3D quantification of the lacunocanalicular network on human femoral diaphysis through synchrotron radiation-based nanoCT.

Osteocytes are the major actors in bone mechanobiology. Within bone matrix, they are trapped close together in a submicrometric interconnected network: the lacunocanalicular network (LCN). The interstitial fluid circulating within the LCN transmits the mechanical information to the osteocytes that convert it into a biochemical signal. Understanding the interstitial fluid dynamics is necessary to better understand the bone mechanobiology. Due to the submicrometric dimensions of the LCN, making it difficult to experimentally investigate fluid dynamics, numerical models appear as a relevant tool for such investigation. To develop such models, there is a need for geometrical and morphological data on the human LCN. This study aims at providing morphological data on the human LCN from measurement of 27 human femoral diaphysis bone samples using synchrotron radiation nano-computed tomography with an isotropic voxel size of 100 nm. Except from the canalicular diameter, the canalicular morphological parameters presented a high variability within one sample. Some differences in terms of both lacunar and canalicular morphology were observed between the male and female populations. But it has to be highlighted that all the canaliculi cannot be detected with a voxel size of 100 nm. Hence, in the current study, only a specific population of large canaliculi that could be characterize. Still, to the authors knowledge, this is the first time such a data set was introduced to the community. Further processing will be achieved in order to provide new insight on the LCN permeability.

Evaluation of imaging setups for quantitative phase contrast nanoCT of mineralized biomaterials.

X-ray nano-tomography with phase contrast (nanoCT) using synchrotron radiation is a powerful tool to non-destructively investigate 3D material properties at the nanoscale. In large bone lesions, such as severe bone fractures, bone cancer or other diseases, bone grafts substituting the lost bone might be necessary. Such grafts can be of biological origin or be composed of a synthetic bone substitute. The long-term functioning of artificial bone substitutes depends on many factors. Synchrotron nanoCT imaging has great potential to contribute to further the understanding of integration of implants into bone tissue by imaging the spatial interaction between bone tissue and implant, and by accessing the interface between implant material and bone tissue. With this aim, a methodology for evaluating the image quality is presented for in-line phase contrast nanoCT images of bone scaffold samples. A PMMA-embedded tricalcium phosphate scaffold was used with both a closed and an open porosity structure and bone ingrowths as a representative system of three known materials. Parameters such as spatial resolution and signal-to-noise ratio were extracted and used to explore and quantitatively compare the effects of implementation choices in the imaging setup, such as camera technology and imaging energy, on the resulting image quality. Increasing the X-ray energy from 17.5 keV to 29.6 keV leads to a notable improvement in image quality regardless of the camera technology used, with the two tested camera setups performing at a comparable level when the recorded intensity was kept constant.

A helium mini-cryostat for the nanoprobe beamline ID16B at ESRF: characteristics and performance.

A helium mini-cryostat has been developed for the hard X-ray nanoprobe ID16B of the European Synchrotron to collect X-ray excited optical luminescence and X-ray fluorescence at low temperature (<10 K). The mini-cryostat has been specifically designed to fit within the strong space restrictions and high-demanding mechanical constraints imposed by the beamline to provide vibration-free operation and maximal thermal stability. This paper reports the detailed design, architecture and technical requirements of the mini-cryostat, and presents the first experimental data measured using the cryogenic equipment. The resulting cryo-system features ultimate thermal stability, fast cool-down and ultra-low vibrations. The simultaneous X-ray fluorescence and X-ray excited optical luminescence data acquired from bulk GaN and core/shell InGaN/GaN multi-quantum wells validated the excellent performance of the cryostat with ultimate resolution, stability and sensitivity.

Medicago truncatula Ferroportin2 mediates iron import into nodule symbiosomes.

Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Ferroportin family members in model legume Medicago truncatula were identified and their expression was determined. Yeast complementation assays, immunolocalization, characterization of a tnt1 insertional mutant line, and synchrotron-based X-ray fluorescence assays were carried out in the nodule-specific M. truncatula ferroportin Medicago truncatula nodule-specific gene Ferroportin2 (MtFPN2) is an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature and in inner nodule tissues, as well as in the symbiosome membranes in the interzone and early-fixation zone of the nodules. Loss-of-function of MtFPN2 alters iron distribution and speciation in nodules, reducing nitrogenase activity and biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that expression in the inner nodule tissues is sufficient to restore the phenotype, while confining MtFPN2 expression to the vasculature did not improve the mutant phenotype. These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen-fixing bacteroids in M. truncatula nodules.

Distribution of nickel and chromium containing particles from tattoo needle wear in humans and its possible impact on allergic reactions.

