The clinical frailty scale - does it predict outcome of the very-old in UK ICUs?

Introduction: The age of patients admitted into critical care in the UK is increasing. Clinical decisions for very-old patients, usually defined as over 80, can be challenging. Clinicians are frequently asked to predict outcomes as part of discussions around the pros and cons of an intensive care unit (ICU) admission. Measures of overall health in old age, such as the clinical frailty scale (CFS), are increasingly used to help guide these discussions. We aimed to understand the characteristics of the very-old critically unwell population in the UK and the associations between frailty and outcome of an ICU admission in our healthcare system (National Health Service, NHS). Methods: Baseline characteristics, ICU interventions and outcomes (ICU- and 30-day mortality) were recorded for sequential admissions of very old patients to UK ICUs as part of the European VIP 1 and 2 studies. Patient characteristics, interventions and outcome measures were compared by frailty group using standard statistical tests. Multivariable logistic regression modelling was undertaken to test association between baseline characteristics, admission type and outcome. Results: 1858 participants were enrolled from 95 ICUs in the UK. The median age was 83. The median CFS was 4 (IQR 3-5). 30-day survival was significantly lower in the frail group (CFS > 4, 58%) compared to vulnerable (CFS = 4, 65%) and fit (CFS < 4 68%, p = .004). Sequential organ failure assessment (SOFA) score, reason for admission and CFS were all independently associated with increased 30-day mortality (p < .01). Conclusion: In the UK, frailty is associated with an increase in mortality at 30-days following an ICU admission. At moderate frailty (CFS 5-6), three out of every five patients admitted survived to 30-days. This is a similar mortality to septic shock.

Enabling urban systems transformations: co-developing national and local strategies.

Transformative urban development is urgent to achieve future sustainable development and wellbeing. Transformation can benefit from shared and cumulative learning on strategies to guide urban development across local to national scales, while also reflecting the complex emergent nature of urban systems, and the need for context-specific and place-based solutions. The article addresses this challenge, drawing on extensive transdisciplinary engagement and National Strategy co-development processes for Australia. This includes generation of two frameworks as boundary objects to assist such transdisciplinary strategy development. An 'enabling urban systems transformation' framework comprises four generic overarching transformation enablers and a set of necessary underpinning urban capacities. This also built cumulatively on other sustainability and urban transformation studies. A complementary 'knowledge for urban systems transformation' framework comprises key knowledge themes that can support an integrated systems approach to mission-focused urban transformations, such as decarbonising cities. The article provides insights on the transdisciplinary processes, urban systems frameworks, and scoping of key strategies that may help those developing transformation strategies from local to national scales. Science highlights • Transdisciplinary national urban strategy development is used to distil generic frameworks and strategy scopes with potential international application. • The frameworks also build on other published framings to support convergent, cumulative and transdisciplinary urban science. • The 'enabling transformations' and 'urban knowledge' frameworks include the perspective of those developing sustainable urban systems strategies. • The enabling framework also informs 'National Urban Policy' and 'Knowledge and Innovation Hub' strategies, and prevailing power imbalances. • The knowledge framework can help frame urban challenges, missions and knowledge programs. Policy and practice recommendations • An urban 'transformation imperative' and 'strategic response' can be co-developed from local to national scales. • Local initiative is crucial to drive urban strategies, but sustained national leadership with coherent policy across sectors and scales is also key. • Diversity in engagement participation and processes generates whole-of-urban-systems and local-to-national perspectives. • Urban solutions are context-specific but generic frameworks can help collaborative issue framing and responses. • Collaborative issue framing informed by generic frameworks can bring broader perspectives to context-specific and contested policy and practice issues. Supplementary Information: The online version contains supplementary material available at 10.1186/s42854-023-00049-9.

Review and comparison of retinal vessel calibre and geometry software and their application to diabetes, cardiovascular disease, and dementia.

