Antony Van Der Ent

Martin D De Jonge

Kathryn M Spiers

Dennis Brueckner

Emmanuelle Montargès-Pelletier

Guillaume Echevarria

Xiaoming Wang

Mei Lei

Rachel Mak

James Lovett

Xiao-Ming Wan

Hugh H Harris

µ-Tomography Reveals Arsenic Three-Dimensional Distribution within Intact Pteris vittata Fronds

Keywords: chemical speciation, elemental distribution, hyperaccumulator, fern

INTRODUCTION

Hyperaccumulator plants are able to regulate potentially toxic concentrations of trace elements in their living tissues [START_REF] Krämer | Metal Hyperaccumulation in Plants[END_REF][START_REF] Pollard | The Genetic Basis of Metal Hyperaccumulation in Plants[END_REF][START_REF] Reeves | A global database for hyperaccumulator plants of metal and metalloid trace elements[END_REF] . Arsenic hyperaccumulation has been described for a number of pteridophytes (ferns) from the genus Pteris (Pteridaceae), most notably Pteris vittata, which accumulates up to 22 600 µg g -1 As [START_REF] Ma | A fern that hyperaccumulates arsenic[END_REF][START_REF] Tu | Effects of arsenate and phosphate on their accumulation by an arsenichyperaccumulator Pteris vittata L[END_REF] and Pityrogramma calomelanos which accumulates up to 16 400 µg g -1 As 6,7 in fronds. Phytoextraction with P. vittata has been successfully applied in the remediation of soil and groundwater As [START_REF] Chen | Accumulation of N, P and K in Pteris vittata L. during phytoremediation: a five-year field study[END_REF][START_REF] Xie | The Arsenic Hyperaccumulator Fern Pteris vittata L[END_REF][START_REF] Iriel | Biosorption of arsenic from groundwater using Vallisneria gigantea plants. Kinetics, equilibrium and photophysical considerations[END_REF] . The As hyperaccumulation mechanisms of P. vittata have elicited intensive research [START_REF] Vetterlein | Pteris vittata Revisited: Uptake of As and ist speciation; impactof P, role of phytochelatins and S[END_REF][START_REF] Poynton | Mechanisms of arsenic hyperaccumulation in Pteris species: root As influx and translocation[END_REF][START_REF] Danh | A Critical Review of the Arsenic Uptake Mechanisms and Phytoremediation Potential of Pteris vittata[END_REF][START_REF] Li | Arsenic, cadmium, copper, manganese, REE, selenium and zinc hyperaccumulator plants from China: a synthesis on the current state of knowledge[END_REF] . Arsenic content in P. vittata follows the order of pinnae > stipe > root, with most As distributed in the pinnae (78-96%). The concentration of As in the pinnae decreases from the base to the apex of the fronds, and As concentrations in midribs is much lower than in the pinnae [START_REF] Lombi | Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata[END_REF] .

The As concentration was relatively high in the midrib compared to the surrounding tissues, and there could be an unloading process of As from midrib to mesophyll tissues [START_REF] Chen | Distributions of arsenic and essential elements in pinnule of arsenic hyperaccumulator Pteris vittata L[END_REF] . An interesting finding is that As concentrations in spores were far lower than in the pinnae, indicating that P. vittata tends to keep As away from reproductive organelles [START_REF] Lombi | Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata[END_REF] . At the cellular level, energy dispersive X-ray spectroscopy combined to scanning electron microscopy (SEM-EDS) identified that As is primarily distributed within the upper and lower epidermis of the pinna in P. vittata [START_REF] Lombi | Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata[END_REF] . Analysis with X-ray fluorescence imaging (µXRF) led to the reporting of even higher As concentration in trichomes compared that prevailing As concentrations in the epidermis [START_REF] Li | Role of trichome of Pteris vittata L. in arsenic hyperaccumulation[END_REF] . At the subcellular level, As was mainly stored in the cytoplasmic supernatant fraction, accounting for 78% of As in the pinnae [START_REF] Chen | Subcellular distribution and compartmentalization of arsenic in Pteris vittata L[END_REF] .

Therefore, As compartmentalisation in trichomes and vacuoles was suggested as a detoxification mechanism in P. vittata [START_REF] Lombi | Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata[END_REF][START_REF] Li | Role of trichome of Pteris vittata L. in arsenic hyperaccumulation[END_REF] . Recently, localization of As in the apoplast near the midrib, in fronds of P. vittata was reported [START_REF] Datta | Evidence for exocellular Arsenic in Fronds of Pteris vittata[END_REF] . A similarity between the As and K distribution at both cellular and subcellular level was found [START_REF] Lombi | Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata[END_REF][START_REF] Huang | Difference of Toxicity and Accumulation of Methylated and Inorganic Arsenic in Arsenic-Hyperaccumulating and -Hypertolerant Plants[END_REF] , implying that K + may act as a counterbalance to As V O 4 3-anions.

