Search

The Jezero crater floor features a suite of related, iron-rich lavas that were examined and sampled by the Mars 2020 rover Perseverance, and whose textures, minerals, and compositions were characterized by the Planetary Instrument for X-ray Lithochemistry (PIXL). This suite, known as the Máaz formation (fm), includes dark-toned basaltic/trachy-basaltic rocks with intergrown pyroxene, plagioclase feldspar, and altered olivine and overlying trachy-andesitic lava with reversely zoned plagioclase phenocrysts in a K-rich groundmass. Feldspar thermal disequilibrium textures indicate that they were carried from their crustal staging area. Bulk and mafic minerals have very high FeO and low MgO to FeO total ratios, which are partially reproduced by thermodynamic models involving high-degree fractional crystallization of a gabbroic assemblage and possibly also assimilation of iron-rich basement. Together, these in situ constraints on petrogenesis provide a uniquely detailed record of intracrustal processes beneath Jezero crater during a time period not represented by Mars samples to date.

Understanding the osteochondral junction, where non-mineralised cartilage and mineralised bone converge, is crucial for joint health. Current sample preparation techniques are insufficient for detailed spatial hyperspectral imaging analysis. Using the enhanced Kawamoto method, we used the super cryo embedding medium's temperature-dependent properties to transfer high-quality tissue samples onto slides for spatial imaging analysis. We transferred osteochondral samples using a tape-free system and successfully tested them in hematoxylin and eosin (HE), Safranin-O, nanomechanical assessments and nano-Fourier transform infrared (FTIR) mapping. This protocol elucidates the structural and elemental gradients, mechanical characteristics and distinctive biochemical layering, making it a useful tool for analysing biochemical properties' co-distribution in healthy and diseased situations., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Placental health and foetal development are dependent upon element homeostasis. Analytical techniques such as mass spectroscopy can provide quantitative data on element concentrations in placental tissue but do not show spatial distribution or co-localisation of elements that may affect placental function. The present study used synchrotron-based X-ray fluorescence microscopy to elucidate element content and distribution in healthy and pathological placental tissue. The X-ray fluorescence microscopy (XFM) beamline at the Australian Synchrotron was used to image trace metal content of 19 placental sections from healthy term ( n = 5, 37-39 weeks), foetal growth-restricted ( n = 3, <32 weeks, birth weight <3rd centile), postdate ( n = 7, >41 completed weeks), and stillbirth-complicated pregnancies ( n = 4, 37-40 weeks). Samples were cryo-sectioned and freeze-dried. The concentration and distribution of fourteen elements were detected in all samples: arsenic, bromine, calcium, chlorine, copper, iron, molybdenum, phosphorous, potassium, rubidium, selenium, strontium, sulphur, and zinc. The elements zinc, calcium, phosphorous, and strontium were significantly increased in stillbirth placental tissue in comparison to healthy-term controls. Strontium, zinc, and calcium were found to co-localise in stillbirth tissue samples, and calcium and strontium concentrations were correlated in all placental groups. Molybdenum was significantly decreased in stillbirth, foetal growth-restricted, and postdate placental tissue in comparison to healthy-term samples ( p < 0.0001). Synchrotron-based XFM reveals elemental distribution within biological samples such as the placenta, allowing for the co-localisation of metal deposits that may have a pathological role. Our pilot study further indicates low concentrations of placental molybdenum in pregnancies complicated by foetal growth restriction, postdate delivery, and stillbirth.

Silicon (Si) is recognized as a promising anode material for next-generation anodes due to its high capacity. However, large volume expansion and active particle pulverization during cycling rapidly deteriorate the battery performance. The relationship between Si anode particle size and particle pulverization, and the structure evolution of Si particles during cycling is not well understood. In this study, a quantitative, time-resolved "operando" small angle X-ray scattering (SAXS) investigation into the morphological change of unwrapped and reduced graphene oxide (rGO) wrapped Si nanoparticles (Si@rGO) is conducted with respect to the operating voltage. The results provide a clear picture of Si particle size change and the role of nonrigid rGO in mitigating Si volume expansion and pulverization. Further, this study demonstrates the advantage of "operando" SAXS in electrochemical environments as compared to other approaches., (© 2023 The Authors. Small Methods published by Wiley‐VCH GmbH.)

