Peter M. Kopittke,a Enzo Lombi,b Antony van der Ent,c Peng Wang,d,2,3 Jamie S. Laird,e Katie L. Moore,f
Daniel P. Persson,g and Søren Hustedg
aUniversity of Queensland, School of Agriculture and Food Sciences, St. Lucia, Queensland 4072, AustraliabUniversity of South Australia, Future Industries Institute, Mawson Lakes, South Australia 5095, AustraliacUniversity of Queensland, Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, St. Lucia,Queensland 4072, AustraliadNanjing Agricultural University, College of Resources and Environmental Sciences, Nanjing 210095, ChinaeUniversity of Melbourne, School of Physics, Parkville, Victoria 3010, AustraliafUniversity of Manchester, School of Materials, Photon Science Institute, Manchester M13 9PL, UnitedKingdomgUniversity of Copenhagen, Department of Plant and Environmental Sciences and Copenhagen Plant ScienceCentre, 1871 Frederiksberg, Denmark
Understanding the distribution of elements in plants is important for researchers across a broad range of fields, including plantmolecular biology, agronomy, plant physiology, plant nutrition, and ionomics. However, it is often challenging to evaluate theapplicability of the wide range of techniques available, with each having its own strengths and limitations. Here, we comparescanning/transmission electron microscopy-based energy-dispersive x-ray spectroscopy, x-ray fluorescence microscopy,particle-induced x-ray emission, laser ablation inductively coupled plasma-mass spectrometry, nanoscale secondary ion massspectroscopy, autoradiography, and confocal microscopy with fluorophores. For these various techniques, we compare theiraccessibility, their ability to analyze hydrated tissues (without sample preparation) and suitability for in vivo analyses, as well asexamining their most important analytical merits, such as resolution, sensitivity, depth of analysis, and the range of elementsthat can be analyzed. We hope that this information will assist other researchers to select, access, and evaluate the approach thatis most useful in their particular research program or application.
Visualizing elements in plants is essential for a broadrange of studies, including those aiming to improveplant nutrition and crop productivity, improving thenutritional content of edible portions of plants for hu-man nutrition, and reducing concentrations of harmfulcontaminants in food and the broader environment.Accordingly, gaining a detailed understanding of thedistribution and chemical forms of target elements inplants is critical in plant molecular biology, agronomy,plant nutrition, plant physiology, and ionomics.A variety of approaches can be used to visualize the
distribution of elements within plants. These tech-niques have their own advantages and disadvantages,and for many researchers, selecting the most appro-priate technique and evaluating the data from indi-vidual techniques can be challenging. For example,these various techniques differ in the range of elementsthat can be analyzed, their detection limits, ability to bequantitative, their resolving power, and whether spec-imens can be examined fresh or frozen hydrated(without sample preparation) or whether dehydration(such as freeze-drying) prior to analysis is required. Inthis review, we aim to compare a suite of techniquesthat are suitable for mapping the distribution of ele-ments within plants. Specifically, we compare scanningelectron microscopy-based energy-dispersive x-ray
1This work was supported by the Department of Industry,Innovation, Science, Research, and Tertiary Education, AustralianGovernment j Australian Research Council and Sonic Essentialsthrough the Linkage Projects funding scheme (grant no.LP130100741) and the Research Training Program, the NaturalScience Foundation of Jiangxi Province (Jiangsu Province NaturalScience Fund) (grant no. BK20180025), and the Fundamental Re-search Funds for the Central Universities (grant no. KJJQ201902).The laboratory XFM instrument was funded through the Universityof Queensland Major Equipment and Infrastructure Advanced Mi-cro-X-Ray Fluorescence Facility for Biological, Medical, Materials Sci-ence, and Geochemistry (grant no. UQMEI1835893). A HousingGrant (to P.M.K.) was provided by the National Bank (Denmark).Parts of this research were undertaken on the XFM beamline at theAustralian Synchrotron, part of the Australian Nuclear Science andTechnology Organisation.
