotomography data, deep learning is used to isolate and assess porosity in artifact-laden tomographies of zebrafish bones. A technical solution is proposed to overcome the halo and shade-off domains in order to reliably obtain the distribution and morphology of the LCN in the tomographic data. Convolutional neural network (CNN) models are utilized with increasing numbers of images, repeatedly validated by `error loss' and `accuracy' metrics. U-Net and Sensor3D CNN models were trained on data obtained from two different synchrotron Zernike phase-contrast transmission X-ray microscopes, the ANATOMIX beamline at SOLEIL (Paris, France) and the P05 beamline at PETRA III (Hamburg, Germany). The Sensor3D CNN model with a smaller batch size of 32 and a training data size of 70 images showed the best performance (accuracy 0.983 and error loss 0.032). The analysis procedures, validated by comparison with human-identified ground-truth images, correctly identified the voids within the bone matrix. This proposed approach may have further application to classify structures in volumetric images that contain non-linear artifacts that degrade image quality and hinder feature identification.

Structure of the myenteric plexus in normal and diseased human ileum analyzed by X-ray virtual histology slices.

BACKGROUNDThe enteric nervous system (ENS) is situated along the entire gastrointestinal tract and is divided into myenteric and submucosal plexuses in the small and large intestines. The ENS consists of neurons, glial cells, and nerves assembled into ganglia, surrounded by telocytes, interstitial cells of Cajal, and connective tissue. Owing to the complex spatial organization of several interconnections with nerve fascicles, the ENS is difficult to examine in conventional histological sections of 3-5 μm.AIMTo examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS.METHODSSix patients were diagnosed with gastrointestinal dysmotility and neuropathy based on routine clinical and histopathological examinations. As controls, full-thickness biopsies were collected from healthy resection ileal regions after hemicolectomy for right colon malignancy. From the paraffin blocks, 4-µm thick sections were prepared and stained with hematoxylin and eosin for localization of the myenteric ganglia under a light microscope. A 1-mm punch biopsy (up to 1 cm in length) centered on the myenteric plexus was taken and placed into a Kapton® tube for mounting in the subsequent investigation. X-ray phase-contrast tomography was performed using two custom-designed laboratory setups with micrometer resolution for overview scanning. Subsequently, selected regions of interest were scanned at a synchrotron-based end-station, and high-resolution slices were reported. In total, more than 6000 virtual slices were analyzed from nine samples.RESULTSIn the overview scans, the general architecture and quality of the samples were studied, and the myenteric plexus was localized. High-resolution scans revealed details, including the ganglia, interganglional nerve fascicles, and surrounding tissue. The ganglia were irregular in shape and contained neurons and glial cells. Spindle-shaped cells with very thin cellular projections could be observed on the surface of the ganglia, which appeared to build a network. In the patients, there were no alterations in the general architecture of the myenteric ganglia. Nevertheless, several pathological changes were observed, including vacuolar degeneration, autophagic activity, the appearance of sequestosomes, chromatolysis, and apoptosis. Furthermore, possible expulsion of pyknotic neurons and defects in the covering cellular network could be observed in serial slices. These changes partly corresponded to previous light microscopy findings.CONCLUSIONThe analysis of serial virtual slices could provide new information that cannot be obtained by classical light microscopy. The advantages, disadvantages, and future possibilities of this method are also discussed.

Laboratory phase-contrast nanotomography of unstained Bombus terrestris compound eyes.

Imaging the visual systems of bumblebees and other pollinating insects may increase understanding of their dependence on specific habitats and how they will be affected by climate change. Current high-resolution imaging methods are either limited to two dimensions (light- and electron microscopy) or have limited access (synchrotron radiation x-ray tomography). For x-ray imaging, heavy metal stains are often used to increase contrast. Here, we present micron-resolution imaging of compound eyes of buff-tailed bumblebees (Bombus terrestris) using a table-top x-ray nanotomography (nano-CT) system. By propagation-based phase-contrast imaging, the use of stains was avoided and the microanatomy could more accurately be reconstructed than in samples stained with phosphotungstic acid or osmium tetroxide. The findings in the nano-CT images of the compound eye were confirmed by comparisons with light- and transmission electron microscopy of the same sample and finally, comparisons to synchrotron radiation tomography as well as to a commercial micro-CT system weredone.

Hard X-ray nano-holotomography with a Fresnel zone plate.