Pteris vittata contains As predominantly as inorganic As species [START_REF] Zhang | Arsenic speciation and distribution in an arsenic hyperaccumulating plant[END_REF] . Regardless of whether As(III) or As(V) was supplied to the plants, the oxidized form As(V) dominated in the roots while As(III) dominated in the rhizomes and fronds [START_REF] Lombi | Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata[END_REF][START_REF] Zhang | Arsenic speciation and distribution in an arsenic hyperaccumulating plant[END_REF][START_REF] Huang | Direct determination of arsenic species in arsenic hyperaccumulator Pteris vittata by EXAFS[END_REF][START_REF] Kertulis | Arsenic speciation and transport in Pteris vittata L. and the effects on phosphorus in the xylem sap[END_REF][START_REF] Mathews | Arsenic transformation in the growth media and biomass of hyperaccumulator Pteris vittata L[END_REF][START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] , suggesting that the reduction of As(V) to As(III) occurs during As translocation. Furthermore, X-ray absorption spectroscopy indicated that, in the pinnae, As(III) was predominantly coordinated with oxygen [START_REF] Huang | Direct determination of arsenic species in arsenic hyperaccumulator Pteris vittata by EXAFS[END_REF][START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] . The precise location where As(V) is reduced to As(III) is still unknown. The reduction of As(V) to As(III) was firstly considered to occur in the pinnae as it was found that As in xylem sap was mainly As(V) [START_REF] Zhang | Arsenic speciation and distribution in an arsenic hyperaccumulating plant[END_REF][START_REF] Kertulis | Arsenic speciation and transport in Pteris vittata L. and the effects on phosphorus in the xylem sap[END_REF] . It was proposed that As(V) is transported through the vascular tissue from the roots to the fronds, where it is reduced to As(III) and stored at high concentrations [START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] . As(V) is reduced mainly in roots and not in the fronds [START_REF] Huang | Direct determination of arsenic species in arsenic hyperaccumulator Pteris vittata by EXAFS[END_REF][START_REF] Mathews | Arsenic transformation in the growth media and biomass of hyperaccumulator Pteris vittata L[END_REF] and root extracts from the P. vittata were able to reduce As(V) to As(III) [START_REF] Duan | Characterization of arsenate reductase in the extract of roots and fronds of Chinese brake fern, an arsenic hyperaccumulator[END_REF] . The reduction of As mainly occurs in the endodermis of roots [START_REF] Lei | First evidence on different transportation modes of arsenic and phosphorus in arsenic hyperaccumulator Pteris vittata[END_REF] . Arsenic is transported in the xylem sap of P. vittata predominantly (93-98%) as As(III), regardless of whether As(V) or As(III) was supplied to the plants, implying that the roots are the main site of As(V) reduction in P. vittata [START_REF] Su | Highly efficient xylem transport of arsenite in the arsenic hyperaccumulator Pteris vittata[END_REF] . Similar reduction of another metalloid, antimony (Sb), from pentavalent Sb to trivalent Sb has been reported in the roots of P. vittata [START_REF] Wan | Interaction of As and Sb in the hyperaccumulator Pteris vittata L.: changes in As and Sb speciation by XANES[END_REF] . However although clearly As(V) is reduced most effectively in root endodermis and then As(III) is transported most effectively in xylem, the identification of As(V) in other tissues means that not all of it is reduced in the root and the As(III) tris-glutathione [As(SR) 3 ] form found in the surroundings of the vasculature suggests that reduction can occur also outside the root [START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] .

Despite all the previously cited publications, no study to date has revealed the As-distribution at the cellular-scale in the P. vittata fronds, and previous data has only shown planar views of As (projected onto a 2D plane). As such no depth information is currently available and the in situ 3D distribution of As needs to be investigated and confirm the 2D visualisation. It is possible that observed differences in As speciation across tissues are due to spatial variations of As in different cell types [START_REF] Lei | First evidence on different transportation modes of arsenic and phosphorus in arsenic hyperaccumulator Pteris vittata[END_REF][START_REF] Caille | Comparison of root absorption, translocation and tolerance of arsenic in the hyperaccumulator Pteris vittata and the nonhyperaccumulator Pteris tremula[END_REF] . Pickering et al. [START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] examined both the gametophyte generation and the sporophyte generation, and found discrete speckles of arsenate localized in parenchymal cells (possibly in vacuoles) in the gametophytes. Another study on a related As-hyperaccumulating fern (Pityrogramma calomelanos) by Kachenko et al. [START_REF] Kachenko | Arsenic hyperaccumulation and localization in the pinnule and stipe tissues of the gold-dust fern (Pityrogramma calomelanos (L.) Link var. austroamericana (Domin) Farw.) using quantitative micro-PIXE spectroscopy[END_REF] showed substantial tissue-level differentiation in As accumulation but could not reveal As chemical speciation.

Conventional synchrotron (planar or 2D) µXRF of plant leaves (whole leaves or cross sections) is a projection of all elements in a sample. As such, a planar view of a plant leaf reveals the elements contained in the adaxial and abaxial epidermis and mesophyll together in a 'compressed' projected 2D plane. Several methods are available for revealing the detailed 3D elemental distributions relevant to complex biological samples. In this article we employ three complementary approaches to access the 3D elemental distributions: mechanical sectioning to access the distributions within a single plane, X-ray fluorescence µ-computed tomography (XFM-µCT) to determine volumetric distributions from a series of projection images without sample sectioning, and confocal imaging to directly access elemental distributions within targeted regions within the specimen. Although we describe these methods here, a more detailed comparison can be found in de Jonge & Vogt 32 and van der Ent et al. [START_REF] Van Der Ent | X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants[END_REF] .

Confocal optics enable a direct-space approach to deliver 3D resolution. A confocal optic confines the field of view of the energy-dispersive detector so that the signal derives from only a small portion of the illuminated column [START_REF] Kanngiesser | A new 3D micro X-ray fluorescence setup -first archaeometric applications[END_REF][START_REF] Janssens | Confocal microscopic X-ray fluorescence at the Hasylab microfocus beamline: characteristics and possibilities[END_REF][START_REF] Vincze | Three dimensional trace element analysis by confocal X-ray microfluorescence imaging[END_REF] . The probe volume is thereby reduced to a spheroid and elemental distribution is mapped in three dimensions by scanning the specimen through the probe volume. The use of confocal X-ray fluorescence methods for the investigation of biological samples has been limited to date, probably due to the increased X-ray dose associated with the confocal geometry in most situations 37 . Such an approach was, however, successfully utilized to decipher the distribution and chemical speciation of selenium in zebrafish larvae [START_REF] Choudhury | Selenium Preferentially Accumulates in the Eye Lens Following Embryonic Exposure: A Confocal X-ray Fluorescence Imaging Study[END_REF] .

Another approach to obtain information on the internal elemental distribution in physically intact samples is X-ray fluorescence µ-computed tomography (XFM-µCT). This technique enables reconstruction of 2D or 3D elemental data from a rotation series of projection images. For typical measurement modalities, µCT requires the measurement of projection images from multiples view around a fixed rotation axis. In order to avoid issues with self-absorption and to simplify the measurement, XFM-µCT is limited to measurements of specimen that are relatively thin in the two dimensions around the rotation axis, such as roots [START_REF] Kopittke | In Situ Distribution and Speciation of Toxic Copper, Nickel, and Zinc in Hydrated Roots of Cowpea[END_REF][START_REF] Lombi | Fast X-Ray Fluorescence Microtomography of Hydrated Biological Samples (JM Schnur[END_REF] , stems and narrow leaves [START_REF] Mcnear | Application of quantitative fluorescence and absorption-edge computed microtomography to image metal compartmentalization in Alyssum murale[END_REF] and seeds [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1[END_REF] , although µCT laminography could be used in flatter objects such as leaves. The confocal approach is more tolerant to self-absorption effects, and especially well-suited to measure depth profiles in extended planar objects such as leaves. Further, the confocal measurement enables direct access to small volumes within a specimen and so is amenable to direct and in situ spectroscopic (XANES) measurements.