The Carajás plateaus in Brazil host endemic epilithic vegetation ("campo rupestre") on top of ironstone duricrusts, known as canga. This capping rock is primarily composed of iron(III) oxide minerals and forms a physically resistant horizon. Field observations reveal an intimate interaction between canga's surface and two native sedges (Rhynchospora barbata and Bulbostylis cangae). These observations suggest that certain plants contribute to the biogeochemical cycling of iron. Iron dissolution features at the root-rock interface were characterised using synchrotron-based techniques, Raman spectroscopy and scanning electron microscopy. These microscale characterisations indicate that iron is preferentially leached in the rhizosphere, enriching the comparatively insoluble aluminium around root channels. Oxalic acid and other exudates were detected in active root channels, signifying ligand-controlled iron oxide dissolution, likely driven by the plants' requirements for goethite-associated nutrients such as phosphorus. The excess iron not uptaken by the plant can reprecipitate in and around roots, line root channels and cement detrital fragments in the soil crust at the base of the plants. The reprecipitation of iron is significant as it provides a continuously forming cement, which makes canga horizons a 'self-healing' cover and contributes to them being the world's most stable continuously exposed land surfaces. Aluminium hydroxide precipitates ("gibbsite cutans") were also detected, coating some of the root cavities, often in alternating layers with goethite. This alternating pattern may correspond with oscillating oxygen concentrations in the rhizosphere. Microbial lineages known to contain iron-reducing bacteria were identified in the sedge rhizospheric microbiome and likely contribute to the reductive dissolution of iron(III) oxides within canga. Drying or percolation of oxygenated water to these anaerobic niches have led to iron mineralisation of biofilms, detected in many root channels. This study sheds light on plants' direct and indirect involvement in canga evolution, with possible implications for revegetation and surface restoration of iron mine sites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The growing amount of W mining waste produced globally is of concern for its proven hazard to the environment and to human health. While uncontrolled biooxidation can result in environmental harm, bioleaching, where pregnant leach solutions are controlled, has been widely used in the mining industry for valuable metals recovery, often from low-grade materials. This bioleaching study was developed to evaluate whether the biogeochemical reprocessing of W tailings could be employed for the decontamination of W-bearing mine waste, combined with valuable metals recovery, i.e., turning a waste into a resource. Using an in-vitro laboratory model, the susceptibility of wolframite [(Fe,Mn)WO 4 ] to acid dissolution during the concomitant oxidation of co-localized sulfidic minerals represented the basic strategy for enhanced W recovery. Encouragingly, geochemistry and synchrotron-based X-ray absorption near edge structure of weathered W tailings demonstrated that early-stage wolframite dissolution occurred. However, W dissolution was limited by the formation of secondary W minerals; weathering produced two secondary W minerals i.e., gallium-rich tungstate and minor sanmartinite [(Zn,Fe)WO 4 ]. The dissolution and re-precipitation of W minerals may provide a strategy for W waste reprocessing if the two processes can be separated by initially putting W into solution, and allowing for its extraction from tailings, followed by its' recovery by secondary W mineral formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The osteochondral interface is a thin layer that connects hyaline cartilage to subchondral bone. Subcellular elemental distribution can be visualised using synchrotron X-ray fluorescence microscopy (SR-XFM) (1 μm). This study aims to determine the relationship between elemental distribution and osteoarthritis (OA) progression based on disease severity. Using modified Mankin scores, we collected tibia plates from 9 knee OA patients who underwent knee replacement surgery and graded them as intact cartilage (non-OA) or degraded cartilage (OA). We used a tape-assisted system with a silicon nitride sandwich structure to collect fresh-frozen osteochondral sections, and changes in the osteochondral unit were defined using quantified SR-XFM elemental mapping at the Australian synchrotron's XFM beamline. Non-OA osteochondral samples were found to have significantly different zinc (Zn) and calcium (Ca) compositions than OA samples. The tidemark separating noncalcified and calcified cartilage was rich in zinc. Zn levels in OA samples were lower than in non-OA samples (P = 0.0072). In OA samples, the tidemark had less Ca than the calcified cartilage zone and subchondral bone plate (P < 0.0001). The Zn-strontium (Sr) colocalisation index was higher in OA samples than in non-OA samples. The lead, potassium, phosphate, sulphur, and chloride distributions were not significantly different (P > 0.05). In conclusion, SR-XFM analysis revealed spatial elemental distribution at the subcellular level during OA development., (© 2023. The Author(s).)

