Distribution of aluminium in hydrated leaves of tea (Camellia sinensis) using synchrotron- and laboratory-based X-ray fluorescence microscopy

Antony van der Ent*a, Peter M. Kopittkeb, David J. Patersonc, Lachlan W. Caseyd and Philip Nti Nkrumaha
aThe University of Queensland, Sustainable Minerals Institute, St Lucia, Queensland 4072, Australia. E-mail: a.vanderent@uq.edu.au
bThe University of Queensland, School of Agriculture and Food Sciences, St Lucia, Queensland 4072, Australia
cAustralian Synchrotron, ANSTO, Clayton, Victoria 3086, Australia
dThe University of Queensland, Centre for Microscopy and Microanalysis, St Lucia, Queensland 4072, Australia

Received 6th December 2019 , Accepted 27th March 2020

First published on 27th March 2020

Aluminium (Al) is highly toxic to plant growth, with soluble concentrations being elevated in the ∼40% of arable soils worldwide that are acidic. Determining the distribution of Al in plant tissues is important for understanding the mechanisms by which it is toxic and how some plants tolerate high concentrations. Synchrotron- and laboratory-based X-ray fluorescence microscopy (XFM) is a powerful technique to quantitatively analyse the distribution of elements, including in hydrated and living plants. However, analysis of light elements (z < phosphorus) is extremely challenging due to signal losses in air, and the unsuitability of vacuum environments for (fresh) hydrated plant tissues. This study uses XFM in a helium environment to avoid Al signal loss to reveal the distribution of Al in hydrated plant tissues of Tea (Camellia sinensis). The results show that Al occurs in localised areas across the foliar surface, whereas in cross-sections Al is almost exclusively concentrated in the apoplastic space above and in between adaxial epidermal cells. This distribution of Al is related to the Al tolerance of this species, and accumulation of phytotoxic elements in the apoplastic space, away from sensitive processes such as photosynthesis in the palisade mesophyll cells, is a common tolerance mechanism reported in many different plant species. This study develops an XFM method on both synchrotron and laboratory sources that overcomes the drawbacks of existing analytical techniques, permitting measurement of light elements down to Al in (fresh) hydrated plant tissues.

Significance to metallomics

Determining the distribution of aluminium in plant tissues is important for understanding both the mechanisms by which aluminium exerts its toxic effect as well as for understanding how some plants are able to tolerate high concentrations of aluminium in the rooting medium. This study used X-ray fluorescence microscopy to reveal the distribution of aluminium in fresh hydrated plant tissues for the first time.

1. Introduction

Aluminium (Al) is the third most abundant element in soils,1 and is not essential to higher plants. In many soils, the Al is of only very low solubility and hence is not toxic. However, solubility increases in acid soils which comprise ca. 4 billion ha of the global ice-free land.2 Elevated concentrations of soluble Al are highly toxic to plant roots. For most crop plants, root growth is reduced markedly by 5–50 μM Al,3 with this being an important growth-limiting factor for crops in acid soils. In contrast, some plants tolerate elevated levels of Al in acidic soils, especially in the highly weathered soils of tropical regions. Aluminium hyperaccumulation (>1000 μg g−1 foliar Al) has been reported in plants from distant phylogenetic origins around the world.4 The most extreme records of Al accumulation are in the Symplocaceae family. The leaves of Symplocos spicata can accumulate a staggering 72[thin space (1/6-em)]300 μg g−1 foliar Al (i.e. 7.23 wt% Al).5 Tea (Camellia sinensis) is another particularly well-known Al hyperaccumulator, and can accumulate up to 30[thin space (1/6-em)]000 μg g−1 foliar Al.6 Most teas (fermented black and non-fermented green) use the flush of young leaves, while for other teas (brick tea), older mature leaves are used. Much remains unknown regarding the ecophysiology mechanisms underlying Al hyperaccumulation. Camellia sinensis is typically cultivated on soils of low pH that are high in soluble Al.7 Aluminium concentrations increase with leaf age, with at least ten-fold greater concentrations in old leaves compared to young leaves.7