Developments in retinal imaging technologies have enabled the quantitative evaluation of the retinal vasculature. Changes in retinal calibre and/or geometry have been reported in systemic vascular diseases, including diabetes mellitus (DM), cardiovascular disease (CVD), and more recently in neurodegenerative diseases, such as dementia. Several retinal vessel analysis softwares exist, some being disease-specific, others for a broader context. In the research setting, retinal vasculature analysis using semi-automated software has identified associations between retinal vessel calibre and geometry and the presence of or risk of DM and its chronic complications, and of CVD and dementia, including in the general population. In this article, we review and compare the most widely used semi-automated retinal vessel analysis softwares and their associations with ocular imaging findings in common systemic diseases, including DM and its chronic complications, CVD, and dementia. We also provide original data comparing retinal calibre grading in people with Type 1 DM using two softwares, with good concordance.

X-ray fluorescence spectroscopy (XRF) for metallome analysis of herbarium specimens.

BACKGROUND: "Herbarium X-ray Fluorescence (XRF) Ionomics" is a new quantitative approach for extracting the elemental concentrations from herbarium specimens using handheld XRF devices. These instruments are principally designed for dense sample material of infinite thickness (such as rock or soil powder), and their built-in algorithms and factory calibrations perform poorly on the thin dry plant leaves encountered in herbaria. While empirical calibrations have been used for 'correcting' measured XRF values post hoc, this approach has major shortcomings. As such, a universal independent data analysis pipeline permitting full control and transparency throughout the quantification process is highly desirable. Here we have developed such a pipeline based on Dynamic Analysis as implemented in the GeoPIXE package, employing a Fundamental Parameters approach requiring only a description of the measurement hardware and derivation of the sample areal density, based on a universal standard. RESULTS: The new pipeline was tested on potassium, calcium, manganese, iron, cobalt, nickel, and zinc concentrations in dry plant leaves. The Dynamic Analysis method can correct for complex X-ray interactions and performs better than both the built-in instrument algorithms and the empirical calibration approach. The new pipeline is also able to identify and quantify elements that are not detected and reported by the device built-in algorithms and provides good estimates of elemental concentrations where empirical calibrations are not straightforward. CONCLUSIONS: The new pipeline for processing XRF data of herbarium specimens has a greater accuracy and is more robust than the device built-in algorithms and empirical calibrations. It also gives access to all elements detected in the XRF spectrum. The new analysis pipeline has made Herbarium XRF approach even more powerful to study the metallome of existing plant collections.

Biomolecular modifications in the sacfry of Mogurnda adspersa in response to copper stress.

Copper (Cu) is one of the most harmful contaminants in fresh-water systems. Fish larvae such as sacfry are particularly vulnerable to metals such as copper (Cu) due to a less-developed excretory organ system and permeable skin that can absorb metals directly from the water. However, the sublethal effects of metals on this life stage are not well understood. This study assessed the sublethal toxicity of Cu on purple-spotted gudgeon sacfry (PSG, Mogurnda adspersa). For this purpose, 96 h Cu toxicity bioassays were performed and toxic effects of Cu on PSG were measured at different levels of biological organization, from the individual (loss of equilibrium, wet weight), to tissue (chemical changes in retinal tissue composition) and molecular responses (whole body amino acid (AA) profiles). The EC10 and EC50 (ECx: effect concentration that affected X% of test organisms) were found to be 12 (9 - 15) µg Cu L-1 and 22 (19 - 24) µg Cu L-1, respectively. Copper stress caused a decrease in total amino acid content and changed the AA profile of PSG compared to the controls. Proton-induced X-ray emission (PIXE) mapping techniques showed accumulation of Cu in the retinal tissues disturbing the distribution of other elements such as zinc, sulfur, phosphorus and potassium. Fourier-transform infrared (FTIR) microspectroscopy of control and Cu treated eye tissues revealed a change in protein secondary structure in retinal tissues in response to Cu accumulation, as well as decreased levels of the molecular retinal, consistent with the degradation of rhodopsin, a key protein in the visual sensory system. This is the first study to demonstrate the multi-level responses of PSG arising from exposure to environmentally realistic Cu concentrations and suggests that AA profiling can serve as a useful tool to assess the impacts of metals on fresh-water organisms.