X-ray phase contrast nanotomography enables imaging of a wide range of samples with high spatial resolution in 3D. Near-field holography, as one of the major phase contrast techniques, is often implemented using X-ray optics such as Kirkpatrick-Baez mirrors, waveguides and compound refractive lenses. However, these optics are often tailor-made for a specific beamline and challenging to implement and align. Here, we present a near-field holography setup based on Fresnel zone plates which is fast and easy to align and provides a smooth illumination and flat field. The imaging quality of different types of Fresnel zone plates is compared in terms of the flat-field quality, the achievable resolution and exposure efficiency i.e. the photons arriving at the detector. Overall, this setup is capable of imaging different types of samples at high spatial resolution of below 100 nm in 3D with access to the quantitative phase information.

Pushing the temporal resolution in absorption and Zernike phase contrast nanotomography: enabling fast in situ experiments.

Hard X-ray nanotomography enables 3D investigations of a wide range of samples with high resolution (<100 nm) with both synchrotron-based and laboratory-based setups. However, the advantage of synchrotron-based setups is the high flux, enabling time resolution, which cannot be achieved at laboratory sources. Here, the nanotomography setup at the imaging beamline P05 at PETRA III is presented, which offers high time resolution not only in absorption but for the first time also in Zernike phase contrast. Two test samples are used to evaluate the image quality in both contrast modalities based on the quantitative analysis of contrast-to-noise ratio (CNR) and spatial resolution. High-quality scans can be recorded in 15 min and fast scans down to 3 min are also possible without significant loss of image quality. At scan times well below 3 min, the CNR values decrease significantly and classical image-filtering techniques reach their limitation. A machine-learning approach shows promising results, enabling acquisition of a full tomography in only 6 s. Overall, the transmission X-ray microscopy instrument offers high temporal resolution inabsorption and Zernike phase contrast, enabling in situ experiments at thebeamline.

Three-Dimensional Correlative Imaging of a Malaria-Infected Cell with a Hard X-ray Nanoprobe.

Benefiting from the recent advances of synchrotron X-ray nanoprobes, we demonstrate three-dimensional (3D) correlative nanoimaging on malaria-infected human red blood cells. By combining X-ray fluorescence tomography and phase contrast nanotomography on the same cell with sub-100 nm pixel size, we establish a routine workflow from the data acquisition, data processing, to tomographic reconstruction. We quantitatively compare the elemental volumes obtained with different reconstruction methods, with the total variation minimization giving the most satisfactory results. We reveal elemental correlations in different cell compartments more reliably on reconstructions as opposed to 2D projections. Finally, we determine for the first time the 3D mass fraction maps of multiple elements at the subcellular level. The estimated total number of Fe atoms and the total mass of red blood cells show very good agreement with previously reported values.

Fast In Situ Nanotomography at ESRF

X-ray computed tomography (CT) is a very powerful technique that provides nondestructively direct access to the three-dimensional morphology of a specimen. It is extensively used in different domains like medicine, paleontology and biology. Today it is even possible to perform real time acquisition to observe in situ damaging and processes occurring in materials. Thanks to continuous improvements in spatial resolution, CT has been recently extended to the nanoscale [1]. It is a very efficient technique to study the performance of many advanced materials, which is mostly determined by the arrangement of their nanostructure [2]. Nevertheless, the combination of fast real time, in situ and CT in the nanometer regime remains a key challenge. We present here, in situ hard X-ray nanotomography experiments with an unprecedented combination of nanometer pixel size and fast acquisition time at high temperature. ID16B is a nano-analysis beamline built during phase I of the European Synchrotron Radiation Facility (ESRF) and opened to users since early 2014. The beamline configuration offers an improved lateral resolution (50x50 nm 2), a monochromatic nanobeam tunable in a large energy range (6-30 kV) with high flux (10 9 to 10 12 ph/s), and large flexibility capable of in situ experiments [3]. Combination of various techniques such as X-ray fluorescence (XRF), X-ray absorption (XANES), X-ray excited optical luminescence (XEOL) and X-ray diffraction (XRD) as well as magnified phase contrast nanotomography allows multi-approach studies to be performed. Taking advantage of the high stability offered on ID16B and magnified phase contrast imaging, we developed a fast in situ nanoimaging setup [4] that allows high temperature experiments to be performed with an unparalleled combination of nanometer pixel size (35nm) and fast acquisition (<10s). An intense X-ray beam (10 12 ph/s) is focused down to a spot size of 50×50 nm 2 using two multilayer-coated Si mirrors in Kirkpatrick-Baez (KB) configuration under pink beam mode (∆E/E=10-2). As illustrated in Figure 1 the sample located in the microheater (200 to 900°C) is positioned out of the focal plane in projection geometry. While rotating the sample, high-resolution images are continuously collected by a PCO edge 5.5 detector (equipped with a CMOS sensor) through an Optique Peter high resolution X-Ray imaging microscope (equipped with an X10 Olympus objective). To date, fastest full tomographic scans are acquired in 7s. Pixel size and field of view depend on the sample position between the focal plane and the detector. Any pixel size between 27 and 650 nm is available and multi-scale measurements are easily achievable. Several real time studies that investigates nanostructure evolution in materials, have been already performed: (i) ceramics sintering (ii) light alloys damage (iii) fuel cells degradation... For example, spherical soda-limes glass particles have been isothermally treated at 670°C, and continuously scanned (timescan:33s) during approximately 2 hours with a pixel size of 100nm. The microstructural evolution during ceramic powders sintering leads to controlled porosity but also formation of defects. 3D observations of the process at the micro-scale already gave important information on the densification process from rearrangement phenomena point of view. While the 3D movie resulting from in situ