Determining the distribution of Al in plant tissues is important for understanding both the mechanisms by which Al exerts its toxic effect as well as for understanding how some plants are able to tolerate high concentrations of Al in the rooting medium. We are aware of several studies that have used a variety of approaches for examining Al in plant tissues. A range of analyses is possible in a vacuum environment if the samples can be fully dehydrated (e.g. lyophilized) or examined in frozen-hydrated state. Synchrotron-based low energy X-ray fluorescence (LEXRF) has been used to investigate leaves of tea8 as well as roots of soybean (Glycine max),9 providing a resolution of ∼0.7 μm. Similarly, micro-beam particle-induced X-ray emission (PIXE) has been used on dehydrated leaves of tea again showing Al localization in epidermal cell walls,10 with co-localization of silicon (Si).11 NanoSIMS has also been used for investigating Al in soybean roots,9 providing excellent subcellular resolution (∼0.3 μm). The requirement for dehydration is a limitation of all of these methods, as this requires extensive sample preparation and carries a significant risk of damage to the structures of interest.12 Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been used by Klug, et al.13 to investigate Al in roots of the Al-accumulator, Fagopyrum esculentum in a hydrated form via a helium (He) environment. However, this caused substantial dehydration of the sample during analysis as the root could not be protected. As a result, the authors were only able to utilize a one-dimensional ablation path across a root cross-section (cf. two-dimensional elemental mapping). Finally, it is important to note that histochemical approaches using either chromogenic ligands or fluorescent probes have been used (for example, see Babourina and Rengel14), including in living plants. However, it is known that histochemical approaches are only able to detect labile forms of Al. For example, it was shown by Eticha, et al.15 that morin is not able to stain the cell-wall bound Al, with this being important when assessing the Al distribution in plant tissues.

In the present study, we aim to develop X-ray fluorescence microscopy (XFM) as an approach for the in situ analyses of Al in hydrated plant tissues. XFM is a powerful technique that can be used to quantitatively analyse the distribution of elements in physically large, intact samples, at room temperature and atmospheric pressure.16–18 Rapid improvements in fluorescence detector systems and other beamline components have reduced the required dwell time and therefore imposed radiation dose to the point where living plants can be analysed (for examples, see19,20). However, the absorption of low-energy fluorescence X-rays in the air-path between detector and sample causes extremely poor sensitivity for light elements where analyses are not conducted in vacuum. Consequently, it has not been practical to investigate light elements beyond P in hydrated and fresh plant tissues. Therefore, in the present study, we sought to develop approaches using synchrotron- and laboratory-based XFM for the analysis of light elements in hydrated tissues, making use of the increased transmission in a He environment while sealing samples between ultra-thin layers of film to counteract accelerated dehydration. We have focussed on Al, given its difficulty of measurement and physiological importance. As such, this study aimed at elucidating the in situ Al distribution in (fresh) hydrated foliar samples of C. sinensis originating from a tea estate in Queensland, Australia. Samples were analysed at the XFM beamline of the Australian Synchrotron21 (high-resolution scans of cross-sections) and using a laboratory-based XFM instrument (whole hydrated leaves) to determine the localisation of Al within tissues and organs respectively.

2. Materials and methods

2.1 Plant material

Tea (C. sinensis) foliar samples were obtained from the Bryn Hill Tea Estate, Malanda, Queensland (Australia). Malanda is located in the Atherton Tablelands at 738 m above sea-level. The annual rainfall is ∼4000 mm, and average temperature range is 17–28 °C in January and 5–22 °C in July. The tea grows on red Ferralsols with a pH of 4.7–5.1 (1[thin space (1/6-em)]:[thin space (1/6-em)]5 soil[thin space (1/6-em)]:[thin space (1/6-em)]water) in which the cation exchange complex is dominated by Al. Whole woody branches (stems ∼1 cm diameter) were excised from 35-year old trees at the estate, wrapped in moistened paper and transported directly to the XFM beamline in Melbourne (see later). Other plant tissues (seed, seed coat, flower, flower bud, young leaves, old leaves and twigs) were oven-dried and used for bulk analyses (see further below).