Multiple Organ Dysfunction in Older Major Trauma Critical Care Patients: A Multicenter Prospective Observational Study.

The objective was to explore the characteristics and outcomes of multiple organ dysfunction syndrome (MODS) in older trauma patients. Background: Severely injured older people present an increasing challenge for trauma systems. Recovery for those who require critical care may be complicated by MODS. In older trauma patients, MODS may not be predictable based on chronological age alone and factors associated with its development and resolution are unclear. Methods: Consecutive adult patients (aged ≥16 years) admitted to 4 level 1 major trauma center critical care units were enrolled and reviewed daily until discharge or death. MODS was defined by a daily total sequential organ failure assessment score of >5. Results: One thousand three hundred sixteen patients were enrolled over 18 months and one-third (434) were aged ≥65 years. Incidence of MODS was high for both age groups (<65 years: 64%, ≥65 years: 70%). There were few differences in severity, patterns, and duration of MODS between cohorts, except for older traumatic brain injury (TBI) patients who experienced a prolonged course of MODS recovery (TBI: 9 days vs no TBI: 5 days, P < 0.01). Frailty rather than chronological age had a strong association with MODS development (odds ratio [OR], 6.9; 95% confidence intervals [CI], 3.0-12.4; P < 0.001) and MODS mortality (OR, 2.1; 95% CI, 1.31-3.38; P = 0.02). Critical care resource utilization was not increased in older patients, but MODS had a substantial impact on mortality (<65 years: 17%; ≥65 years: 28%). The majority of older patients who did not develop MODS survived and had favorable discharge outcomes (home discharge ≥65 years NoMODS: 50% vs MODS: 15%; P < 0.01). Conclusions: Frailty rather than chronological age appears to drive MODS development, recovery, and outcome in older cohorts. Early identification of frailty after trauma may help to predict MODS and plan care in older trauma.

Multicenter phase II trial (SWOG S1609, cohort 51) of ipilimumab and nivolumab in metastatic or unresectable angiosarcoma: a substudy of dual anti-CTLA-4 and anti-PD-1 blockade in rare tumors (DART).

PURPOSE: Angiosarcoma is a rare aggressive endothelial cell cancer with high mortality. Isolated reports suggest immune checkpoint inhibition efficacy in angiosarcoma, but no prospective studies have been published. We report results for angiosarcoma treated with ipilimumab and nivolumab as a cohort of an ongoing rare cancer study. METHODS: This is a prospective, open-label, multicenter phase II clinical trial of ipilimumab (1 mg/kg intravenously every 6 weeks) plus nivolumab (240 mg intravenously every 2 weeks) for metastatic or unresectable angiosarcoma. Primary endpoint was objective response rate (ORR) per RECIST 1.1. Secondary endpoints include progression-free (PFS) and overall survival, and toxicity. A two-stage design was used. RESULTS: Overall, there were 16 evaluable patients. Median age was 68 years (range, 25-81); median number of prior lines of therapy, 2. Nine patients had cutaneous and seven non-cutaneous primary tumors. ORR was 25% (4/16). Sixty per cent of patients (3/5) with primary cutaneous scalp or face tumors attained a confirmed response. Six-month PFS was 38%. Altogether, 75% of patients experienced an adverse event (AE) (at least possibly related to drug) (25% grade 3-4 AE); 68.8%, an immune-related AE (irAE) (2 (12.5%), grade 3 or 4 irAEs (alanine aminotransferase/aspartate aminotransferase increase and diarrhea)). There were no grade 5 toxicities. One of seven patients in whom tumor mutation burden (TMB) was assessed showed a high TMB (24 mutations/mb); that patient achieved a partial response (PR). Two of three patients with PDL1 immunohistochemistry assessed had high PDL1 expression; one achieved a PR. CONCLUSION: The combination of ipilimumab and nivolumab demonstrated an ORR of 25% in angiosarcoma, with three of five patients with cutaneous tumors of the scalp or face responding. Ipilimumab and nivolumab warrant further investigation in angiosarcoma. TRIAL REGISTRATION NUMBER: NCT02834013.