Self-absorption is a combination of two effects that reduce the quantitative nature of reconstructions obtained by XFM-µCT and affect the visualization of element distribution: the absorption of the incident X-ray photons and the re-absorption of the fluorescent X-ray photons (in particular lowenergy photons) by the specimen (for a review see Vogt & de Jonge 32 ). Self-absorption is usually dominated by the latter of these effects, and this in turn has a greater impact on the quantitative determinations of the elements with low energy x-ray photons for K-shell emission this corresponds to with lower-Z elements. Self-absorption places a practical upper limit on the specimen dimensions, so that, for example, Ni can be mapped in hydrated biological samples up to ∼800 µm thick with reasonable accuracy (Kα line at 7.47 keV), but this increases up to ∼1500 µm for As (Kα line at 10.54 keV) assuming a tolerable limit of 50% self-absorption.

X-ray fluorescence microscopy (XFM) beamlines also offer the unique ability to perform in situ Xray absorption spectroscopy (XAS) to obtain information on the chemical speciation of elements (As in this case) in plant tissues without preparations and avoiding the possibility for artefacts resulting from those preparations. Moreover, the use of confocal optics allows examination of As chemical speciation at depth in distinct cell types within intact plant tissues and hence elucidation of the spatially resolved chemical speciation and elemental distribution in physically intact samples without the need for destructive sample preparation [START_REF] Van Der Ent | X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants[END_REF][START_REF] Kopittke | Synchrotron-Based X-Ray Fluorescence Microscopy as a Technique for Imaging of Elements in Plants[END_REF] .

The complexity of As distribution and chemical speciation requires a range of different methodological approaches to understand the 3D distribution at the cellular level and also for the speciation. This study uses a combination of different synchrotron X-ray methodological approaches to interrogate As distribution and chemical speciation in Pteris vittata tissues, which are summarised in Table 1. To date, depth-resolved information has not been available, and this study aims not only to unravel the As pathway in the As hyperaccumulating fern Pteris vittata, but also to demonstrate how a set of advanced synchrotron X-ray microscopy techniques can be complementary in revealing, at tissue and cellular levels, the distribution and chemical speciation of As in this hyperaccumulator plant.

MATERIALS AND METHODS

Cultivation and collection of plant tissues and soils

Tissue samples were obtained in one of two ways. Cultured tissue samples were obtained from P.

vittata grown in soil at the University of Queensland. The potting mix was spiked with an As(V) solution supplied as Na 2 HAsO 4 •7H 2 O prior to transplanting (dose rate was 1000 mg kg -1 as As). The plants were grown in a greenhouse for two months with an 11-hr daily photoperiod, a photon flux of >370 µmol m -2 s -1 , a day-night temperature range of 20-29°C and relative humidity of ~70%. Tissue samples of P. vittata were also collected from a contaminated site in Shimen, Hunan Province in south China. This site is heavily contaminated by As due to realgar ore smelting activities in the past (N29 o 39'33.88" E111 o 2'43.1") with a soil As concentration of 313 µg g -1 . The live plants, together with the soil attached to the roots, were transported from Hunan to Beijing, and maintained in the laboratory in Beijing. Tissue samples intended for synchrotron XFM analysis were excised with a razor blade and immediately shock-frozen using the metal mirror freezing technique (n which the sample is rapidly pressed against a block of solid copper cooled by at -196°C liquid N 2 . The samples were then wrapped in Al-foil and transported in a cryogenic container (at least -190°C) for storage and transport. Samples were also freeze-dried in a freeze-dryer (LABCONCO FreeZone 6) starting at -50°C until room temperature in 48 hours. Slow freeze-drying has been shown to avoid, or at least, reduce elemental re-distribution and tissue shrinkage [START_REF] Tylko | X-ray microanalysis of biological material in the frozen-hydrated state by PIXE[END_REF][START_REF] Wang | Micro-PIXE analyses of frozen-hydrated semi-thick biological sections Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms[END_REF] but may cause subcellular migration of dissolved elements to the nearest solid surface. The influence of freeze-drying on element chemical speciation is still contentious and depends on several factors such as the element itself, its chemical speciation and initial oxidation state [START_REF] George | X-ray-induced photo-chemistry and X-ray absorption spectroscopy of biological samples[END_REF][START_REF] Wang | Minimizing experimental artefacts in synchrotron based X-ray analyses of Fe speciation in plant tissues of rice plants[END_REF] .

Some physical cross-sections were prepared from the cultivated P. vittata fronds. After shock freezing (as described earlier), the samples were cryo-transferred on dry ice (-80°C) into the cryochamber of the microtome (maintained at -30°C) and mounted on a plate by application of chilled (-8°C) polypropylene glycol for physical support. The fronds were then cut to 30 µm sections using a cooled blade (-20 °C), and the sections transferred to pre-cooled 1 mL cryo-containers and freezedried.

Plant tissue bulk analysis

Plant tissue samples for bulk chemical analysis were gathered from the cultured ferns after two months, and from the freeze-dried fern samples from China. The samples were dried at 70°C for five days in a dehydrating oven, subsequently ground and a 200-mg fraction digested using 4 mL concentrated nitric acid (70%) and 1 mL hydrogen peroxide (30%) in a digestion microwave oven (Milestone Start D) for a 45-minute programme. After cooling, the sample was diluted to 30 mL with TDI water. The aliquots were analysed on ICP-AES (Varian Vista Pro II) for P, Mg, Ca, K, S and As.

Histochemistry and bright field microscopy

Frond samples were fixed in 3% glutaraldehyde (in PBS), sectioned with a vibratome (Leica VT1000s) and stained with Toluidine Blue. The sections (20-40 µm thickness) were then examined and photographed with bright field microscope (Nikon Diskovery Spinning Disk Confocal with Andor Zyla 4.2 camera).

Scanning Electron Microscopy with X-ray microanalysis (SEM-EDS)

Freeze-dried leaf samples were sputter-coated with carbon (25 nm) and mounted on stubs. The samples were then imaged with Scanning Electron Microscopy (SEM) with X-ray microanalysis (Energy dispersive X ray spectroscopy or EDS) on a JEOL JSM-6610 instrument. Images were obtained for 100-1000 x magnifications at 5-15 kV with lower accelerator energies for imaging with secondary electrons only and higher accelerator energies for imaging using backscattered electrons.