X-ray fluorescence spectroscopy (XRF) is a powerful technique for the in vivo assessment of plant tissues. However, the potential X-ray exposure damages might affect the structure and elemental composition of living plant tissues, leading to artefacts in the recorded data. Herein, we exposed in vivo soybean (Glycine max (L.) Merrill) leaves to several X-ray doses through a polychromatic benchtop microprobe X-ray fluorescence spectrometer, modulating the photon flux density by adjusting either the beam size, current, or exposure time. Changes in the irradiated plant tissues' structure, ultrastructure, and physiology were investigated through light and transmission electron microscopy (TEM). Depending on X-ray exposure dose, decreased K and X-ray scattering intensities and increased Ca, P, and Mn signals on soybean leaves were recorded. Anatomical analysis indicated the necrosis of epidermal and mesophyll cells on the irradiated spots, where TEM images revealed the collapse of cytoplasm and cell wall breaking. Furthermore, the histochemical analysis detected the production of reactive oxygen species and the inhibition of chlorophyll autofluorescence in these areas. Under certain X-ray exposure conditions, e.g. high photon flux density and long exposure time, XRF measurements may affect the soybean leaves structures, elemental composition, and cellular ultrastructure, inducing programmed cell death. Our characterization shed light on the plant's responses to the X-ray-induced radiation damage and might help to establish proper X-ray radiation limits and novel strategies for in vivo benchtop-XRF analysis of vegetal materials., (© The Author(s) 2023. Published by Oxford University Press.)

Wolframite [(Fe,Mn)WO 4 ] tailings represent a hazardous waste that can pose a threat to the environment, humans, animals and plants. The present study aims to conduct a high-resolution depth profile characterization of wolframite tailings from Wolfram Camp, North Queensland, Australia, to understand the biogeochemical influences on W mobilization. Several indigenous Fe- and S-oxidizing bacteria (e.g., Streptococcus pneumoniae and Thiomonas delicata) in wolframite tailings were found highly associated with W, As, and rare earth elements. Biooxidation of metal sulfides, i.e., pyrite, molybdenite and bismuthinite, produced sulfuric acid, which accelerated the weathering of wolframite, mobilizing tungstate (WO 4 2- ). Using synchrotron-based X-ray fluorescence microscopy (XFM) and W L-edge X-ray absorption near-edge spectroscopy (µ-XANES) analysis, wolframite was initially transformed into Na- and Bi- tungstate as well as tungstic acid (partial weathering) followed by the formation of Ga- and Zn- tungstate after extensive weathering, i.e., the wolframite had disappeared. While W (VI) was the major W species in wolframite tailings, minor W(0) and W(II), and trace W(IV) were also detected. The major contaminant in the Wolfram Camp tailings was As. Though wolframite tailings are hazardous waste, the toxicity of W was unclear. Tungsten waste still has industrial value; apart from using them as substitution material for cement and glass production, there is interest in reprocessing W waste for valuable metal recovery. If the environmental benefits are taken into consideration, i.e., preventing the release of toxic metals into surrounding waterways, reprocessing may be economic., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Collocated crystal sizes and mineral identities are critical for interpreting textural relationships in rocks and testing geological hypotheses, but it has been previously impossible to unambiguously constrain these properties using in situ instruments on Mars rovers. Here, we demonstrate that diffracted and fluoresced x-rays detected by the PIXL instrument (an x-ray fluorescence microscope on the Perseverance rover) provide information about the presence or absence of coherent crystalline domains in various minerals. X-ray analysis and multispectral imaging of rocks from the Séítah formation on the floor of Jezero crater shows that they were emplaced as coarsely crystalline igneous phases. Olivine grains were then partially dissolved and filled by finely crystalline or amorphous secondary silicate, carbonate, sulfate, and chloride/oxychlorine minerals. These results support the hypothesis that Séítah formation rocks represent olivine cumulates altered by fluids far from chemical equilibrium at low water-rock ratios.