Facilitating diabetic retinopathy screening using automated retinal image analysis in underresourced settings.

AIM: To evaluate an automated retinal image analysis (ARIA) of indigenous retinal fundus images against a human grading comparator for the classification of diabetic retinopathy (DR) status. METHODS: Indigenous Australian adults with type 2 diabetes (n = 410) from three remote and very remote primary-care services in the Northern Territory, Australia, underwent teleretinal DR screening. A single, central retinal fundus photograph (opportunistic mydriasis) for each eye was later regraded using a single ARIA and a UK human grader and national DR classification system. The sensitivity and specificity of ARIA were assessed relative to the comparator. Proportionate agreement and a Kappa statistic were also computed. RESULTS: Retinal images from 391 and 393 participants were gradable for 'Any DR' by the human grader and ARIA grader, respectively. 'Any DR' was detected by the human grader in 185 (47.3%) participants and by ARIA in 202 (48.6%) participants (agreement =88.0%, Kappa = 0.76,), whereas proliferative DR was detected in 31 (7.9%) and 37 (9.4%) participants (agreement = 98.2%, Kappa = 0.89,), respectively. The ARIA software had 91.4 (95% CI, 86.3-95.0) sensitivity and 85.0 (95% CI, 79.3-89.5) specificity for detecting 'Any DR' and 96.8 (95% CI, 83.3-99.9) sensitivity and 98.3 (95% CI, 96.4-99.4) specificity for detecting proliferative DR. CONCLUSIONS: This ARIA software has high sensitivity for detecting 'Any DR', hence could be used as a triage tool for human graders. High sensitivity was also found for detection of proliferative DR by ARIA. Future versions of this ARIA should include maculopathy and referable DR (CSME and/or PDR). Such ARIA software may benefit diabetes care in less-resourced regions.

Rare earth elements, aluminium and silicon distribution in the fern Dicranopteris linearis revealed by µPIXE Maia analysis.

BACKGROUND: The fern Dicranopteris linearis is a hyperaccumulator of rare earth elements (REEs), aluminium (Al) and silicon (Si). However, the physiological mechanisms of tissue-level tolerance of high concentrations of REE and Al, and possible interactions with Si, are currently incompletely known. METHODS: A particle-induced X-ray emission (µPIXE) microprobe with the Maia detector, scanning electron microscopy with energy-dispersive spectroscopy and chemical speciation modelling were used to decipher the localization and biochemistry of REEs, Al and Si in D. linearis during uptake, translocation and sequestration processes. RESULTS: In the roots >80 % of REEs and Al were in apoplastic fractions, among which the REEs were most significantly co-localized with Si and phosphorus (P) in the epidermis. In the xylem sap, REEs were nearly 100 % present as REEH3SiO42+, without significant differences between the REEs, while 24-45 % of Al was present as Al-citrate and only 1.7-16 % Al was present as AlH3SiO42+. In the pinnules, REEs were mainly concentrated in necrotic lesions and in the epidermis, and REEs and Al were possibly co-deposited within phytoliths (SiO2). Different REEs had similar spatial localizations in the epidermis and exodermis of roots, the necrosis, veins and epidermis of pinnae of D. linearis. CONCLUSIONS: We posit that Si plays a critical role in REE and Al tolerance within the root apoplast, transport within the vascular bundle and sequestration within the blade of D. linearis.

Imaging the eye and its relevance to diabetes care.