X-ray fluorescence microscopy (XFM)

The X-ray fluorescence microscopy (XFM) beamline of the Australian Synchrotron employs an invacuum undulator to produce a brilliant X-ray beam of 4.1-20 keV with a focus down to 1000 nm. A Si(111) monochromator and a pair of Kirkpatrick-Baez (KB) mirrors deliver a monochromatic focused incident beam onto the specimen [START_REF] Paterson | The x-ray fluorescence microscopy beamline at the Australian synchrotron[END_REF] . The P06 beamline of PETRA III (Deutsches Elektronen-Synchrotron; DESY) is also equipped with Si(111) monochromator and KB mirrors [START_REF] Schroeder | A Cryogenic Sample Environment for the Analysis of Frozen Hydrated Biological Tissue at the Hard X-ray Micro/Nano-Probe Beamline P06 at PETRA III[END_REF] producing Xray beam of 5-23 keV with a focus down to 300 nm. Both beamlines are equipped with a Maia detector consisting of a large detector array to maximize the detection of fluorescence signal. The Maia detector allows for high overall count-rates and uses an annular detector geometry, where the beam passes though the detector and strikes the sample at normal incidence [START_REF] Kirkham | The Maia Spectroscopy Detector System: Engineering for Integrated Pulse Capture, Low-Latency Scanning and Real-Time Processing[END_REF][START_REF] Siddons | Maia X-ray Microprobe Detector Array System[END_REF] . This geometry enables a large solid angle (1.2 steradian) to maximize detected signal and reducing the dose and potential damage to a specimen [START_REF] Ryan | Elemental X-ray imaging using the Maia detector array: The benefits and challenges of large solid-angle[END_REF] . Maia is designed for event-mode data acquisition, where each detected X-ray event is recorded, tagged by detector number in the array, position in the scan and other metadata [START_REF] Ryan | Maia X-ray fluorescence imaging: Capturing detail in complex natural samples[END_REF] . This approach eliminates readout delays and enables short pixel times (typically On the XFM beamline, the confocal µXRF and µXAS experiments used a polycapillary optic fitted on the SDD detector allowing for a depth resolution (dz) of ~10 µm (energy and hence elementdependant), while the beam spot size using the KB mirrors was ~2 µm. The resolution of the polycapillary optic limits the z-resolution (along beam axis) of confocal tomography, while the beam spot size limits the x-y-resolution. Accordingly, samples can be raster imaged with an effective probe volume probe of volume of ~10 × 2 × 2 µm. The scan was carried out in the x-z plane which samples the two smallest dimension of the pinnule -a virtual section -so that the y-axis is not probed, with a The µ-XANES spectra were measured using the Maia detector array with a ~1.7 ms per pixel dwell time.

down
Arsenic 'hotspots' were identified from the confocal µXRF images as regions of interest for volumetric XANES point measurements. Of particular interest is the reduction of arsenate to arsenite as As species cross from the vascular system to the leaf tissues, so regions were selected in the (tissues listed in decreasing proximity to main vascular system) midvein, endodermis, mesophyll and epidermis as well as regions in the secondary veins (vein bundles and surrounding protective sheaths) and sporangia (reproductive parts of the fern). In order to obtain spatial coordination, first coarse confocal µXRF maps were made of the sample. Volumetric XANES point measurements were acquired using the confocal optic attached to a Vortex SDD detector (as described above) and measured across the As K-edge (11 877 eV) at 100 energies spanning the energy range 11 802 to 12 017 eV in 0.25 eV steps with 0.5s dwell.

Energy calibration at the XFM beamline was achieved using a sample of solid sodium arsenite diluted in boron nitride. The "white line" peak energy of this sample was assumed to be 11874.4 eV.

The 2D µXRF and single-slice µXRF tomography measurements carried out in the microprobe of P06 at DESY were performed with a beam size of 532 × 444 nm at an incident energy of 14 keV.

Considering a minimum step size of 0.5 µm and comparing it to the size of the beam, the resolution of the resulting images can be treated as being equal to the step size. Samples consisted of a freestanding rachis and two pinnule margins, one mounted between Ultralene films for 2D measurements and the other mounted inside a Kapton capillary for tomography. All scans were conducted in continuous-scanning mode to minimize dead-time between the exposures with dwell times between 1 ms and 10 ms. In the case of single-slice tomography, the rotation stage was the slow scanning axis and the longer scans of the rachis were divided into eight interlaced sub-scans to account for possible vertical drift of the sample. All the samples were either freeze-dried (rachis) or dehydrated (pinnule margin) and so a cryo-stream was not employed.

Sample preparation and measurement conditions

All frozen-hydrated samples were scanned in using a cryo-stream, whereas the freeze-dried samples were scanned at room temperature (20°C). Individual freeze-dried pinnules were glued to carbon sticks with cyanoacrylate (Fig 1b,c), but larger samples (i.e. the intact 'fresh/live' frond) were sandwiched between two sheets of Kapton thin film (4 µm) stretched over a Perspex frame (Fig 1d)

magnetically attached to the x-y motion stage at atmospheric temperature (~20°C). The samples were scanned 'on-the-fly' with a 1-5 ms per pixel dwell at an incident energy of 15.8 keV. The frozenhydrated samples were transferred under the N 2 cryo-stream (-100°C) and were maintained at dry ice temperature during transfer -as such they were kept under cryogenic conditions from collection through to the completion of measurement.

Data analysis

The Maia X-ray fluorescence data were analysed using GeoPIXE [START_REF] Ryan | Quantitative analysis of PIXE spectra in geoscience applications[END_REF][START_REF] Ryan | Nuclear Microprobe -Synchrotron Synergy: Towards Integrated Quantitative Real-time Elemental Imaging using PIXE and SXRF[END_REF] , and images were generated using the Dynamic Analysis method [START_REF] Ryan | Dynamic analysis: on-line quantitative PIXE microanalysis and its use in overlap-resolved elemental mapping[END_REF][START_REF] Ryan | Quantitative trace element imaging using PIXE and the nuclear microprobe[END_REF] . Unfortunately, due to a technical error associated with the data writing to the cluster storage the raw data of a number of 2D µXRF elemental maps were irretrievably lost. However, real-time (RT) processing data from GeoPIXE were locally retained, and the corresponding pseudo-elemental maps are included in the Supplementary Information.