BACKGROUND: Allergic reactions to tattoos are amongst the most common side effects occurring with this permanent deposition of pigments into the dermal skin layer. The characterization of such pigments and their distribution has been investigated in recent decades. The health impact of tattoo equipment on the extensive number of people with inked skin has been the focus of neither research nor medical diagnostics. Although tattoo needles contain high amounts of sensitizing elements like nickel (Ni) and chromium (Cr), their influence on metal deposition in skin has never been investigated. RESULTS: Here, we report the deposition of nano- and micrometer sized tattoo needle wear particles in human skin that translocate to lymph nodes. Usually tattoo needles contain nickel (6-8%) and chromium (15-20%) both of which prompt a high rate of sensitization in the general population. As verified in pig skin, wear significantly increased upon tattooing with the suspected abrasive titanium dioxide white when compared to carbon black pigment. Additionally, scanning electron microscopy of the tattoo needle revealed a high wear after tattooing with ink containing titanium dioxide. The investigation of a skin biopsy obtained from a nickel sensitized patient with type IV allergy toward a tattoo showed both wear particles and iron pigments contaminated with nickel. CONCLUSION: Previously, the virtually inevitable nickel contamination of iron pigments was suspected to be responsible for nickel-driven tattoo allergies. The evidence from our study clearly points to an additional entry of nickel to both skin and lymph nodes originating from tattoo needle wear with an as yet to be assessed impact on tattoo allergy formation and systemic sensitization.

ID16B: a hard X-ray nanoprobe beamline at the ESRF for nano-analysis.

Within the framework of the ESRF Phase I Upgrade Programme, a new state-of-the-art synchrotron beamline ID16B has been recently developed for hard X-ray nano-analysis. The construction of ID16B was driven by research areas with major scientific and societal impact such as nanotechnology, earth and environmental sciences, and bio-medical research. Based on a canted undulator source, this long beamline provides hard X-ray nanobeams optimized mainly for spectroscopic applications, including the combination of X-ray fluorescence, X-ray diffraction, X-ray excited optical luminescence, X-ray absorption spectroscopy and 2D/3D X-ray imaging techniques. Its end-station re-uses part of the apparatus of the earlier ID22 beamline, while improving and enlarging the spectroscopic capabilities: for example, the experimental arrangement offers improved lateral spatial resolution (∼50 nm), a larger and more flexible capability for in situ experiments, and monochromatic nanobeams tunable over a wider energy range which now includes the hard X-ray regime (5-70 keV). This paper describes the characteristics of this new facility, short-term technical developments and the first scientific results.

Fate of Ag-NPs in Sewage Sludge after Application on Agricultural Soils.

The objective of this work was to investigate the fate of silver nanoparticles (Ag-NPs) in a sludge-amended soil cultivated with monocot (Wheat) and dicot (Rape) crop species. A pot experiment was performed with sludges produced in a pilot wastewater treatment plant containing realistic Ag concentrations (18 and 400 mg kg(-1), 14 mg kg(-1) for the control). Investigations focused on the highest dose treatment. X-ray absorption spectroscopy (XAS) showed that Ag2S was the main species in the sludge and amended soil before and after plant culture. The second most abundant species was an organic and/or amorphous Ag-S phase whose proportion slightly varied (from 24% to 36%) depending on the conditions. Micro and nano X-ray fluorescence (XRF) showed that Ag was preferentially associated with S-rich particles, including organic fragments, of the sludge and amended soils. Ag was distributed as heteroaggregates with soil components (size ranging from ≤0.5 to 1-3 µm) and as diffused zones likely corresponding to sorbed/complexed Ag species. Nano-XRF evidenced the presence of mixed metallic sulfides. Ag was weakly exchangeable and labile. However, micronutrient mobilization by plant roots and organic matter turnover may induce Ag species interconversion eventually leading to Ag release on longer time scales. Together, these data provide valuable information for risk assessment of sewage sludge application on agricultural soils.

X-ray nanotomography using near-field ptychography.

Propagation-based imaging or inline holography in combination with computed tomography (holotomography) is a versatile tool to access a sample's three-dimensional (3D) micro or nano structure. However, the phase retrieval step needed prior to tomographic reconstruction can be challenging especially for strongly absorbing and refracting samples. Near-field ptychography is a recently developed phase imaging method that has been proven to overcome this hurdle in projection data. In this work we extend near-field ptychography to three dimensions and we show that, in combination with tomography, it can access the nano structure of a solid oxide fuel cell (SOFC). The quality of the resulting tomographic data and the structural properties of the anode extracted from this volume were compared to previous results obtained with holotomography. This work highlights the potential of 3D near-field ptychography for reliable and detailed investigations of samples at the nanometer scale, with important applications in materials and life sciences among others.