Diabetes is a major cause of vision loss globally, yet this devastating complication is largely preventable. Early detection and treatment of diabetic retinopathy necessitates screening. Ocular imaging is widely used clinically, both for the screening and management of diabetic retinopathy. Common eye conditions, such as glaucoma, cataracts and retinal vessel thrombosis, and signs of systemic conditions, such as hypertension, are frequently revealed. As well as imaging by a skilled clinician during an eye examination, non-ophthalmic clinicians, such as general practitioners, endocrinologists, nurses and trained health workers, can also can carry out diabetic eye screening. This process usually comprises local imaging with remote grading, mostly human grading. However, grading incorporating artificial intelligence is emerging. In a clinical research context, retinal vasculature analyses using semi-automated software in many populations have identified associations between retinal vessel geometry, such as vessel caliber, and the risk of diabetic retinopathy and other chronic complications of type 1 and type 2 diabetes. Similarly, evaluation of corneal nerves by corneal confocal microscopy is revealing diabetes-related abnormalities, and associations with and predictive power for other chronic diabetes complications. As yet, the value of retinal vessel geometry and corneal confocal microscopy measures at an individual level is uncertain. In this article, targeting non-ocular clinicians and researchers, we review existent and emerging ocular imaging and grading tools, including artificial intelligence, and their associations between ocular imaging findings and diabetes and its chronic complications.

Biogenicity of Spicular Geyserite from Te Kopia, New Zealand: Integrated Petrography, High-Resolution Hyperspectral and Elemental Analysis.

Hyperspectral and micro X-ray fluorescence (µXRF) imagery were used to derive maps of mineralogy and elemental chemistry from a sample of a siliceous hot spring deposit, or sinter, collected from a landslide breccia deposit at the base of the Paeroa fault, which bounds the eastern Taupo Rift at Te Kopia, Taupo Volcanic Zone, New Zealand. The sample is of a known biogenic sinter layer from a paleo-vent area of a recently extinct alkali chloride hot spring. The aim of the study was to distinguish it from other horizons derived from nonbiogenic sources, which is of relevance to early and extraterrestrial life research, specifically to help assess the potential reliability of morphology as an indicator of biology in the geological record. In particular, the distribution of opal, a common mineral in hot springs deposits that is known to preserve microbial features, and the relative abundances of Al-OH clay and water (OH and H2O) were mapped from hyperspectral imagery and element distributions defined by µXRF element mapping. Layers within the sinter sample composed of spicular geyserite-a type of micro-columnar stromatolite-showed contrasting mineralogy and water content in comparison with interspicular clastic sediment. Whereas clay was found to be concentrated in the interspicular sediment, high water contents characterized the spicules. µXRF imagery also showed differences in the composition of the two components of the spicule-bearing layers, with interspicular sediment being enriched in K, Ti, Fe, and Rb relative to the spicules, which are enriched in Ga. The contrasting nature of the mapped components highlights the detailed upward-branching nature of the spicules, identical to those found in living microstromatolites. These discriminants show that the spicular component can be discerned from the geological background through hyperspectral and µXRF mapping and used to define morphological features that may survive burial diagenesis and metamorphism as a biosignature in deep time rocks.

The XFM beamline at the Australian Synchrotron.

The X-ray fluorescence microscopy (XFM) beamline is an in-vacuum undulator-based X-ray fluorescence (XRF) microprobe beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the 4-27 keV energy range, permitting K emission to Cd and L and M emission for all other heavier elements. With a practical low-energy detection cut-off of approximately 1.5 keV, low-Z detection is constrained to Si, with Al detectable under favourable circumstances. The beamline has two scanning stations: a Kirkpatrick-Baez mirror microprobe, which produces a focal spot of 2 µm × 2 µm FWHM, and a large-area scanning `milliprobe', which has the beam size defined by slits. Energy-dispersive detector systems include the Maia 384, Vortex-EM and Vortex-ME3 for XRF measurement, and the EIGER2 X 1 Mpixel array detector for scanning X-ray diffraction microscopy measurements. The beamline uses event-mode data acquisition that eliminates detector system time overheads, and motion control overheads are significantly reduced through the application of an efficient raster scanning algorithm. The minimal overheads, in conjunction with short dwell times per pixel, have allowed XFM to establish techniques such as full spectroscopic XANES fluorescence imaging, XRF tomography, fly scanning ptychography and high-definition XRF imaging over large areas. XFM provides diverse analysis capabilities in the fields of medicine, biology, geology, materials science and cultural heritage. This paper discusses the beamline status, scientific showcases and future upgrades.

