Implications of X-ray beam profiles on qualitative and quantitative synchrotron micro-focus X-ray fluorescence microscopy.

Synchrotron radiation X-ray fluorescence microscopy is frequently used to investigate the spatial distribution of elements within a wide range of samples. Interrogation of heterogeneous samples that contain large concentration ranges has the potential to produce image artefacts due to the profile of the X-ray beam. The presence of these artefacts and the distribution of flux within the beam profile can significantly affect qualitative and quantitative analyses. Two distinct correction methods have been generated by referencing the beam profile itself or by employing an adaptive-thresholding procedure. Both methods significantly improve qualitative imaging by removing the artefacts without compromising the low-intensity features. The beam-profile correction method improves quantitative results but requires accurate two-dimensional characterization of the X-ray beam profile.

The Spectroscopy Village at Diamond Light Source.

This manuscript presents the current status and technical details of the Spectroscopy Village at Diamond Light Source. The Village is formed of four beamlines: I18, B18, I20-Scanning and I20-EDE. The village provides the UK community with local access to a hard X-ray microprobe, a quick-scanning multi-purpose XAS beamline, a high-intensity beamline for X-ray absorption spectroscopy of dilute samples and X-ray emission spectroscopy, and an energy-dispersive extended X-ray absorption fine-structure beamline. The optics of B18, I20-scanning and I20-EDE are detailed; moreover, recent developments on the four beamlines, including new detector hardware and changes in acquisition software, are described.

Metal Tolerance Protein 8 Mediates Manganese Homeostasis and Iron Reallocation during Seed Development and Germination.

Metal accumulation in seeds is a prerequisite for germination and establishment of plants but also for micronutrient delivery to humans. To investigate metal transport processes and their interactions in seeds, we focused on METAL TOLERANCE PROTEIN8 (MTP8), a tonoplast transporter of the manganese (Mn) subclade of cation diffusion facilitators, which in Arabidopsis (Arabidopsis thaliana) is expressed in embryos of seeds. The x-ray fluorescence imaging showed that expression of MTP8 was responsible for Mn localization in subepidermal cells on the abaxial side of the cotyledons and in cortical cells of the hypocotyl. Accordingly, under low Mn availability, MTP8 increased seed stores of Mn, required for efficient seed germination. In mutant embryos lacking expression of VACUOLAR IRON TRANSPORTER1 (VIT1), MTP8 built up iron (Fe) hotspots in MTP8-expressing cells types, suggesting that MTP8 transports Fe in addition to Mn. In mtp8 vit1 double mutant seeds, Mn and Fe were distributed in all cell types of the embryo. An Fe transport function of MTP8 was confirmed by its ability to complement Fe hypersensitivity of a yeast mutant defective in vacuolar Fe transport. Imbibing mtp8-1 mutant seeds in the presence of Mn or subjecting seeds to wet-dry cycles showed that MTP8 conferred Mn tolerance. During germination, MTP8 promoted reallocation of Fe from the vasculature. These results indicate that cell type-specific accumulation of Mn and Fe in seeds depends on MTP8 and that this transporter plays an important role in the generation of seed metal stores as well as for metal homeostasis and germination efficiency under challenging environmental conditions.

Slow lung clearance and limited translocation of four sizes of inhaled iridium nanoparticles.

BACKGROUND: Concerns have been expressed that inhaled nanoparticles may behave differently to larger particles in terms of lung clearance and translocation, with potential implications for their toxicity. Studies undertaken to investigate this have typically involved limited post-exposure periods. There is a shortage of information on longer-term clearance and translocation patterns and their dependence on particle size, which this study aimed to address. METHODS: Rats were exposed (<3 h) nose-only to aerosols of spark-generated radioactive iridium-192 nanoparticles of four sizes: 10 nm, 15 nm, 35 nm and 75 nm (count median diameter) (aerosol mass concentrations 17, 140, 430, and 690 µg/m3, respectively). The content of iridium-192 in the whole animal, organs, tissues, and excreta was measured at various times post-exposure to ≥ 1 month. Limited toxicological investigations were undertaken for the 10 nm aerosol using bronchoalveolar lavage fluid. Elemental maps of tissue samples were produced using laser ablation inductively coupled plasma mass spectrometry and synchrotron micro-focus x-ray fluorescence. The chemical speciation of the iridium was explored using synchrotron micro focus x-ray near-edge absorption spectroscopy. RESULTS: Long-term lung retention half-times of several hundred days were found, which were not dependent on particle size. There was significant variation between individual animals. Analysis of bronchoalveolar lavage fluid for the 10 nm aerosol indicated a limited inflammatory response resolving within the first 7 days. Low levels of, particle size dependent, translocation to the kidney and liver were found (maximum 0.4% of the lung content). Any translocation to the brain was below the limits of detection (i.e. < 0.01% of the lung content). The kidney content increased to approximately 30 days and then remained broadly constant or decreased, whereas the content in the liver increased throughout the study. Laser ablation inductively coupled plasma mass spectrometry analysis indicated homogeneous iridium distribution in the liver and within the cortex in the kidney. CONCLUSIONS: Slow lung clearance and a pattern of temporally increasing concentrations in key secondary target organs has been demonstrated for inhaled iridium aerosol particles < 100 nm, which may have implications for long-term toxicity, especially in the context of chronic exposures.