2.2 Bulk chemical analysis of plant samples

The plant material samples were oven-dried at 70 °C for 5 d. Each sample was weighed, ground to fine powder and digested using 4 mL HNO3 (70%) in a microwave oven (Milestone Start D) for a 45 min programme and diluted to 40 mL with ultrapure water. The digestates were analysed with inductively coupled plasma atomic emission spectroscopy (ICP-AES) with a Thermo Scientific iCAP 7400 instrument for the elements sodium (Na), magnesium (Mg), Al, phosphorus (P), sulphur (S), potassium (K), calcium (Ca), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu) and zinc (Zn) in radial and axial modes depending on the element and expected analyte concentration. All elements were calibrated with a four-point curve covering analyte ranges in the samples. In-line internal addition standardization using yttrium was used to compensate for matrix-based interferences. Quality controls included matrix blanks, certified reference material (Sigma-Aldrich Periodic table mix 1 for ICP TraceCERT®, 33 elements, 10 mg L−1 in HNO3), Standard Reference Material (NIST Apple 1515 digested with HNO3), and internal reference material (powdered Rinorea bengalensis leaves digested with HNO3).

2.3 Synchrotron-based XFM

The X-ray fluorescence microscopy (XFM) beamline of the Australian Synchrotron (ANSTO, Australia) employs an in-vacuum undulator to produce a brilliant X-ray beam of 4.1–20 keV (flux up to 4.0 × 1011 photons s−1) with a focus down to ∼1 μm. A Si(111) monochromator and a Kirkpatrick–Baez (K/B) pair of mirrors delivers a monochromatic focused beam onto the specimen.21 The beamline is equipped with a large-area (42 mm2) silicon drift detector (SDD) (Hitachi Vortex EM) coupled to ultra-fast processing electronics with a FalconX system operating in list-mode to GeoPIXE. In order to maximize Al fluorescence (Al absorption edge is 1.56 keV) by greater X-ray cross-section, an incident beam energy of 5.70 keV was chosen to excite elements of interest up to Ca (the Ca absorption edge is 4.04 keV). The SDD was fitted with a carbon collimator cone with a central opening of 2.5 mm diameter. Helium was purged (∼1 L min−1) through the side of the cone and out from the opening, thereby creating a helium path between sample-detector. The cone opening to sample distance was kept at the minimum value possible (∼1 mm). The effectiveness of the helium purge path could be verified by the presence of argon (Ar) fluorescence in the acquired spectrum. Samples were scanned in a 45-degree geometry relative to the incident beam. The flux at sample typically was 5.0 × 1010 photons s−1.

2.4 Laboratory-based XFM

The microXRF facility at The University of Queensland employs a modified IXRF ATLAS X system, with access to two 50 W X-ray sources fitted with polycapillary focussing optics: XOS microfocus molybdenum (Mo)-target tube producing 17.4 keV X-rays (flux of 2.2 × 108 ph s−1) focussing to 25 μm full-width half-max (FWHM) and a rhodium-target tube producing 20.2 keV (flux of 1.0 × 107 ph s−1) focussing to 5 μm FWHM. The system is fitted with two KETEK H150 SDDs of 170 mm2 collimated to 150 mm2 coupled to a XIA Mercury X4 signal processing unit. Typical energy resolution is <145 eV with a maximum input count rates of 2 M counts per second. The motion stage can address areas up to 300 × 300 mm. Measurements were conducted at atmospheric temperature (∼20 °C), using the Mo 25 μm X-ray source at a 40 kV, 1000 μA, with a risetime of 0.25 μs and a per-pixel dwell of 100 ms. The analysis chamber was purged with He until Ar fluorescence was not detectable prior to measurement, and a continuous flow of 1 L min−1 was maintained for the duration of the scan.