Formation of micro-spherulitic barite in association with organic matter within sulfidized stromatolites of the 3.48 billion-year-old Dresser Formation, Pilbara Craton.

The shallow marine and subaerial sedimentary and hydrothermal rocks of the ~3.48 billion-year-old Dresser Formation are host to some of Earth's oldest stromatolites and microbial remains. This study reports on texturally distinctive, spherulitic barite micro-mineralization that occur in association with primary, autochthonous organic matter within exceptionally preserved, strongly sulfidized stromatolite samples obtained from drill cores. Spherulitic barite micro-mineralization within the sulfidized stromatolites generally forms submicron-scale aggregates that show gradations from hollow to densely crystallized, irregular to partially radiating crystalline interiors. Several barite micro-spherulites show thin outer shells. Within stromatolites, barite micro-spherulites are intimately associated with petrographically earliest dolomite and nano-porous pyrite enriched in organic matter, the latter of which is a possible biosignature assemblage that hosts microbial remains. Barite spherulites are also observed within layered barite in proximity to stromatolite layers, where they are overgrown by compositionally distinct (Sr-rich), coarsely crystalline barite that may have been sourced from hydrothermal veins at depth. Micro-spherulitic barite, such as reported here, is not known from hydrothermal systems that exceed the upper temperature limit for life. Rather, barite with near-identical morphology and micro-texture is known from zones of high bio-productivity under low-temperature conditions in the modern oceans, where microbial activity and/or organic matter of degrading biomass controls the formation of spherulitic aggregates. Hence, the presence of micro-spherulitic barite in the organic matter-bearing Dresser Formation sulfidized stromatolites lend further support for a biogenic origin of these unusual, exceptionally well-preserved, and very ancient microbialites.

Endosperm prevents toxic amounts of Zn from accumulating in the seed embryo - an adaptation to metalliferous sites in metal-tolerant Biscutella laevigata.

Seed germination represents the first crucial stage in the life cycle of a plant, and the seed must contain all necessary transition elements for the development and successful establishment of the seedling. Problematically, seed development and germination are often hampered by elevated metal(loid) concentrations in industrially polluted soils, making their revegetation a challenging task. Biscutella laevigata L. (Brassicaceae) is a rare perennial pseudometallophyte that can tolerate high concentrations of trace metal elements. Yet, the strategies of this and other plant species to ensure reproductive success at metalliferous sites are poorly understood. Here we characterized several parameters of germination and used synchrotron X-ray fluorescence microscopy to investigate the spatial distribution and concentration of elements within B. laevigata seeds from two metallicolous and two non-metallicolous populations. We find that average germination time was shorter and the seed weight was lower in the metallicolous compared to the non-metallicolous populations. By allowing for at least two generations within one growth season, relatively fast germination at metalliferous sites accelerates microevolutionary processes and likely enhances the potential of metallicolous accessions to adapt to environmental stress. We also identified different strategies of elemental accumulation within seed tissues between populations. Particularly interesting patterns were observed for zinc, which was found in 6-fold higher concentrations in the endosperm of metallicolous compared to non-metallicolous populations. This indicates that the endosperm protects the seed embryo from accumulating toxic concentrations of metal(loid)s, which likely improves reproductive success. Hence, we conclude that elemental uptake regulation by the seed endosperm is associated with enhanced metal tolerance and adaptation to metalliferous environments in B. laevigata.