Binder-Free Cnt Cathodes for Li-O2 Batteries with More Than One Life.

Li-O2 batteries (LOB) performance degradation ultimately occurs through the accumulation of discharge products and irreversible clogging of the porous electrode during the cycling. Electrode binder degradation in the presence of reduced oxygen species can result in additional coating of the conductive surface, exacerbating capacity fading. Herein, a facile method to fabricate free-standing is established, binder-free electrodes for LOBs in which multi-wall carbon nanotubes form cross-linked networks exhibiting high porosity, conductivity, and flexibility. These electrodes demonstrate high reproducibility upon cycling in LOBs. After cell death, efficient and inexpensive methods to wash away the accumulated discharge products are demonstrated, as reconditioning method. The second life usage of these electrodes is validated, without noticeable loss of performance. These findings aim to assist in the development of greener high energy density batteries while reducing manufacturing and recycling costs.

Imaging Voids and Defects Inside Li-Ion Cathode LiNi0.6Mn0.2Co0.2O2 Single Crystals.

Li-ion battery cathode active materials obtained from different sources or preparation methods often exhibit broadly divergent performance and stability despite no obvious differences in morphology, purity, and crystallinity. We show how state-of-the-art, commercial, nominally single crystalline LiNi0.6Mn0.2Co0.2O2 (NMC-622) particles possess extensive internal nanostructure even in the pristine state. Scanning X-ray diffraction microscopy reveals the presence of interlayer strain gradients, and crystal bending is attributed to oxygen vacancies. Phase contrast X-ray nano-tomography reveals two different kinds of particles, welded/aggregated, and single crystal like, and emphasizes the intra- and interparticle heterogeneities from the nano- to the microscale. It also detects within the imaging resolution (100 nm) substantial quantities of nanovoids hidden inside the bulk of two-thirds of the overall studied particles (around 3000), with an average value of 12.5%v per particle and a mean size of 148 nm. The powerful combination of both techniques helps prescreening and quantifying the defective nature of cathode material and thus anticipating their performance in electrode assembly/battery testing.

Unraveling the synergistic effects of Cu-Ag tandem catalysts during electrochemical CO2 reduction using nanofocused X-ray probes.

Controlling the selectivity of the electrocatalytic reduction of carbon dioxide into value-added chemicals continues to be a major challenge. Bulk and surface lattice strain in nanostructured electrocatalysts affect catalytic activity and selectivity. Here, we unravel the complex dynamics of synergistic lattice strain and stability effects of Cu-Ag tandem catalysts through a previously unexplored combination of in situ nanofocused X-ray absorption spectroscopy and Bragg coherent diffraction imaging. Three-dimensional strain maps reveal the lattice dynamics inside individual nanoparticles as a function of applied potential and product yields. Dynamic relations between strain, redox state, catalytic activity and selectivity are derived. Moderate Ag contents effectively reduce the competing evolution of H2 and, concomitantly, lead to an enhanced corrosion stability. Findings from this study evidence the power of advanced nanofocused spectroscopy techniques to provide new insights into the chemistry and structure of nanostructured catalysts.

Synchrotron microanalysis techniques applied to potential photovoltaic materials.

X-ray synchrotron radiation techniques are used to characterize photovoltaic-related semiconductors. Micro-X-ray-fluorescence and X-ray beam induced current mapping of multicrystalline silicon photovoltaic cells show metallic impurities accumulating at the interface of crystallographic defects, and current variations over the cell that are attributed to bulk defects and structural variation of the silicon. Similarly, studies on a single-crystal GaAs using X-ray fluorescence and X-ray excited optical luminescence show an inhomogeneous As distribution correlated with the photoluminescence signal, with higher As concentration regions having stronger photoluminescence signal. Both examples show how the combination of synchrotron microanalysis techniques can contribute to a better understanding of the optical properties of photovoltaic materials.

Status of the hard X-ray microprobe beamline ID22 of the European Synchrotron Radiation Facility.

The ESRF synchrotron beamline ID22, dedicated to hard X-ray microanalysis and consisting of the combination of X-ray fluorescence, X-ray absorption spectroscopy, diffraction and 2D/3D X-ray imaging techniques, is one of the most versatile instruments in hard X-ray microscopy science. This paper describes the present beamline characteristics, recent technical developments, as well as a few scientific examples from recent years of the beamline operation. The upgrade plans to adapt the beamline to the growing needs of the user community are briefly discussed.