Self-absorption, which can lead to inaccuracy in the determination of elemental concentration by XRM, especially in 'thicker' samples, depends on the composition of the material, on the thickness of the sample, on the localisation of the element to be detected and on the element itself (energy of its X-ray fluorescence lines), on the distance between sample and the detector, on the energy of the incident beam. Assuming that fern tissue has a similar composition and density to that of cellulose (C 6 H 10 O 5 ) with an average thickness of 300 µm (a pinnule blade is somewhat thinner at ~80 µm whereas a rachis is thicker at ~500 µm) and a density of 1.0 g cm -3 then it is assumed that the As fluorescence (K alpha line et 10.5 KeV) maximum absorption on escape is ~10%.

Confocal XRF imaging data were analysed with MAPS. Because the (element-dependant) transmission efficiency of the polycapillary optic is not known, and because of the different geometric orientations of the sample (resulting in varying X-ray flux on the sample), the confocal elemental maps were not quantified with respect to concentrations of particular elements. The concentrations are hence plotted on a relative scale.

Analysis of the XFM-µCT data was achieved by alignment steps using consistency and crosscorrelation methods to correct for horizontal drift in the sinograms followed by a tomographic reconstruction step. The high angular sampling of the rachis allowed reconstruction using a filtered back-projection algorithm. However, the pinnule sinograms were recorded with lower angular sampling and so were reconstructed with a maximum-likelihood expectation-maximization algorithm based on the functions of the scikit-image image processing library.

Fluorescence-XANES mapping gave a 3D image stack of spatial dimensions (x, y) and an X-ray incident energy-resolved dimension E. Initially, the XANES images were aligned using a wellfeatured spectator element. Alternatively, one could consider each pixel (x, y) of the 477 × 260-pixel image stack to be associated with a unique XANES spectrum. The energy variation in the fluorescence spectra were identified using principal component analysis (PCA) allowing for up to six principal components, with the calculations performed using the Multivariate ANalysis Tool for Spectromicroscopy (MANTiS, Lerotic). Pixels possessing similar spectra were grouped together by k-means clustering, identifying three distinct clusters associated with the leaf, vein and image background (Figure 10). The cluster associated with the background of the image has been subtracted from composite image in Figure 10.

RESULTS.

Bulk chemistry and anatomical features

Although P. vittata can reach up to 22 600 µg g -1 As dry weight in its fronds [START_REF] Ma | A fern that hyperaccumulates arsenic[END_REF] , the cultured plants display 1354 µg g -1 and field collected plants from China have 8580 µg g -1 As on a dry weight basis (Table 2). The concentration of Ca is relatively high in the pinnae with up to 10 300 µg g -1 , whereas the K concentration is extremely high in both the pinnae and rachis ranging from 12 300 to 33 000 µg g -1 . The concentration of P is exceptionally high, especially in the samples from China with up to 62 700 µg g -1 in the pinnae and up to 67 000 µg g -1 in the rachis. Concentrations of Cu, Zn and Fe are unremarkable. Toluidine blue stained P. vittata sections of the rachis (Fig. 2) show the typical anatomy of a pteridophyte with the C-shaped elliptical vascular bundles surrounded by three-cell thick pericycle and endodermis cells. The mesophyll is not differentiated into palisade and spongy parenchyma, and mesophyll is absent from the mid-rib. Scanning Electron Microscopy (SEM) images show details of the sporangium with sporophytes (Fig. 3). Each sorus contains clusters of sporangia protected by the upper indusial flap ('false indusium') that is formed by the margins of the pinnule. The abundant presence of fertile cells is evident with the sporangium showing individual sori enclosed by large annulus cells.

Planar elemental imaging, confocal 'virtual' and computed tomography slices

The 2D projection µXRF elemental maps of the live frond (Fig. 4) show substantial enrichment of As in the vascular bundles, particularly in the rachis, but also in the veins of the pinnule. There is a depletion of As in the interveinal areas of the pinnule. The localisation of K is similar except that its concentration in the rachis is higher than the mean As concentration in fronds. In contrast, Ca is depleted in the rachis, but enriched in the veins of the pinnule particularly towards the terminal ends.

Calcium is also enriched in damaged (chloric) parts of the fronds, mainly along the margins. These differences are more pronounced in the high-resolution scans of the pinnule apex (Fig. 5), with Ca depleted in the veins but enriched along the pinnule margins. The distribution of As and K are again similar although the distribution of K extends into the lamina. In the freeze-dried pinnule portion towards the terminus (Suppl. Fig. S1 A,B), the distribution of As mirrors that of the frozen-hydrated samples with substantial enrichment in the vascular bundles, especially in the central vein. In fertile pinnules (Suppl. Fig. 1, C, D) strong accumulation of As in the sorus and sporangia is visible.

Further detail can be gleaned from scans of the sori (Suppl. Fig. 2). Apart from As enriched in the indusium, the strongest accumulation occurs in the annulus of each sporangium. Some of the individual spores are also rich in As (see top-right panel, Fig. 9), as are hairs in the sorus (for example bottom right panel Fig. 9). Enrichment in the hairs that guard the sporangium at the margin of the fronds has been noted previously [START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] .

Higher resolution 2D µXRF images (up to 0.5 µm) were collected of the (freeze-dried) pinna margin containing the indusium covering the sporangia, which reveals that As is enriched predominantly in the vascular bundles connecting to the sporangia, but little in the sori themselves (Suppl. Figs. 6,7, and 8). K is enriched along the indusium edge, and Zn highly localised in the mature sori.

Fluorescence computed µ-tomography slices and projections of an intact pinna (Fig. 8 and9, 0.5 µm pixels) similarly reveal As enrichment in the vascular structures, especially in nodes, but also in some sori.

Elemental maps of As, Br, Ca and Rb obtained from confocal 'virtual' cross-sections of a pinnule are shown in Suppl. Fig. 9. The self-absorption effect as a function of element z is clearly visible from the Ca elemental map with a 'shadow' evident behind the thicker midrib. For that reason, we have plotted maps of (high Z) Rb and Br to serve as proxies for K and Cl respectively. The elemental distribution of Rb is similar to As, which was expected on the basis of the earlier observed similarities between As and K. Arsenic is localised in the adaxial (upper) epidermis extending into the palisade mesophyll. Arsenic is also strongly enriched in the phloem of the (sub)lateral veins.