The impact of steeping, germination and hydrothermal processing of wheat (Triticum aestivum L.) grains on phytate hydrolysis and the distribution, speciation and bio-accessibility of iron and zinc elements.

Chelation of iron and zinc in wheat as phytates lowers their bio-accessibility. Steeping and germination (15 °C, 120 h) lowered phytate content from 0.96% to only 0.81% of initial dry matter. A multifactorial experiment in which (steeped/germinated) wheat was subjected to different time (2-24 h), temperature (20-80 °C) and pH (2.0-8.0) conditions showed that hydrothermal processing of germinated (15 °C, 120 h) wheat at 50 °C and pH 3.8 for 24 h reduced phytate content by 95%. X-ray absorption near-edge structure imaging showed that it indeed abolished chelation of iron to phytate. It also proved that iron was oxidized during steeping, germination and hydrothermal processing. It was further shown that zinc and iron bio-accessibility were respectively 3 and 5% in wheat and 27 and 37% in hydrothermally processed wheat. Thus, hydrothermal processing of (germinated) wheat paves the way for increasing elemental bio-accessibility in whole grain-based products.

Publisher Correction: Natural gold particles in Eucalyptus leaves and their relevance to exploration for buried gold deposits.

This corrects the article DOI: 10.1038/ncomms3614.

Sustainable urban systems: Co-design and framing for transformation.

Rapid urbanisation generates risks and opportunities for sustainable development. Urban policy and decision makers are challenged by the complexity of cities as social-ecological-technical systems. Consequently there is an increasing need for collaborative knowledge development that supports a whole-of-system view, and transformational change at multiple scales. Such holistic urban approaches are rare in practice. A co-design process involving researchers, practitioners and other stakeholders, has progressed such an approach in the Australian context, aiming to also contribute to international knowledge development and sharing. This process has generated three outputs: (1) a shared framework to support more systematic knowledge development and use, (2) identification of barriers that create a gap between stated urban goals and actual practice, and (3) identification of strategic focal areas to address this gap. Developing integrated strategies at broader urban scales is seen as the most pressing need. The knowledge framework adopts a systems perspective that incorporates the many urban trade-offs and synergies revealed by a systems view. Broader implications are drawn for policy and decision makers, for researchers and for a shared forward agenda.

X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants.

Contents Summary 432 I. Introduction 433 II. Preparation of plant samples for X-ray micro-analysis 433 III. X-ray elemental mapping techniques 438 IV. X-ray data analysis 442 V. Case studies 443 VI. Conclusions 446 Acknowledgements 449 Author contributions 449 References 449 SUMMARY: Hyperaccumulators are attractive models for studying metal(loid) homeostasis, and probing the spatial distribution and coordination chemistry of metal(loid)s in their tissues is important for advancing our understanding of their ecophysiology. X-ray elemental mapping techniques are unique in providing in situ information, and with appropriate sample preparation offer results true to biological conditions of the living plant. The common platform of these techniques is a reliance on characteristic X-rays of elements present in a sample, excited either by electrons (scanning/transmission electron microscopy), protons (proton-induced X-ray emission) or X-rays (X-ray fluorescence microscopy). Elucidating the cellular and tissue-level distribution of metal(loid)s is inherently challenging and accurate X-ray analysis places strict demands on sample collection, preparation and analytical conditions, to avoid elemental redistribution, chemical modification or ultrastructural alterations. We compare the merits and limitations of the individual techniques, and focus on the optimal field of applications for inferring ecophysiological processes in hyperaccumulator plants. X-ray elemental mapping techniques can play a key role in answering questions at every level of metal(loid) homeostasis in plants, from the rhizosphere interface, to uptake pathways in the roots and shoots. Further improvements in technological capabilities offer exciting perspectives for the study of hyperaccumulator plants into the future.