Phase and absorption retrieval using incoherent X-ray sources.

X-ray phase contrast imaging has overcome the limitations of X-ray absorption imaging in many fields. Particular effort has been directed towards developing phase retrieval methods: These reveal quantitative information about a sample, which is a requirement for performing X-ray phase tomography, allows material identification and better distinction between tissue types, etc. Phase retrieval seems impossible with conventional X-ray sources due to their low spatial coherence. In the only previous example where conventional sources have been used, collimators were employed to produce spatially coherent secondary sources. We present a truly incoherent phase retrieval method, which removes the spatial coherence constraints and employs a conventional source without aperturing, collimation, or filtering. This is possible because our technique, based on the pixel edge illumination principle, is neither interferometric nor crystal based. Beams created by an X-ray mask to image the sample are smeared due to the incoherence of the source, yet we show that their displacements can still be measured accurately, obtaining strong phase contrast. Quantitative information is extracted from only two images rather than a sequence as required by several coherent methods. Our technique makes quantitative phase imaging and phase tomography possible in applications where exposure time and radiation dose are critical. The technique employs masks which are currently commercially available with linear dimensions in the tens of centimeters thus allowing for a large field of view. The technique works at high photon energy and thus promises to deliver much safer quantitative phase imaging and phase tomography in the future.

In Situ Microfocus Chemical Computed Tomography of the Composition of a Single Catalyst Particle During Hydrogenation of Nitrobenzene in the Liquid Phase.

Heterogeneous catalysis performed in the liquid phase is an important type of catalytic process which is rarely studied in situ. Using microfocus X-ray fluorescence and X-ray diffraction computed tomography (µ-XRF-CT, µ-XRD-CT) in combination with X-ray absorption near-edge spectroscopy (XANES), we have determined the active state of a Mo-promoted Pt/C catalyst (NanoSelect) for the liquid-phase hydrogenation of nitrobenzene under standard operating conditions. First, µ-XRF-CT and µ-XRD-CT reveal the active state of Pt catalyst to be reduced, noncrystalline, and evenly dispersed across the support surface. Second, imaging of the Pt and Mo distribution reveals they are highly stable on the support and not prone to leaching during the reaction. This study demonstrates the ability of chemical computed tomography to image the nature and spatial distribution of catalysts under reaction conditions.

Combining µXANES and µXRD mapping to analyse the heterogeneity in calcium carbonate granules excreted by the earthworm Lumbricus terrestris.

The use of fluorescence full spectral micro-X-ray absorption near-edge structure (µXANES) mapping is becoming more widespread in the hard energy regime. This experimental method using the Ca K-edge combined with micro-X-ray diffraction (µXRD) mapping of the same sample has been enabled on beamline I18 at Diamond Light Source. This combined approach has been used to probe both long- and short-range order in calcium carbonate granules produced by the earthworm Lumbricus terrestris. In granules produced by earthworms cultured in a control artificial soil, calcite and vaterite are observed in the granules. However, granules produced by earthworms cultivated in the same artificial soil amended with 500 p.p.m. Mg also contain an aragonite. The two techniques, µXRD and µXANES, probe different sample volumes but there is good agreement in the phase maps produced.

A multi-modal high pressure and high temperature reaction cell for combined x-ray spectroscopy, scattering, and imaging.

A free-standing and compact reaction cell for combined in situ/operando x-ray spectroscopy, scattering, and imaging measurements at high pressures and high temperatures is described. The cell permits measurements under realistic operating conditions (up to 50 bar and 1000 °C), under static and flow conditions (up to 100 ml/min), over a wide range of hard x-ray energies, variable detection modes (transmission, fluorescence, and scattering), and at all angles of rotation. An operando XAS, x-ray fluorescence, x-ray computed tomography, and x-ray diffraction computed tomography case study on the reduction of a heterogeneous catalyst is presented to illustrate the performance of the reaction cell.

Correlating activities and defects in (photo)electrocatalysts using in-situ multi-modal microscopic imaging.