Confocal cross-sections of the central part of the pinnule with the midrib reveal that As is strongly accumulated in the endodermis and pericycle surrounding the phloem (Fig. 6). Arsenic is also strongly enriched in abaxial (lower) epidermal cells of the midrib (but not extending to the lamina).

Detailed confocal scans of the pinnule margins show accumulation of As in the indusium as well as in the sporangia, particularly the annulus (Suppl. Fig 10).

The elemental distribution of As in freeze-dried physical cross-sections scanned in 2D (Suppl. Fig. 34) also shows strong enrichment in the endodermis and pericycle surrounding the vascular bundles, as well as in the hypodermis extending into the cortex, ostensibly in the apoplastic space between the cells. Again, As is accumulated in the indusium and in the annulus of the sporangia. A scan of a physically sectioned freeze-dried rachis (Suppl. Fig. 5) shows the accumulation of As in the endodermis and pericycle surrounding the phloem. Fluorescence computed µ-tomography with 0.5 µm pixel size was undertaken on a freeze-dried rachis (Fig. 7) and reveals a very similar As enrichment pattern in the endodermis and pericycle surrounding the vascular bundles.

XANES imaging and volumetric XANES spot analysis

2D XANES imaging was performed on a frozen-hydrated pinnule. In addition, volumetric XANES measurements were acquired from spatial regions of interest in pinnae in both freeze-dried samples recorded at room temperature (Suppl. Figs. 11 and12) and frozen-hydrated state under a cryostream using a confocal optic.

The cluster spectra associated with the pinnule and vascular bundle areas respectively were compared to reference spectra of relevant As standards (figure 10C). On the basis of the energy position of the absorption maxima, and comparison to the spectrum of solid sodium arsenite recorded during the same beamtime run, cluster 1 and 2 spectra were assumed to correspond to As(III) (as arsenite) and As(V) (as arsenate), respectively. Both spectra are slightly broadened in comparison with reference spectra reproduced from previous study 25 that were recorded with higher energy resolution (0.2 instead of 0.5 eV). However, the spectrum extracted from cluster 2 in Fig 10 ,    primarily representing the main vascular bundle zone, displays an even broader absorption edge, displaying a shoulder at lower energy consistent with the peak energy of the arsenite model spectrum. This broadening is likely due to the presence of a mixture of As(III) and (V) species in the vascular bundles, where the relative abundance of each species is consistent across the cluster.

Volumetric XANES point measurements from a selection of different anatomical features returned spectra that were distinct from the model compound spectra. Absorption peak energies were aligned more closely to those of the As(III) standards, even in tissues where As(V) would be expected from previous work and from the 2D XANES imaging in this work (Figure 10). For instance, it is expected that the vascular bundles (regions G and H, Suppl Fig. 11) would exhibit As(V) prior to crossing the endodermis, however these bundles display reduced As(III) as stated from the peak energy. The peak height to background edge jump ratio in several of the volumetric XANES spectra is drastically attenuated relative to the model spectra. The results of the volumetric XANES analysis suggest that this approach delivers too high of a radiation dose to the sample to be reliable in determining the speciation of As in the specimen without drastic photoreduction, even in samples cooled under a liquid N 2 cryostream. In fact, point XANES spectra from freeze-dried tissues (Suppl Fig 12), which should be less susceptible to photoreduction due to the absence of water, show higher peak energies in some cases compared to those spectra from frozen tissue, strongly implicating photodamage in the unusual appearance of these spectra.

The XANES spectra collected using either 2D XANES imaging or confocal point XANES are compared below to model As compound spectra previously reported [START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] . We note that the spectral resolution of the beamline used in this work is significantly poorer than that from the previous work;

as such, any comparison should be interpreted with caution as the spectra from this study will be broadened significantly compared to the model compound spectra.

DISCUSSION

The use of confocal optics has made it possible to examine As in the depth profile of the tissues in physically intact samples with high aspect ratio, without the need for destructive sample preparation, and this approach has provided new information on the mechanisms underlying As hyperaccumulation in P. vittata. Confocal cross-sections of the central part of the pinnule with the midrib have revealed that As is strongly accumulated in the endodermis and pericycle surrounding the phloem. XFM µ-CT revealed a very similar As enrichment pattern in the endodermis and pericycle surrounding the vascular bundles. As such, the various technical approaches lead to conclusions that are entirely consistent with each other.

Confocal optics also offer the ability to perform volumetric XANES to obtain spatially resolved information on the chemical speciation within intact tissues. However, it eventuated that volumetric XANES causes substantial photodamage due to the very high radiation dose of this approach (60s total exposure per point), even if the sample is cryogenically cooled. Therefore, observations indicate that significant interpretation regarding As chemical form should not be drawn from the confocal volumetric XANES measurements, and reiterate the utility of 2D XANES imaging to provide chemical information with lower X-ray dose (0.0017s total exposure per pixel, or ~300 less than then confocal XANES measurements), albeit without the capacity to provide information in the third dimension in this case. The results of the 2D XANES imaging are congruent with previous knowledge of As speciation in P. vittata [START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] , which in part validates the novel confocal imaging in this work as it demonstrates that the sample preparation has not modified the distribution of the primary arsenic species in the tissues. Published 2D XANES imaging studies showed that different chemical forms of As (As(III) and As-(V)) occur across the plant organs [START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF] . Depth information of As chemical speciation in different cell types within tissues of P. vittata was not available and the knowledge of the distribution across different cells types within the fronds and rachis was limited [START_REF] Wan | Micro-distribution of arsenic species in tissues of hyperaccumulator Pteris vittata L[END_REF] . In this work we have demonstrated that observed differences in As speciation across tissues are related to spatial variation of As concentration in different cell types.