2Author for contact: [email protected] authorA.v.d.E. wrote the section on SEM- and TEM-EDS; P.M.K.,
A.v.d.E., E.L., and P.W. wrote the section on XFM; A.v.d.E. andJ.S.L. wrote the microPIXE section; D.P.P. and S.H. wrote the sectionon LA-ICP-MS; K.L.M. wrote the section on NanoSIMS; E.L. wrotethe section on autoradiography; P.M.K. and A.v.d.E. wrote the sec-tion on laser confocal microscopy; P.M.K. coordinated the overalldrafting of the article; all authors edited and approved the final ver-sion of the article.
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spectroscopy (SEM-EDS) and transmission electronmicroscopy-based energy-dispersive x-ray spectros-copy (TEM-EDS), x-ray fluorescence microscopy(XFM), particle-induced x-ray emission (microPIXE),laser ablation inductively coupled plasma-mass spec-trometry (LA-ICP-MS), nanoscale secondary ion massspectroscopy (NanoSIMS), autoradiography, and con-focal microscopy using fluorophores as element-specific labels. For all experimental approaches, it isnecessary to consider the methods used for samplepreparation. However, this is not discussed in detailhere; rather, the reader is referred to other reviews.
This review builds upon and complements previousreviews, such as those of van der Ent et al. (2018b), whoexamined the use of x-ray-based approaches in hyper-accumulator plants, Kopittke et al. (2018), who exam-ined synchrotron-based approaches in plants, Perssonet al. (2016a), who examined LA-ICP-MS, Zhao et al.(2014), who discussed the types of research questionsenabled by synchrotron-based approaches and massspectrometry approaches, and Moore et al. (2012a),who examined NanoSIMS and complementaryapproaches for examining elemental distribution inplants. In this review, we aim to provide a compre-hensive comparison of the advantages and disadvan-tages of the most frequently used techniques andmethodologies, thereby enabling readers to select theapproach that is most applicable for use in their par-ticular experiment.
IMPORTANCE OF VISUALIZING ELEMENTSIN PLANTS
The study of the distribution of elements in plants iscritical for many research questions. These are brieflyconsidered below before discussing the approaches thatcan be used to measure elements and their distributionin plants.
Functional Characterization in Plant Molecular Biology
Studies that link elemental imaging with geneticapproaches are critical for characterizing genes thatinfluence elemental homeostasis. Such studies provideopportunities for the analyses of gene 3 environmentinteractions in planta, for example, by comparingtransporter phenotypes. The approaches described be-low have been used for functional characterization inmolecular biology, with most studies focusing on mi-cronutrients, although some also focused on macronu-trients. For example, using a mutant of Arabidopsis(Arabidopsis thaliana) unable to synthesize the metalchelator nicotianamine, it was found with LA-ICP-MSthat the mutant accumulated Zn and Mn in the tissuessurrounding the vascular cylinder, while Fe was con-fined to the cortical cell walls in the mutant despitebeing primarily in the epidermis of the wild type(Persson et al., 2016a). In another recent study, XFMwas used to investigate Arabidopsis, finding thatMETAL TOLERANCE PROTEIN8 (MTP8) determinedthe distribution of Fe andMn in seeds (Chu et al., 2017).In this latter study, the use of XFM to image elementdistribution in vivo avoided potential issues associatedwith GFP imaging in quiescent seed tissue. In anotherexample, XFM was used by Punshon et al. (2013) toshow differences in Ca localization and speciation in acalcium oxalate deficient5 (cod5) mutant of Medicagotruncatula. These authors reported that knockout ofCOD5 prevented biogenic crystal formation by alteringCa distribution and the form of Ca oxalate. Finally, Kimet al. (2006) examined Fe in Arabidopsis seeds, findingthat when VACUOLAR IRON TRANSPORTER1(VIT1) is disrupted, Fe did not accumulate in the pro-vascular strands of the embryo.