Photo(electro)catalysts use sunlight to drive chemical reactions such as water splitting. A major factor limiting photocatalyst development is physicochemical heterogeneity which leads to spatially dependent reactivity. To link structure and function in such systems, simultaneous probing of the electrochemical environment at microscopic length scales and a broad range of timescales (ns to s) is required. Here, we address this challenge by developing and applying in-situ (optical) microscopies to map and correlate local electrochemical activity, with hole lifetimes, oxygen vacancy concentrations and photoelectrode crystal structure. Using this multi-modal approach, we study prototypical hematite (α-Fe2O3) photoelectrodes. We demonstrate that regions of α-Fe2O3, adjacent to microstructural cracks have a better photoelectrochemical response and reduced back electron recombination due to an optimal oxygen vacancy concentration, with the film thickness and extended light exposure also influencing local activity. Our work highlights the importance of microscopic mapping to understand activity, in even seemingly homogeneous photoelectrodes.

A quantitative, non-interferometric X-ray phase contrast imaging technique.

We present a quantitative, non-interferometric, X-ray differential phase contrast imaging technique based on the edge illumination principle. We derive a novel phase retrieval algorithm which requires only two images to be acquired and verify the technique experimentally using synchrotron radiation. The technique is useful for planar imaging but is expected to be important for quantitative phase tomography also. The properties and limitations of the technique are studied in detail.

Quantitative 3D elemental microtomography of Cyclotella meneghiniana at 400-nm resolution.

X-ray fluorescence tomography promises to map elemental distributions in unstained and unfixed biological specimens in three dimensions at high resolution and sensitivity, offering unparalleled insight in medical, biological, and environmental sciences. X-ray fluorescence tomography of biological specimens has been viewed as impractical-and perhaps even impossible for routine application-due to the large time required for scanning tomography and significant radiation dose delivered to the specimen during the imaging process. Here, we demonstrate submicron resolution X-ray fluorescence tomography of a whole unstained biological specimen, quantifying three-dimensional distributions of the elements Si, P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn in the freshwater diatom Cyclotella meneghiniana with 400-nm resolution, improving the spatial resolution by over an order of magnitude. The resulting maps faithfully reproduce cellular structure revealing unexpected patterns that may elucidate the role of metals in diatom biology and of diatoms in global element cycles. With anticipated improvements in data acquisition and detector sensitivity, such measurements could become routine in the near future.

Intermediates during the Nucleation of Platinum Nanoparticles by a Reaction with Ethylene Glycol: Operando X-ray Absorption Spectroscopy Studies with a Microfluidic Cell.

Using operando X-ray absorption spectroscopy in a continuous-flow microfluidic cell, we have investigated the nucleation of platinum nanoparticles from aqueous hexachloroplatinate solution in the presence of the reducing agent ethylene glycol. By adjusting flow rates in the microfluidic channel, we resolved the temporal evolution of the reaction system in the first few seconds, generating the time profiles for speciation, ligand exchange, and reduction of Pt. Detailed analysis of the X-ray absorption near-edge structure and extended X-ray absorption fine structure spectra with multivariate data analysis shows that at least two reaction intermediates are involved in the transformation of the precursor H2PtCl6 to metallic platinum nanoparticles, including the formation of clusters with Pt-Pt bonding before complete reduction to Pt nanoparticles.

Multi-resolution Correlative Ultrastructural and Chemical Analysis of Carious Enamel by Scanning Microscopy and Tomographic Imaging.

Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.

Noninterferometric phase-contrast images obtained with incoherent x-ray sources.

We report on what are believed to be the first full-scale images obtained with the coded aperture concept, which uses conventional x-ray sources without the need to collimate/aperture their output. We discuss the differences in the underpinning physical principles with respect to other methods, and explain why these might lead to a more efficient use of the source. In particular, we discuss how the evaluation of the first imaging system provided promising indications on the method's potential to detect details invisible to conventional absorption methods, use an increased average x-ray energy, and reduce exposure times-all important aspects with regards to real-world implementations.

The nature of Pu-bearing particles from the Maralinga nuclear testing site, Australia.

The high-energy release of plutonium (Pu) and uranium (U) during the Maralinga nuclear trials (1955-1963) in Australia, designed to simulate high temperature, non-critical nuclear accidents, resulted in wide dispersion µm-sized, radioactive, Pu-U-bearing 'hot' particles that persist in soils. By combining non-destructive, multi-technique synchrotron-based micro-characterization with the first nano-scale imagining of the composition and textures of six Maralinga particles, we find that all particles display intricate physical and chemical make-ups consistent with formation via condensation and cooling of polymetallic melts (immiscible Fe-Al-Pu-U; and Pb ± Pu-U) within the detonation plumes. Plutonium and U are present predominantly in micro- to nano-particulate forms, and most hot particles contain low valence Pu-U-C compounds; these chemically reactive phases are protected by their inclusion in metallic alloys. Plutonium reworking was observed within an oxidised rim in a Pb-rich particle; however overall Pu remained immobile in the studied particles, while small-scale oxidation and mobility of U is widespread. It is notoriously difficult to predict the long-term environmental behaviour of hot particles. Nano-scale characterization of the hot particles suggests that long-term, slow release of Pu from the hot particles may take place via a range of chemical and physical processes, likely contributing to on-going Pu uptake by wildlife at Maralinga.