After the Zn-Ni-Cd hyperaccumulator Noccaea caerulescens, P. vittata is the most well-studied hyperaccumulator. As a consequence, much is now known about the uptake and metabolic regulation of As in this species [START_REF] Datta | Evidence for exocellular Arsenic in Fronds of Pteris vittata[END_REF][START_REF] Zhang | Arsenic speciation and distribution in an arsenic hyperaccumulating plant[END_REF][START_REF] Huang | Direct determination of arsenic species in arsenic hyperaccumulator Pteris vittata by EXAFS[END_REF][START_REF] Duan | Characterization of arsenate reductase in the extract of roots and fronds of Chinese brake fern, an arsenic hyperaccumulator[END_REF][START_REF] Pickering | Localizing the Biochemical Transformations of Arsenate in a Hyperaccumulating Fern[END_REF][START_REF] Wan | Micro-distribution of arsenic species in tissues of hyperaccumulator Pteris vittata L[END_REF][START_REF] Caille | Comparison of root absorption, translocation and tolerance of arsenic in the hyperaccumulator Pteris vittata and the nonhyperaccumulator Pteris tremula[END_REF][START_REF] Wan | Interaction of As and Sb in the hyperaccumulator Pteris vittata L.: changes in As and Sb speciation by XANES[END_REF] . Nevertheless, many open questions remain, specifically surrounding the adaptive advantages for As hyperaccumulation in this species, and about the physiological functions of As(V) to As(III) conversion for storage and translocation in the fronds. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap').

Supporting Information

Planar µXRF maps of As distribution in dehydrated Pteris vittata pinnule (Figure S1). Planar µXRF maps of As distribution in dehydrated Pteris vittata pinnule (Figure S2). Planar µXRF maps of As and K of a dehydrated cross-sectioned Pteris vittata pinnule (central portion) (Figure S3). Planar µXRF maps of As and K of a dehydrated cross-sectioned Pteris vittata pinnule (Figure S4). Planar µXRF maps of As and K of a dehydrated cross-sectioned Pteris vittata rachis (Figure S5). Planar Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap'). Scale bars denote µm.

  to 0.1 ms) and large pixel counts to be achieved for high definition imaging (typically 10-100 M pixels). The confocal experiment used a custom-made polycapillary collimating optic (XOS, input focal distance 2.5 mm, input field of view 10.0 µm, transmission efficiency optimized for 17.4 keV radiation) fitted on a Vortex SDD detector (Hitachi Vortex EM-90, 80mm 2 , minimum energy of 1.6 keV, energy resolution 120 eV, operated in 90-degree geometry). The experimental setup at the XFM beamline the Australian Synchrotron, showing both the Maia detector and SDD detector with confocal optic, is shown in Fig 1.

1

  second per pixel dwell time. The confocal µ-XRF images were recorded using a beam incident energy of 15.8 keV The fluorescence 2D XANES imaging at the XFM beamline was performed on a portion of a frozenhydrated pinnule, spanning the midvein and one margin containing sporangia. The fluorescence 2D XANES imaging consisted of forming 'stacks' of µXRF images by scanning the sample area multiple times at successive incident energies. As such, the sample was raster-scanned over a 2.380 ×. 1.295 mm window with a 5 µm step size, resulting in a µ-XANES image stack of 477 ×. 260 pixels. The sample was scanned over the As K-edge (11 877 eV) at 112 energies from 11 800 to 12 100 eV using 2 eV steps across the pre-edge region (11 800 to 11 855 eV), 0.5 eV steps across the XANES region (11 855 to 11 900 eV) and 20 eV across the post-edge region (11 900 to 12 100 eV).

Figure 1 .

 1 Figure 1. Experimental setup at the XFM beamline of the Australian Synchrotron: [A] Schematic diagram of confocal probe, SDD -silicon drift detector (image by Gary Ruben) [B] frozen-hydrated sample under the cryo-stream in front of the Maia detector; [C] Frozen-hydrated samples probed with the confocal optic; [D] Maia planar imaging at room temperature of a large live frond still attached to the plant (with rhizome in pouch with moist cotton). Typical dimensions of the probe beam are ~1 µm, those of the confocal field of view are ~10 um, and the specimen thickness is in the >100 µm domain.

Figure 2 .

 2 Figure 2. Toluidine blue stained Pteris vittata sections: [clockwise from top left] section of the rachis; section of the central part of the frond; unstained and stained sections of the sporophytes attached to the frond. Abbreviations: LE lower epidermis, UE upper epidermis, C cortex, P pamenta, VB vascular bundle, Ct cuticle, H hypodermis, M mesophyll, X xylem, E endodermis, Pe pericycle, Ph phloem, R receptacle, I false indusium, Ha hair, Ys young sporangium, Ms mature sporangium, A annulus, S stomium.

Figure 3 .

 3 Figure 3. Scanning Electron Microscopy (SEM) images of Pteris vittata tissues: [A] sporangium enclosures within the margin of the frond (indusium); [B] detail of sporophytes showing [C] vascular bundles of the rachis; [D] detail of central xylem bundles.

Figure 4 .

 4 Figure 4. Planar µXRF maps of Ca, K and As of a live Pteris vittata frond examined at room temperature. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap').

Figure 5 .

 5 Figure 5. Planar µXRF maps of Ca, K, As and Compton Scatter of the terminal ends of a live Pteris vittata frond examined at room temperature. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap').

Figure 6 .

 6 Figure 6. Confocal µXRF virtual section elemental maps of As in Pteris vittata fronds (central part/rachis). Panels: [A] freeze-dried f examined at room temperature; [B] frozen-hydrated examined under the cryostream; [C] freeze-dried f examined at room temperature; [D] frozen-hydrated examined under the cryostream. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap').

Figure 7

 7 Figure 7 Single-slice µXRF tomogram of a freeze-dried Pteris vittata rachis examined at room temperature showing As and Compton signals. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap').

Figure 8 .

 8 Figure 8. Single-slice µXRF tomograms of a freeze-dried Pteris vittata pinnule margin inside a Kapton capillary examined at room temperature showing As and Compton signals. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap').

Figure 9 .

 9 Figure 9. 2D µXRF maps of a freeze-dried Pteris vittata pinnule margin inside a Kapton capillary examined at room temperature showing As and Compton signals from different angles. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap').

Figure 10 .

 10 Figure 10. Left: As elemental map of a portion of Pteris vittata pinnule including midvein and leaf margin, taken at the top energy of the 2D XANES imaging scan (12.1 keV). Middle: composite of cluster maps generated by k-means clustering analysis with k = 3. Cluster 1 is shown and blue and cluster 2 is shown in red, the third cluster associated with the image background has been set to white. Right: the spectra of cluster 1 and cluster 2 reconstructed from principal components compared to reference As standards. The sample is frozen-hydrated examined under the cryostream.