Improving Plant Nutrition and Productivity
Understanding element distribution in plants is alsoimportant for improving plant mineral nutrition andproductivity. As an example, consider the applicationof foliar fertilizers to improve plant growth in soilscontaining low levels of plant-available nutrients. Themechanisms by which foliar-applied nutrients moveacross the leaf surface and are translocated and assim-ilated remain unclear. To examine this research ques-tion, Zn fertilizer was applied to the surface of a leaf ofwheat (Triticum aestivum) and changes in leaf Zn con-centrations were measured in vivo for up to 24 h(Doolette et al., 2018). Although some translocation of
the foliar-applied Zn was observed, it was found thatthe Zn had only limited mobility regardless of the formof Zn applied. Similar results were also reported byTian et al. (2015). In a similar manner, autoradiographyhas been used to examine the translocation of foliar-applied Zn over time in vivo in whole plants of wheat(Read et al., 2019). These authors found that the use of65Zn-labeled compounds allowed for time-resolvedanalyses of Zn distribution in live plants, reportingthat 65Zn was translocated throughout the plant (in-cluding to the grain, where it is important for humannutrition) following its foliar application.
Improving Human Nutrition through Foodstuffs
To improve human nutrition through higher qualityfoods, an understanding of nutrient concentration anddistribution within foodstuffs is essential. This is be-cause the nutritive value of foods depends not onlyupon the total elemental concentration but also its dis-tribution and molecular speciation. Accordingly, bio-fortification strategies need to consider both thedistribution and speciation of nutrients within thefoodstuff tissue. As an example of a study aiming toimprove human nutrition, grains of buckwheat (Fag-opyrum esculentum) were examined using microPIXE(Pongrac et al., 2011). These authors found that the in-ner layers of the pericarp were enriched in K, Mn, Ca,and Fe while the outer layer was enriched in Na, Mg, P,S, and Al, and that by altering the milling approach itwas possible to alter the nutritional content of the grain.Furthermore, for both wheat and rice (Oryza sativa) grain,it is known that while micronutrients tend to accumulatein the bran layers (i.e. the aleurone, the tegument, and thepericarp), those elements that are generally more mobilewithin the phloem (such as K,Mg, P, Fe, Zn, andCu) tendto accumulate to higher concentrations in the aleuronelayer (De Brier et al., 2015). In this regard, Wang et al.(2011) used LA-ICP-MS to examine the stable Zn iso-tope 70Zn in wheat grain, finding that there are two bar-riers to Zn transport in wheat grain: between the stemtissue rachis and the grain and between the maternal andfilial tissues in the grain.Not only is distribution important in influencing the
nutritional value of foods, but it is also necessary tounderstand how the colocalization of different elementswithin foods impacts on nutrient availability to humans.For example, colocalization of micronutrients with P (of-ten present as phytate) likely reduces micronutrientavailability in the human gut, with such colocalizationobserved in sweetcorn and maize (Zea mays; Cheah et al.,2019) and wheat (Moore et al., 2012b).
Understanding Toxic Elements in Plants andTolerance Mechanisms
Understanding the behavior of toxic elements inplants, their impact on plant growth, their translocation
through the plant and accumulation in human food-stuffs, and the mechanisms that plants use to toleratethese toxicants is of critical importance. First, to illus-trate the importance of understanding elemental dis-tribution in crop plants, consider the problem of Altoxicity. Soluble concentrations of Al are elevated in theacid soils that constitute approximately 3.95 billion haof the global ice-free land (Eswaran et al., 1997). Al-though Al is highly toxic to plant root growth, muchremains unknown about how it exerts its toxic effects.In this regard, NanoSIMS has been used to examine Aldistribution in root tissues of soybean (Glycine max),finding that Al accumulates almost entirely in thewalls ofcells in the rhizodermis and outer cortex (Kopittke et al.,2015). These authors reported that this Al in the cell wallof young, elongating roots was toxic and caused a rapidreduction in root elongation. Interestingly, in tea (Camelliasinensis), a known accumulator of Al, much of the Al ac-cumulated in the cell walls of the leaves, representing apotential tolerance mechanism (Tolrà et al., 2011). Asanother example, the accumulation of As in foods is ofinterest due to the consumption of this carcinogen byhumans. The distribution of As in roots of Arabidopsiswas examined using XFM, confirming the localization ofa new arsenate reductase (HAC) that limits As accumu-lation in the tissues (Chao et al., 2014).Another area of major research interest has been in
the use of imaging techniques to understand howhyperaccumulating plants are able to tolerate highconcentrations of metal(loid)s in their tissues. Nihyperaccumulator plants (which make up the majority ofhyperaccumulator plants known globally) have been themost intensively studied (Reeves et al., 2018). In mostspecies studied to date, Ni is concentrated in the epider-mal cell vacuoles of the leaves (Küpper et al., 2001; Bhatiaet al., 2004; Kachenko et al., 2008; van der Ent et al., 2017).Hyperaccumulation spans several length scales, fromwhole plants down to organs, tissues, individual cells,cellular organelles, and transporter molecules, and infor-mation at all of these scales is important for understand-ing the mechanisms associated with hyperaccumulation(van der Ent et al., 2017).