Detection and Characterization of TiO2 Nanomaterials in Sludge from Wastewater Treatment Plants of Chihuahua State, Mexico.

TiO2 nanoparticles (TiO2-NPs) have a wide range of industrial applications (paintings, sunscreens, food and cosmetics) and is one of the most intensively used nanomaterials worldwide. Leaching from commercial products TiO2-NPs are predicted to significantly accumulate in wastewater sludges, which are then often used as soil amendment. In this work, sludge samples from four wastewater treatment plants of the Chihuahua State in Mexico were obtained during spring and summer (2017). A comprehensive characterization study was performed by X-ray based (laboratory and synchrotron) techniques and electron microscopy. Ti was detected in all sludge samples (1810-2760 mg/kg) mainly as TiO2 particles ranging from 40 nm up to hundreds of nm. Micro-XANES data was analyzed by principal component analysis and linear combination fitting enabling the identification of three predominant Ti species: anatase, rutile and ilmenite. Micro-XANES from the smaller Ti particles was predominantly anatase (68% + 32% rutile), suggesting these TiO2-NPs originate from paintings and cosmetics. TEM imaging confirmed the presence of nanoscale Ti with smooth surface morphologies resembling engineered TiO2-NPs. The size and crystalline phase of TiO2-NPs in the sludge from this region suggest increased reactivity and potential toxicity to agro-systems. Further studies should be dedicated to evaluating this.

Assessing implications of nanoplastics exposure to plants with advanced nanometrology techniques.

Despite the increasing attention given to the impacts of nanoplastics in terrestrial environments, there is limited data about the effects on plants, and the quantitative information on uptake. In the present study, wheat plants grown in hydroponics were exposed to Pd-doped nanoplastics. This allowed us to quantify nanoplastics uptake and translocation to the shoots. Visualization of nanoplastics in roots was performed with synchrotron micro X-ray fluorescence (µXRF). Nanoplastics accumulated on the root epidermis, especially at the root tip and in root maturation zones. A close relationship between plant roots, rhizodeposits and nanoplastics behaviour was shown. Reinforcement of the cell wall in roots was evidenced using Fourier transform infrared spectroscopy (FTIR) and synchrotron-computed microtomography (µCT). Synchrotron-computed nanotomography (nanoCT) evidenced the presence of globular structures but they could not be identified as nanoplastics since they were observed both in the control and treated roots. By utilizing the inorganic tracer in the doped-nanoplastics, this study paves the road for elucidating interactions in more complex systems by using an integrative approach combining classical phytotoxicity markers with advanced nanometrology techniques.

Structural and chemical variations in Mg-calcite skeletal segments of coralline red algae lead to improved crack resistance.

The calcareous alga Jania sp. is an articulated coralline red seaweed that is abundant in the shallow waters of oceans worldwide. We have previously demonstrated that its structure is highly intricate and exhibits hierarchical organization across multiple length scales from the macro to the nano scale. Moreover, we have proven that the inner pores of its structure are helical, conveying the alga greater compliance as compared to a cylindrical configuration. Herein, we reveal new insights into the structure of Jania sp., particularly, its crystallographic variations and the internal elemental distribution of Mg and Ca. We show that the high-Mg calcite cell wall nanocrystals of Jania sp. are arranged in layers with alternating Mg contents. Moreover, we show that this non-homogenous elemental distribution assists the alga in preventing fracture caused by crack propagation. We further reveal that each one of the cell wall nanocrystals in Jania sp. is not a single crystal as was previously thought, but rather comprises Mg-rich calcite nanoparticles demonstrating various crystallographic orientations, arranged periodically within the layered structure. We also show that these Mg-rich nanoparticles are present in yet another species of the coralline red algae, Corallina sp., pointing to the generality of this phenomenon. To the best of our knowledge this is a first report on the existence of Mg-rich nanoparticles in algal mineralized tissue. We envisage that our findings on the bio-strategy found in the algae to enhance their fracture toughness will have an impact on the design of structures with superior mechanical properties. STATEMENT OF SIGNIFICANCE: Understanding the structure-property relation in biomineralized tissues is of great importance in unveiling Nature's material design strategies, which form the basis for the development of novel structural materials. Crystallographic and elemental variations in the skeletal parts of the coralline red algae and their cumulative contribution to prevention of mechanical failure are yet poorly studied. Herein, we reveal that the high-Mg calcite cell wall nanocrystals of Jania sp. are arranged in layers with alternating Mg concentrations and that this organization facilitates crack deflection, thereby preventing catastrophic fracture. We further discovered that the nanocrystals contain incoherent Mg-rich nanoparticles and suggest that they form via spinodal decomposition of the Mg-ACC precursor and self-arrange periodically throughout the alga's mineralized cell wall, a phenomenon most likely to be widespread in high-Mg calcite biomineralization.