µXRF

  maps of the K, As, Zn and Compton signals of a dehydrated Pteris vittata pinnule margin (Figure S6). Planar µXRF maps of the K, As, Zn and Compton signals of a dehydrated Pteris vittata pinnule margin (Figure S7). Planar µXRF maps of the K, As, Zn and Compton signals of a dehydrated Pteris vittata pinnule margin (Figure S8). Confocal µXRF maps of As, Br, Ca and Rb in a freeze-dried Pteris vittata pinnule showing the varying degrees of self-absorption depending on element z (Figure S9). Confocal µXRF maps of As in Pteris vittata pinnule (terminal end/sporangium) (Figure S10). As fluorescence map from confocal µXRF imaging of P. vittata sporangia recorded using the Vortex SDD from an incident beam energy of 15.8 keV, showing the locations of the volumetric XANES measurements taken at the sporangia, As fluorescence map from confocal µXRF imaging of P. vittata midvein, volumetric XANES spectra taken from the ROIs identified (FigureS11). Supplementary Figure12. As fluorescence map from confocal µXRF imaging of P. vittata sporangia, volumetric XANES spectra taken from the ROIs identified (FigureS12). Bright-field microscopy images of transversely cut sections of Pteris vittata samples (FigureS13).

Figure 1 .

 1 Figure 1. Experimental setup at the XFM beamline of the Australian Synchrotron: [A] Schematic diagram of confocal probe, SDD -silicon drift detector (prepared by Gary Ruben) [B] frozenhydrated sample under the cryo-stream in front of the Maia detector; [C] Frozen-hydrated samples probed with the confocal optic; [D] Maia planar imaging at room temperature of a large live frond still attached to the plant (with rhizome in pouch with moist cotton). Typical dimensions of the probe beam are ~1 µm, those of the confocal field of view are ~10 um, and the specimen thickness is in the >100 µm domain.

Figure 2 .

 2 Figure 2. Toluidine blue stained Pteris vittata sections: [clockwise from top left] section of the rachis; section of the central part of the frond; unstained and stained sections of the sporophytes attached to the frond. Abbreviations: LE lower epidermis, UE upper epidermis, C cortex, P pamenta, VB vascular bundle, Ct cuticle, H hypodermis, M mesophyll, X xylem, E endodermis, Pe pericycle, Ph phloem, R receptacle, I false indusium, Ha hair, Ys young sporangium, Ms mature sporangium, A annulus, S stomium. Scale bars denote µm.

Figure 3 .

 3 Figure 3. Scanning Electron Microscopy (SEM) images of Pteris vittata tissues: [A] sporangium enclosures within the margin of the frond (indusium); [B] detail of sporophytes showing [C] vascular bundles of the rachis; [D] detail of central xylem bundles. Scale bars denote µm.

Figure 4 .

 4 Figure 4. Planar µXRF maps of Ca, K and As of a live Pteris vittata of a partial frond with pinnae examined at room temperature. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap'). Scale bars denote µm.

Figure 5 .

 5 Figure 5. Planar µXRF maps of Ca, K, As and Compton Scatter of the terminal ends of a live Pteris vittata pinnules examined at room temperature. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap'). Scale bars denote µm.

Figure 6 .

 6 Figure 6. Confocal µXRF virtual section elemental maps of As in Pteris vittata pinnules (central part/rachis). Panels: [A] freeze-dried f examined at room temperature; [B] frozen-hydrated examined under the cryostream; [C] freeze-dried f examined at room temperature; [D] frozen-hydrated examined under the cryostream. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap'). Scale bars denote µm.

Figure 7

 7 Figure 7 Single-slice µXRF tomogram of a freeze-dried Pteris vittata rachis examined at room temperature showing As and Compton signals. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap'). Scale bars denote µm.

Figure 8 .

 8 Figure 8. Single-slice µXRF tomograms of a freeze-dried Pteris vittata pinnule margin inside a Kapton capillary examined at room temperature showing As and Compton signals. The same sample at different depths is shown. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap'). Scale bars denote µm.

Figure 9 .

 9 Figure 9. 2D µXRF maps of a freeze-dried Pteris vittata pinnule margin inside a Kapton capillary examined at room temperature showing As and Compton signals from different angles. Relative concentrations are depicted by shading with brighter colours equating to higher concentrations ('heatmap'). Scale bars denote µm.

Figure 10 .

 10 Figure 10. Left: As elemental map of a portion of Pteris vittata pinnule including midvein and leaf margin, taken at the top energy of the 2D XANES imaging scan (12.1 keV). Middle: composite of cluster maps generated by k-means clustering analysis with k = 3. Cluster 1 is shown and blue and cluster 2 is shown in red, the third cluster associated with the image background has been set towhite. Right: the spectra of cluster 1 and cluster 2 reconstructed from principal components compared to reference As standards. As the energy calibration of the standards was not performed at the beamline, both cluster spectra have been shifted down in energy by 1 eV to align the peaks more closely with those of the standards. The sample is frozen-hydrated examined under the cryostream.

TABLE CAPTIONS Table 1 .

 CAPTIONS1 Comparison of synchrotron X-ray methodological approaches that are available to an experimenter at XFM beamlines to interrogate As distribution and chemical speciation in Pteris vittata tissues. The exact values will change with the beamline.

Table 2 .

 2 Bulk elemental concentrations in fronds of Pteris vittata (values as ranges and means in µg g -1 dry weight) with ICP-AES.

Acknowledgements

This research was undertaken on the X-Ray Fluorescence Microscopy beamline of the Australian Synchrotron (part of ANSTO), Victoria, Australia. This work was supported by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE). Beamtime at beamline P06 at PETRA III was granted within the in-house research program of DESY, a member of the Helmholtz-Association. We would like to thank Jan Garrevoet for assistance during the experiments. We thank the Centre for Microscopy and Microanalysis, The University of Queensland. A. van der Ent was the recipient of a Discovery Early Career Researcher Award (DE160100429) from the Australian Research Council. H. Harris acknowledges the Australian Research Council for financial support (DP140100176). The French National Research Agency through the national "Investissements d'avenir" program (ANR-10-LABX-21, LABEX RESSOURCES21) and through the ANR-14-CE04-0005 Project "Agromine" is acknowledged for funding support to A. van der Ent. Finally, the Program "Bingwei" Excellent Talent in the Institute of Geographic Sciences and Natural Resources Research, CAS supported this work.

Author contributions

M.L and X-M.W collected the P. vittata samples in the field in China. A.vdE cultivated live P. vittata in Australia. X-M.W prepared freeze-dried samples for the synchrotron analysis. A.