The anatomy of the osteochondral junction is complex because several tissue components exist as a unit, including uncalcified cartilage (with superficial, middle, and deep layers), calcified cartilage, and subchondral bone. Furthermore, it is difficult to study because this region is made up of a variety of cell types and extracellular matrix compositions. Using X-ray fluorescence microscopy, we present a protocol for simultaneous elemental detection on fresh frozen samples. We transferred the osteochondral sample using a tape-assisted system and successfully tested it in synchrotron X-ray fluorescence. This protocol elucidates the distinct distribution of elements at the human knee's osteochondral junction, making it a useful tool for analyzing the co-distribution of various elements in both healthy and diseased states., (© 2022. The Author(s).)

The geological units on the floor of Jezero crater, Mars, are part of a wider regional stratigraphy of olivine-rich rocks, which extends well beyond the crater. We investigated the petrology of olivine and carbonate-bearing rocks of the Séítah formation in the floor of Jezero. Using multispectral images and x-ray fluorescence data, acquired by the Perseverance rover, we performed a petrographic analysis of the Bastide and Brac outcrops within this unit. We found that these outcrops are composed of igneous rock, moderately altered by aqueous fluid. The igneous rocks are mainly made of coarse-grained olivine, similar to some martian meteorites. We interpret them as an olivine cumulate, formed by settling and enrichment of olivine through multistage cooling of a thick magma body.

Over the last decade ptychography has progressed rapidly from a specialist ultramicroscopy technique into a mature method accessible to non-expert users. However, to improve scientific value ptychography data must reconstruct reliably, with high image quality and at no cost to other correlative methods. Presented here is the implementation of high-speed ptychography used at the Australian Synchrotron on the XFM beamline, which includes a free-run data collection mode where dead time is eliminated and the scan time is optimized. It is shown that free-run data collection is viable for fast and high-quality ptychography by demonstrating extremely high data rate acquisition covering areas up to 352 000 µm 2 at up to 140 µm 2  s -1 , with 13× spatial resolution enhancement compared with the beam size. With these improvements, ptychography at velocities up to 250 µm s -1 is approaching speeds compatible with fast-scanning X-ray fluorescence microscopy. The combination of these methods provides morphological context for elemental and chemical information, enabling unique scientific outcomes., (open access.)

Tumours are abnormal growths of cells that reproduce by redirecting essential nutrients and resources from surrounding tissue. Changes to cell metabolism that trigger the growth of tumours are reflected in subtle differences between the chemical composition of healthy and malignant cells. We used LA-ICP-MS imaging to investigate whether these chemical differences can be used to spatially identify tumours and support detection of primary colorectal tumours in anatomical pathology. First, we generated quantitative LA-ICP-MS images of three colorectal surgical resections with case-matched normal intestinal wall tissue and used this data in a Monte Carlo optimisation experiment to develop an algorithm that can classify pixels as tumour positive or negative. Blinded testing and interrogation of LA-ICP-MS images with micrographs of haematoxylin and eosin stained and Ki67 immunolabelled sections revealed Monte Carlo optimisation accurately identified primary tumour cells, as well as returning false positive pixels in areas of high cell proliferation. We analysed an additional 11 surgical resections of primary colorectal tumours and re-developed our image processing method to include a random forest regression machine learning model to correctly identify heterogenous tumours and exclude false positive pixels in images of non-malignant tissue. Our final model used over 1.6 billion calculations to correctly discern healthy cells from various types and stages of invasive colorectal tumours. The imaging mass spectrometry and data analysis methods described, developed in partnership with clinical cancer researchers, have the potential to further support cancer detection as part of a comprehensive digital pathology approach to cancer care through validation of a new chemical biomarker of tumour cells., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Examining chemical and structural characteristics of micro-features in complex tissue matrices is essential for understanding biological systems. Advances in multimodal chemical and structural imaging using synchrotron radiation have overcome many issues in correlative imaging, enabling the characterization of distinct microfeatures at nanoscale resolution in ex vivo tissues. We present a nanoscale imaging method that pairs X-ray ptychography and X-ray fluorescence microscopy (XFM) to simultaneously examine structural features and quantify elemental content of microfeatures in complex ex vivo tissues. We examined the neuropathological microfeatures Lewy bodies, aggregations of superoxide dismutase 1 (SOD1) and neuromelanin in human post-mortem Parkinson's disease tissue. Although biometals play essential roles in normal neuronal biochemistry, their dyshomeostasis is implicated in Parkinson's disease aetiology. Here we show that Lewy bodies and SOD1 aggregates have distinct elemental fingerprints yet are similar in structure, whilst neuromelanin exhibits different elemental composition and a distinct, disordered structure. The unique approach we describe is applicable to the structural and chemical characterization of a wide range of complex biological tissues at previously unprecedented levels of detail., Competing Interests: Authors have no competing interests to declare., (This journal is © The Royal Society of Chemistry.)