2.5 Specimen preparation and mounting and XFM analysis

For the synchrotron-based XFM, samples were prepared as small (1 × 1 cm2) foliar fragments leaves that were excised from the living donor plant immediately before the analysis and sealed between one sheet of polypropylene thin-film (Ultralene®, 4 μm thickness) stretched over a plastic frame, and another 1.5 μm Etnom® thin film on top facing the detector. This special film has negligible absorption of Al fluorescent X-rays. The tight sandwich reduced air pockets and prevents dehydration of the sample during analysis. Furthermore, tissue samples of the petiole and leaf blade were sectioned with a razor blade to ∼0.5 mm thick sections (using a ‘dry knife’ method to avoid elemental deportment and losses), and within 30 seconds mounted between the thin film sandwich as described above. All XFM analyses were conducted immediately after mounting the sample.

For the laboratory-based XFM, whole, hydrated leaves were excised from the living donor plant immediately before the analysis and sealed between one sheet of polypropylene thin-film (4 μm thickness) stretched over a plastic frame, and another 1.5 μm Etnom® thin film facing the detector.

2.6 Data processing

The synchrotron XFM data event stream was analysed using the Dynamic Analysis method22,23 as implemented in GeoPIXE.24 The matrix file used for the spectra fitting was an assumed plant material composition of C7.3O33H59N0.7S0.8 with a density of 0.90 g cm−3 and considering one layer of Ultralene (4 μm) and one layer of Etnom (1.5 μm).

In the laboratory system, elemental maps were generated in the Iridium software package (IXRF systems) from the sum of the counts at the position of the principal peak for each element, producing single-channel maps of relative concentration, internally normalised from 0 to 255 for minimum-to-maximum signal.

3. Results

3.1 Bulk elemental concentrations in plant parts

Chemical analysis after acid digestion (Tables 1 and 2) confirmed that older (basal) leaves have significantly high Al concentrations (7170–9850 μg g−1, mean of 8160 μg g−1) than younger (apical) leaves (715–4810 μg g−1, mean of 2780 μg g−1). Aluminium concentrations were lower in other plant parts, and increased in the order flower < seed < flower bud < seed coat < twigs < young leaves < old leaves (Fig. 1). Potassium concentrations were consistently high in all plant parts, reaching up to 27[thin space (1/6-em)]500 μg g−1 in the seed coat, and a mean of 16[thin space (1/6-em)]500 μg g−1 in the young leaves, but relatively lower in the old leaves (mean of 7230 μg g−1). Calcium concentrations are also high in plant parts, with up to 15[thin space (1/6-em)]000 μg g−1 in young leaves and up to 11[thin space (1/6-em)]200 μg g−1 in old leaves. Magnesium concentrations are highest in the young leaves with up to 11[thin space (1/6-em)]200 μg g−1 (mean of 5800 μg g−1). Phosphorus concentrations are highest in the flower (mean of 2860 μg g−1) and seed (mean of 2350 μg g−1), but up to 2540 μg g−1 in young leaves. Iron (mean of 95 μg g−1 in old leaves) and Cr (<11.5 μg g−1 in young leaves) concentrations are unremarkable, while Cu concentrations are in the expected ranges for healthy plants (mean of 8.5 μg g−1 and 6.0 μg g−1 in young and old leaves respectively).25 Zinc concentrations too are in expected ranges for normal plants, with the highest concentrations record in the flower buds (up to 45 μg g−1), whereas the old leaves contained on average 25 μg g−1.25 Nickel concentrations are slightly enriched, at 4.3–9.7 μg g−1 (mean of 5.9 μg g−1) in old leaves. The leaves have relatively high concentrations of Mn with 915–3600 μg g−1 in young leaves and 1230–1920 μg g−1 in old leaves. The latter is visible in necrotic spots characteristic for Mn phyto-toxicity.26
Table 1 Bulk elemental concentrations based on dry weight (μg g−1) in various plant parts of Camellia sinensis determined by ICP-AES after acid digestion (macro-elements). Values are reported in ranges and means, and <LOD depicts below limit of detection
Plant part n Al Na K Ca Mg P
Seed 2 130–140 130–305 16[thin space (1/6-em)]500–16[thin space (1/6-em)]100 840–1540 1590–1910 2250–2450
135 215 16[thin space (1/6-em)]300 1190 1750 2350
Seed coat 2 20–680 95–1260 800–27[thin space (1/6-em)]400 105–3610 90–3290 50–1530
350 675 14[thin space (1/6-em)]100 1860 1690 790
Flower 2 75–85 745–865 25[thin space (1/6-em)]900–26[thin space (1/6-em)]600 2190–2800 2810–3120 2460–3260
80 805 26[thin space (1/6-em)]200 2490 2960 2860
Flower bud 8 7.0–650 420–2900 8330–22[thin space (1/6-em)]500 2430–4530 3190–4970 1470–3350
230 1280 16[thin space (1/6-em)]700 3440 3840 2420
Young leaves 10 715–4810 205–440 5270–23[thin space (1/6-em)]200 5750–15[thin space (1/6-em)]000 380–11[thin space (1/6-em)]200 1360–2540
2780 305 16[thin space (1/6-em)]500 8320 5800 2030
Old leaves 14 7170–9850 235–565 5810–9930 7190–11[thin space (1/6-em)]200 2790–5370 865–3400
8160 390 7230 8930 3900 1680
Twigs 7 280–430 360–970 6380–8560 3610–5130 1410–2960 820–1230
365 560 7600 4300 2050 1000