Geochemical investigations of noble metal-bearing ores: Synchrotron-based micro-analyses and microcosm bioleaching studies.

Auriferous sulphide ores often incorporate micro-fine (or invisible) gold and silver particles in a manner making their extraction difficult. Nobel metals are lost in the tailings due to the refractory nature of these ores. Bioleaching is an environment-friendly alternative to the commonly used and toxic cyanidation protocols for gold extraction from refractory ores. In this paper, we investigate gold and silver bioleaching from porphyry and epithermal mineralisation systems, using iron-oxidizing bacteria Acidithiobacillus ferrooxidans. The invisible Au, sequestered in refractory ores, was characterised in situ by synchrotron micro X-Ray Fluorescence (SR-µ-XRF) and X-ray Absorption Spectroscopy (XAS), offering information on Au unaltered speciation at the atomistic level within the ore matrices and at a micro-scale spatial resolution. The SR-µ-XRF and XAS results showed that 10-20 µm sized elemental Au(0) nuggets are sequestered in pyrite, chalcopyrite, arsenopyrite matrices and at the interface of a mixture of pyrite and chalcopyrite. Moreover, the preliminary bioleaching experiments of the two types of ores, showed that Acidithiobacillus ferrooxidans can catalyse the dissolution of natural heterogeneous Fe-rich geo-matrices, sequestering Au and Ag and releasing particulate phases or partially solubilising them within 60 days. These results provide an understanding of noble metal sequestration and speciation within natural ores and a demonstration of the application of synchrotron-based micro-analysis in characterizing economic trace metals in major mineral structures. This work is a contribution to the ongoing efforts towards finding feasible and greener solutions of noble metal extraction protocols.

More than the Eye Can See: Shedding New Light on SARS-CoV-2 Lateral Flow Device-Based Immunoassays.

Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen-antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to ∼10 000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.

Source size and temporal coherence requirements of coded aperture type x-ray phase contrast imaging systems.

There is currently much interest in developing X-ray Phase Contrast Imaging (XPCI) systems which employ laboratory sources in order to deploy the technique in real world applications. The challenge faced by nearly all XPCI techniques is that of efficiently utilising the x-ray flux emitted by an x-ray tube which is polychromatic and possesses only partial spatial coherence. Techniques have, however, been developed which overcome these limitations. Such a technique, known as coded aperture XPCI, has been under development in our laboratories in recent years for application principally in medical imaging and security screening. In this paper we derive limitations imposed upon source polychromaticity and spatial extent by the coded aperture system. We also show that although other grating XPCI techniques employ a different physical principle, they satisfy design constraints similar to those of the coded aperture XPCI.

The relationship between wave and geometrical optics models of coded aperture type x-ray phase contrast imaging systems.

X-ray phase contrast imaging is a very promising technique which may lead to significant advancements in medical imaging. One of the impediments to the clinical implementation of the technique is the general requirement to have an x-ray source of high coherence. The radiation physics group at UCL is currently developing an x-ray phase contrast imaging technique which works with laboratory x-ray sources. Validation of the system requires extensive modelling of relatively large samples of tissue. To aid this, we have undertaken a study of when geometrical optics may be employed to model the system in order to avoid the need to perform a computationally expensive wave optics calculation. In this paper, we derive the relationship between the geometrical and wave optics model for our system imaging an infinite cylinder. From this model we are able to draw conclusions regarding the general applicability of the geometrical optics approximation.

Limitations imposed by specimen phase gradients on the design of grating based x-ray phase contrast imaging systems.

X-ray phase contrast imaging is a very promising technique that may lead to significant advancements in a variety of fields, perhaps most notably, medical imaging. The radiation physics group at University College London is currently developing an x-ray phase contrast imaging technique that works with laboratory x-ray sources. This system essentially measures the degree to which photons are refracted by regions of an imaged object. The amount of refraction that may be expected to be encountered in practice impacts strongly upon the design of the imaging system. In this paper, we derive an approximate expression between the properties of archetypal imaged objects encountered in practice and the resulting distribution of refracted photons. This is used to derive constraints governing the design of the system.