Helical Microstructures of the Mineralized Coralline Red Algae Determine Their Mechanical Properties.

Through controlled biomineralization, organisms yield complicated structures with specific functions. Here, Jania sp., an articulated coralline red alga that secretes high-Mg calcite as part of its skeleton, is in focus. It is shown that Jania sp. exhibits a remarkable structure, which is highly porous (with porosity as high as 64 vol%) and reveals several hierarchical orders from the nano to the macroscale. It is shown that the structure is helical, and proven that its helical configuration provides the alga with superior compliance that allows it to adapt to stresses in its natural environment. Thus, the combination of high porosity and a helical configuration result in a sophisticated, light-weight, compliant structure. It is anticipated that the findings on the advantages of such a structure are likely to be of value in the design or improvement of lightweight structures with superior mechanical properties.

X-ray nano-tomography of complete scales from the ultra-white beetles Lepidiota stigma and Cyphochilus.

High resolution X-ray nano-tomography experiments are often limited to a few tens of micrometer size volumes due to detector size. It is possible, through the use of multiple overlapping tomography scans, to produce a large area scan which can encompass a sample in its entirety. Mounting and positioning regions to be scanned is highly challenging and normally requires focused ion beam approaches. In this work we have imaged intact beetle scale cells mounted on the tip of a needle using a micromanipulator stage. Here we show X-ray holotomography data for single ultra-white scales from the beetles Lepidiota stigma (L. stigma) and Cyphochilus which exhibit the most effective scattering of white light in the literature. The final thresholded matrices represent a scan area of 25 × 70 × 362.5 µm and 25 × 67.5 × 235µm while maintaining a pixel resolution of 25 nm. This tomographic approach allowed the internal structure of the scales to be captured completely intact and undistorted by the sectioning required for traditional microscopy techniques.

3D visualisation of hepatitis B vaccine in the oral delivery vehicle SBA-15.

Developing a technology that enables oral vaccines to work efficiently remains a considerable effort since a number of difficulties must be addressed. The key objective being to ensure the safe passage through the harsh conditions within the gastrointestinal tract, promoting delivery that induces enhanced immune response. In the particular case of hepatitis B, the oral formulation in the nanostructured silica SBA-15 is a viable approach. As a result of its porous structure, low toxicity and structural stability, SBA-15 is capable to protect and release the hepatitis B surface antigen (HBsAg), used in the vaccination scheme, at the desired destination. Furthermore, when compared to the currently used injection based delivery method, better or similar antibody response has been observed. However, information about the organisation of the antigen protein remains unknown. For instance, HBsAg is too large to enter the 10 nm ordered mesopores of SBA-15 and has a tendency to agglomerate when protected by the delivery system. Here we report on the pH dependence of HBsAg aggregation in saline solution investigated using small angle X-rays scattering that resulted in an optimisation of the encapsulation conditions. Additionally, X-ray microscopy combined with neutron and X-ray tomography provided full 3D information of the HBsAg clustering (i.e. agglomeration) inside the SBA-15 macropores. This method enables the visualisation of the organisation of the antigen in the interior of the delivery system, where agglomerated HBsAg coexists with its immunological effective uniformly distributed counterpart. This new approach, to be taken into account while preparing the formulation, can greatly help in the understanding of clinical studies and advance new formulations.

From spinodal decomposition to alternating layered structure within single crystals of biogenic magnesium calcite.

As organisms can form crystals only under ambient conditions, they demonstrate fascinating strategies to overcome this limitation. Recently, we reported a previously unknown biostrategy for toughening brittle calcite crystals, using coherently incorporated Mg-rich nanoprecipitates arranged in a layered manner in the lenses of a brittle star, Ophiocoma wendtii. Here we propose the mechanisms of formation of this functional hierarchical structure under conditions of ambient temperature and limited solid diffusion. We propose that formation proceeds via a spinodal decomposition of a liquid or gel-like magnesium amorphous calcium carbonate (Mg-ACC) precursor into Mg-rich nanoparticles and a Mg-depleted amorphous matrix. In a second step, crystallization of the decomposed amorphous precursor leads to the formation of high-Mg particle-rich layers. The model is supported by our experimental results in synthetic systems. These insights have significant implications for fundamental understanding of the role of Mg-ACC material transformation during crystallization and its subsequent stability.