Objective: Damage to locus ceruleus neurons could play a part in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis because of impairment of the blood-brain barrier and enhanced neuroinflammation. The locus ceruleus has connections throughout the brain and spinal cord, so the characteristic widespread multifocal pathology in these disorders could be due to damage to different subsets of locus ceruleus neurons. Previous studies have shown that only certain locus ceruleus neurons accumulate the neurotoxic metal mercury. To find out if concentrations of other toxic metals or of essential trace elements also vary between individual locus ceruleus neurons, we used synchrotron X-ray fluorescence microscopy on frozen sections of locus ceruleus neurons taken from people with multiple sclerosis, in whom the locus ceruleus is structurally intact., Materials and Methods: Paraffin embedded sections containing the locus ceruleus from seven people with multiple sclerosis were stained with autometallography that demonstrates accumulations of mercury, silver and bismuth. These were compared to maps of multiple elements obtained from frozen sections of locus ceruleus neurons from the same people using X-ray fluorescence microscopy. Neurons in the anterior pons from three of these donors were used as internal controls., Results: Autometallography staining was observed in scattered locus ceruleus neurons from three of the seven donors. X-ray fluorescence microscopy showed variations among individual locus ceruleus neurons in levels of mercury, selenium, iron, copper, lead, bromine, and rubidium. Variations between donors of locus ceruleus neuronal average levels of mercury, iron, copper, and bromine were also detected. Anterior pons neurons contained no mercury, had varied levels of iron, and had lower copper levels than locus ceruleus neurons., Conclusions: Individual human locus ceruleus neurons contain varying levels of toxic metals and essential trace elements. In contrast, most toxic metals are absent or at low levels in nearby anterior pons neurons. The locus ceruleus plays a role in numerous central nervous system functions, including maintaining the blood-brain-barrier and limiting neuroinflammation. Toxic metals, or alterations in essential trace metals within individual locus ceruleus neurons, could be one factor determining the non-random destruction of locus ceruleus neurons in normal aging and neurodegenerative diseases, and subsequently the sites of the widespread multifocal central nervous system pathology in these disorders., Competing Interests: The authors have declared that no competing interests exist.

Background and Aims: X-ray fluorescence microscopy (XFM) is a powerful technique to elucidate the distribution of elements within plants. However, accumulated radiation exposure during analysis can lead to structural damage and experimental artefacts including elemental redistribution. To date, acceptable dose limits have not been systematically established for hydrated plant specimens., Methods: Here we systematically explore acceptable dose rate limits for investigating fresh sunflower (Helianthus annuus) leaf and root samples and investigate the time-dose damage in leaves attached to live plants., Key Results: We find that dose limits in fresh roots and leaves are comparatively low (4.1 kGy), based on localized disintegration of structures and element-specific redistribution. In contrast, frozen-hydrated samples did not incur any apparent damage even at doses as high as 587 kGy. Furthermore, we find that for living plants subjected to XFM measurement in vivo and grown for a further 9 d before being reimaged with XFM, the leaves display elemental redistribution at doses as low as 0.9 kGy and they continue to develop bleaching and necrosis in the days after exposure., Conclusions: The suggested radiation dose limits for studies using XFM to examine plants are important for the increasing number of plant scientists undertaking multidimensional measurements such as tomography and repeated imaging using XFM., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Electrochemical water splitting to generate hydrogen has been identified as a possible solution to the storage of intermittent renewable energy. However there are still challenges remaining in the development of stable electrocatalysts for the oxygen evolution half-reaction. Here we investigate the effects that the oxygen evolution reaction (OER) has on an electrodeposited Ni(OH)2 catalyst operated under alkaline conditions. The electrocatalyst was characterised by established methods including cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy both before and after the OER to identify changes that may have occurred in the structure and/or composition of the catalyst. In addition, synchrotron X-ray absorption near edge structure mapping was used to generate spatially resolved maps of the species present within the Ni(OH)2 catalyst and how they change in a heterogeneous manner into a NiO species after the OER. When compared to the morphological data it suggests that changes in the morphology after the OER can be correlated to the formation of NiO within the newly formed clusters that were generated across the electrocatalyst.