Table 2 Bulk elemental concentrations (μg g−1) in various plant parts of Camellia sinensis determined by ICP-AES after acid digestion (trace-elements). Values are reported in ranges and means, and <LOD depicts below limit of detection
Plant part n Fe Cr Mn Ni Cu Zn
Seed 2 15–25 0.8–0.9 95–105 4.8–6.2 5.6–6.2 15–30
20 0.8 100 5.5 5.9 25
Seed coat 2 <LOD–19 0.4–2.0 25–695 <LOD–2.0 0.7–5.3 0.3–11
19 1.2 360 2.0 3.0 5.5
Flower 2 20–35 3.2–4.4 435–490 5.9–7.9 15–20 20–30
30 3.8 460 6.9 18 25
Flower bud 8 20–35 2.0–19 580–950 <LOD–11.5 15–40 10–45
25 11 730 6.5 25 25
Young leaves 10 45–80 2.5–11.3 915–3600 3.2–10.4 5.5–15 15–30
65 4.5 1520 5.6 8.5 20
Old leaves 14 80–120 4.9–8.2 1230–1920 4.3–9.7 4.2–8.2 15–34
100 6.0 1480 5.9 6.2 23
Twigs 7 25–55 1.8–3.2 485–980 5.1–8.9 7.2–11.3 10–35
40 2.2 630 6.8 10 22

image file: c9mt00300b-f1.tif
Fig. 1 Bulk elemental concentrations of aluminium based on dry weight (μg g−1) in different parts of Camellia sinensis branches analysed by ICP-AES after acid digestion.

3.2 Distribution of aluminium and other elements in whole leaves

Hydrated young (apical) and old (basal) whole leaves were analysed using laboratory (lower resolution – 25 μm – of whole leaves) and synchrotron (higher resolution – 15 μm – of foliar portions) XFM. The results of the laboratory XFM analysis show that Al is distributed in the interveinal areas with enrichment in small patches (Fig. 2). Aluminium concentrations are higher in the old leaf (left panel in Fig. 2), evidenced from the higher intensity of the Al signal. Phosphorus is distributed in narrow areas along the midvein in the young leaf and in patches towards the margin in the old leaf. Potassium is distributed relatively evenly in the young leaf, although it is slightly lower in the basal part of the midvein in leaf. However, in the old leaf K is highly enriched in an area running perpendicular from the apex towards the top margin of the leaf. This obvious K enrichment coincides with notable lower Al in this area. Calcium is most enriched in areas adjacent to the midvein in the young leaf and in small hotspots (appearing as crystalline deposits) toward the leaf margin in the old leaf, and co-localized with Mn in these hotspots. Synchrotron XFM analyses at higher resolution of portions of an old (hydrated) leaf shows that Al distribution is highly localised in irregular shaped lesion-like ‘plaques’ in interveinal areas of the leaf (Fig. 3). Judging by the signal intensity gradients (ranging from extremely high in the centre of largest deposits to lower in the margins), Al is likely present in very superficial deposits. In the large plaques Al enrichment occurs in the outer margins of large Ca hotspots. Calcium enrichment occurs in numerous smaller (∼5 μm diameter) and larger (∼50 μm diameter) hotspots (Fig. 3). The smaller plaques (possibly Ca-oxalate crystalline deposits) occur predominantly along the secondary veins of the leaf, whereas the larger plaques are found in the interveinal areas.
image file: c9mt00300b-f2.tif
Fig. 2 Laboratory-based XFM elemental maps of Al, P, K, Ca and Mn of hydrated excised whole leaves of Camellia sinensis showing an young leaf (left panels) and old leaf (right panels). The maps measure 36.27 × 13.43 mm (left) and 89.70 × 25.85 mm (right).