Nondestructive Multiscale X-Ray Tomography by Combining Microtomography and High-Energy Phase-Contrast Nanotomography

Nondestructive Multiscale X-Ray Tomography by Combining Microtomography and High-Energy Phase-Contrast Nanotomography

Nanoscale quantification of intracellular element concentration by X-ray fluorescence microscopy combined with X-ray phase contrast nanotomography

We present here a correlative X-ray microscopy approach for quantitative single cell imaging of molar concentrations. By combining the elemental content provided by X-ray fluorescence microscopy and the morphology information extracted from X-ray phase nanotomography, we determine the intracellular molarity distributions. This correlative method was demonstrated on a freeze-dried human phagocytic cell to obtain the absolute elemental concentration maps of K, P, and Fe. The cell morphology results showed a very good agreement with atomic-force microscopy measurements. This work opens the way for non-destructive single cell chemical analysis down to the sub-cellular level using exclusively synchrotron radiation techniques. It will be of high interest in the case where it is difficult to access the morphology using atomic-force microscopy, for example, on frozen-hydrated cells or tissues.

Multimodal correlative investigation of the interplaying micro-architecture, chemical composition and mechanical properties of human cortical bone tissue reveals predominant role of fibrillar organization in determining microelastic tissue properties.

The mechanical competence of bone is crucially determined by its material composition and structural design. To investigate the interaction of the complex hierarchical architecture, the chemical composition and the resulting elastic properties of healthy femoral bone at the level of single bone lamellae and entire structural units, we combined polarized Raman spectroscopy (PRS), scanning acoustic microscopy (SAM) and synchrotron X-ray phase contrast nano tomography (SR-nanoCT). In line with earlier studies, mutual correlation analysis strongly suggested that the characteristic elastic modulations of bone lamellae within single units are the result of the twisting fibrillar orientation, rather than compositional variations, modulations of the mineral particle maturity, or mass density deviations. Furthermore, we show that predominant fibril orientations in entire tissue units can be rapidly assessed from Raman parameter maps. Coexisting twisted and oscillating fibril patterns were observed in all investigated tissue domains. Ultimately, our findings demonstrate in particular the potential of combined PRS and SAM measurements in providing multi-scalar analysis of correlated fundamental tissue properties. In future studies, the presented approach can be applied for non-destructive investigation of small pathologic samples from bone biopsies and a broad range of biological materials and tissues. Bone is a complex structured composite material consisting of collagen fibrils and mineral particles. Various studies have shown that not only composition, maturation, and packing of its components, but also their structural arrangement determine the mechanical performance of the tissue. However, prominent methodologies are usually not able to concurrently describe these factors on the micron scale and complementary tissue characterization remains challenging. In this study we combine X-ray nanoCT, polarized Raman imaging and scanning acoustic microscopy and propose a protocol for fast and easy assessment of predominant fibril orientations in bone. Based on our site-matched analysis of cortical bone, we conclude that the elastic modulations of bone lamellae are mainly determined by the fibril arrangement.

Quantification of a Single Aggregate Inner Porosity and Pore Accessibility Using Hard X-ray Phase-Contrast Nanotomography

The 3D structure of three individual aggregates composed of 165 nm polystyrene primary particles is revealed nondestructively by hard X-ray phase-contrast synchrotron nanotomography. Three-dimensional image analysis allows us for the first time to obtain the complex inner porosity of the entire aggregate. It is demonstrated that despite their rather compact structure, characterized by a fractal dimension equal to 2.7, the produced aggregates are still porous, with porosity increasing with its size. Generated pores have diameters from 100 nm to 3 μm and are almost completely interconnected.