Biological X-ray fluorescence microscopy (XFM) is an important tool for determining quantitative distributions of bioinorganics and essential trace elements. Here we present a new analysis approach for rapid nanoscale ptychographic imaging and simultaneous chemical mapping of large radiation sensitive specimens without image degradation associated with probe evolution.

A unique combination of sensitivity, resolution, and penetration make X-ray fluorescence imaging (XFI) ideally suited to investigate trace elemental distributions in the biological context. XFI has gained widespread use as an analytical technique in the biological sciences, and in particular enables exciting new avenues of research in the field of neuroscience. In this study, elemental mapping by XFI was applied to characterise the elemental content within neuronal cell layers of hippocampal sub-regions of mice and rats. Although classical histochemical methods for metal detection exist, such approaches are typically limited to qualitative analysis. Specifically, histochemical methods are not uniformly sensitive to all chemical forms of a metal, often displaying variable sensitivity to specific "pools" or chemical forms of a metal. In addition, histochemical methods require fixation and extensive chemical treatment of samples, creating the strong likelihood for metal redistribution, leaching, or contamination. Direct quantitative elemental mapping of total elemental pools, in situ within ex vivo tissue sections, without the need for chemical fixation or addition of staining reagents is not possible with traditional histochemical methods; however, such a capability, which is provided by XFI, can offer an enormous analytical advantage. The results we report herein demonstrate the analytical advantage of XFI elemental mapping for direct, label-free metal quantification, in situ within ex vivo brain tissue sections. Specifically, we definitively characterise for the first time, the abundance of Fe within the pyramidal cell layers of the hippocampus. Localisation of Fe to this cell layer is not reproducibly achieved with classical Perls histochemical Fe stains. The ability of XFI to directly quantify neuronal elemental (P, S, Cl, K, Ca, Fe, Cu, Zn) distributions, revealed unique profiles of Fe and Zn within anatomical sub-regions of the hippocampus i.e., cornu ammonis 1, 2 or 3 (CA1, CA2 or CA3) sub-regions. Interestingly, our study reveals a unique Fe gradient across neuron populations within the non-degenerating and pathology free rat hippocampus, which curiously mirrors the pattern of region-specific vulnerability of the hippocampus that has previously been established to occur in various neurodegenerative diseases.

Analytical approaches that preserve the endogenous state of the examined system are essential for the in vivo study of bioinorganics. X-ray fluorescence microscopy of biological samples can map elements in vivo at subcellular resolutions in tissue samples and multicellular organisms. However, X-ray irradiation induces modifications that accumulate with dose. Consequently, the utility of X-ray fluorescence microscopy is intrinsically limited by the radiation damage it causes and the degree to which it alters the target features of interest. Identification of the dose threshold, below which the integrity of the specimen and its elemental distribution is preserved, is required to ensure valid interpretation of concentrations. Here we use the nematode, Caenorhabditis elegans, to explore these issues using three chemical-free specimen preparations: lyophilization, cryofixation, and live. We develop quantitative methods for investigating damage and present dose limits for each preparation pertaining to the micrometer-scale spatial distribution of specific analytes (potassium, calcium, manganese, iron, and zinc), and discuss dose-appropriate guidelines for X-ray fluorescence microscopy of microscale biological samples.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered 'random'. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.