image file: c9mt00300b-f3.tif
Fig. 3 Synchrotron-based XFM elemental maps of Al, Ca, K and P of hydrated excised leaf portions of Camellia sinensis.

3.3 Distribution of aluminium and other elements tissue sections

Hydrated old (basal) leaf and petiole cross-sections were analysed using synchrotron XFM at high resolution (1–2 μm). The results show that Al is exclusively concentrated in the apoplastic space above and in between adaxial (top) epidermal cells (Fig. 4, 5 and Fig. S2, S3, ESI). No Al is detectable within the other cell types (palisade, mesophyll, lower epidermis) of the leaves. Calcium is mainly localised in (crystalline) deposits surrounding the sclerenchyma around secondary veins. Potassium occurs throughout all cell types within the leaves.
image file: c9mt00300b-f4.tif
Fig. 4 Synchrotron-based XFM elemental maps of Al, Ca, K and P of hydrated leaf cross-sections of Camellia sinensis.

image file: c9mt00300b-f5.tif
Fig. 5 Synchrotron-based XFM elemental maps of Al, Ca, K and P of hydrated leaf cross-sections of Camellia sinensis.

4. Discussion

The results from this study on hydrated C. sinensis leaves agree with previous studies on freeze-dried and frozen-hydrated leaves,6–8,10,11 with Al localization in the apoplastic space and cell walls around the upper epidermal cells. In addition, this study has revealed Al distribution in hydrated whole leaves, showing that Al is mainly localized in plaques in interveinal areas (Fig. 3 and Fig. S1, ESI). Accumulation of Al in root cell walls can be directly toxic to some plants.9,27 However, the distribution of Al in the leaves of C. sinensis observed in the present study is related to the Al tolerance of this species. Presumably Al3+ is binding to the cell wall due to its high negative charge (also found in roots, see Kopittke et al.,9 where Al bounds very strongly). The Al tolerance mechanism employed by C. sinensis differs from Al tolerance in other species, such as buckwheat, which stores Al in vacuoles in the leaf.28 It also differs to other elements such as Ni and Cd which accumulate in vacuoles in hyperaccumulators.29 Thus, our finding that Al is in the leaf epidermal cells suggests that this is important for Al tolerance in this plant species, and confirms the findings by Carr et al.7 and Tolrà et al.8 that Al compartmentation in leaf cell walls may be the major mechanism responsible for Al detoxification in this species. Notably, accumulation of phytotoxic elements in the leaf apoplastic space, away from sensitive processes such as photosynthesis in the palisade mesophyll cells, is a common tolerance mechanism reported in many different (hyperaccumulator) plant species.30,31 It remains unclear why Al accumulation in roots of soybean is directly related to its toxicity9 yet its accumulation in leaves of tea appears to be related to its tolerance. Perhaps it is because, in the roots, it is accumulating in the young tissues and preventing root elongation within 5 min. In the case of C. sinensis, Al possibly slowly accumulates in older leaves that are not actively growing, but this hypothesis requires testing.

Previously, it was not practical to investigate light elements beyond P in hydrated plants using XFM. Attempts to use other methods such as scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS) could produce only very low-quality maps of Al in leaf tissues of tea (see Carr et al.7). This present study has shown that it is feasible to measure light elements down to Al in hydrated plants using minor adaptations to existing X-ray detector setups via continuous He purge. Further beamline adaptations, such as a simple enclosure surrounding the stage and detectors, may remove additional air from the measurement path and significantly improve acquisition rates. This would enable substantially higher throughput and lower radiation doses imposed on samples.

The possibility of radiation-induced damage in XFM analysis (especially in fresh hydrated samples) is an important consideration that may limit the information sought from the analysis.12,32 Such damage was not observed in laboratory-based XFM analysis, as its reduced flux of 2.2 × 108 photons s−1 and large spot size delivers a dose of just 6.6 Gy even at the relatively long dwell time of 100 ms. In a recent study radiation dose limits for XFM analysis were assessed, and in hydrated plant tissues dose-limits are 4.1 kGy, before damage occurs.32 However, in the synchrotron-based XFM analysis, radiation doses were substantially higher (>1.8 × 103 Gy) due to the use of greater flux, and the low-energy (5.7 keV) of the incident beam used to maximise Al excitation also increased absorption by the plant tissue. Managing this trade-off is not trivial and will have important implications for low-energy XFM analysis of hydrated samples. This can be entirely mitigated through the use of cryogenic analysis in frozen-hydrated state which does not incur any apparent damage even at enormous doses (e.g. 587 kGy) as demonstrated by Jones et al.32 in plant samples. Unfortunately, few XFM beamlines have cryogenic capabilities beyond a nitrogen cryostream, which is suitable only for very small specimens (<2 mm) and not compatible with a helium purge for low-energy X-ray detection.

This study was ‘helped’ by the fact that Al is distributed in C. sinensis exclusively in the upper ∼10 μm of the intact whole leaf, which was confirmed through the analysis of cross-sections (Fig. 4, 5 and Fig. S3, ESI). Aluminium fluorescence is not only strongly absorbed by air, but also by the cellulose matrix of plant material samples. Escaping Al fluorescent X-rays would not arise from more than ∼50 μm into the sample, which corresponds to approximately 2–3 cell layers in C. sinensis. For other species, and other light elements, physical tissue or plant organ cross-sections are essential to obtain reliable results in in situ elemental distribution. The use of freeze-dried tissue samples will further improve detection of Al (and other elements z < P) due to reduction of X-ray scattering due to water content. This in turn may make it possible to analyse Al concentrations in ‘normal’ plants (e.g. not hyperaccumulator plants), such as common crops.

We have demonstrated that it is possible to analyse light elements in fresh hydrated plant tissues via XFM using a He environment, requiring only minor modifications to conventional measurement setups. This provides an in situ information, and offers results true to biological conditions of the living plant.

Conflicts of interest

There are no conflicts to declare.


This research was undertaken on the X-Ray Fluorescence Microscopy beamline at the Australian Synchrotron, part of ANSTO, and at the Centre for Microscopy and Microanalysis at the University of Queensland, a node of Microscopy Australia. This work was supported by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE). The laboratory XFM instrument has been funded through the UQ Major Equipment and Infrastructure “Advanced micro-X-ray Fluorescence (μ-XRF) facility for biological, medical, materials science and geochemistry” (UQMEI1835893). We thank Margaret Wilson of Bryn Hill Tea Estate for providing the Tea plant samples.


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Electronic supplementary information (ESI) available: Fig. S1: Synchrotron-based XFM elemental maps of Al, Ca and K of a hydrated leaf portion of Camellia sinensis. Fig. S2: Synchrotron-based XFM elemental maps of Al, Ca, K and P of a hydrated petiole cross-section of Camellia sinensis. Fig. S3: Synchrotron-based XFM elemental maps of Al, Ca, K, S, P and Cl of a hydrated leaf cross-section of Camellia sinensis. See DOI: 10.1039/c9mt00300b

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