With x-ray fluorescence-based approaches, elementsare detected based upon their characteristic fluorescentx-rays. These fluorescent x-rays are generated bypassing the specimen through a focused beam of high-energy x-rays (XFM), electrons (SEM- and TEM-EDS),or protons (PIXE). This beam excites a range of differentelements (depending on the energy of the incidentx-rays, electrons, or protons), which are detected andquantified by a detector to determine elemental con-centrations in the specimen. The movement of thespecimen through the incident beam in x-y creates araster map in which each point represents a pixel withconcentration data (or relative element intensity) for a
range of elements. High-energy x-rays (greater than 15keV) have great penetrative power and will passthrough plant specimens (both sectioned tissues andpotentially even through entire, intact plant tissues),whereas electrons and protons will only penetrate 5 to50 mm into a specimen. In principle, the incident x-raybeam does not destroy the sample, hence the methodis typically considered nondestructive. However, asx-rays are ionizing radiation, depending on the energyand dwell on the sample, damage might occur due tothe formation of free radicals, which are highly reactiveand damaging to the tissue being analyzed. Further-more, the incident x-ray beams do not generate heat inthe specimen, in contrast to electron and proton beams,which consist of particles and have a far greater po-tential to damage the specimen during scanning.Obtaining sufficient element sensitivity while keepingdwell low enough not to cause beam-induced damagecan be challenging in PIXE (Laird et al., 2019).
SEM- and TEM-Based EDS
Using SEM- and TEM-based EDS (Figs. 1 and 2),samples are scanned using an incident electron beam inorder to produce the characteristic fluorescent x-rays.
These approaches are the most commonly usedmethods for examining elemental distribution in planttissues.
Given that most SEM- and TEM-based EDS systemsoperate under a high vacuum, the plant tissue specimenmust be totally dehydrated (and coated with carbon tomake it conductive for electrons) prior to analysis(Fig. 2). However, where a cryo-SEM system is avail-able, it is possible to examine frozen plant tissue spec-imens in the hydrated state. Appropriate specimenpreparation for cryo-SEM and cryotransfer remainsextremely challenging technically, and detection limitsare poorer than for dehydrated specimens. In addition,it is also increasingly possible to analyze living plantsusing environmental SEM, although there are issueswith sample size restrictions and beam damage(Danilatos, 1981; McGregor and Donald, 2010). In themajority of studies, specimen dehydration (typically byfreeze-drying or lyophilization) is required, and this hasthe potential to cause artifacts due to elemental redis-tribution (see van der Ent et al. [2018b] for a full dis-cussion of considerations).
When imaging a specimen, SEM can typically ach-ieve a resolution of approximately 1 to 50 nm. How-ever, when examining elemental composition using
Figure 1. Comparison of seven broad techniques used for examining element distribution in plants. All values are indicative oftypical systems.
SEM-based EDS, the resolution is considerably poorer,due to the interaction of the electrons with the sample,typically being on the order of 2 to 5 mm andworseningwith increasing accelerating voltage. For TEM-basedEDS, the resolution is better than for SEM-based EDSbecause the use of TEM requires the plant tissues to becut as ultrathin sections (approximately 60–100 nm inthickness), thereby greatly reducing problems associ-ated with the depth of penetration. Thus, for TEM-based EDS, it is possible to achieve a resolution ofapproximately 100 nm.A comparatively large range of elements can be
detected use SEM- and TEM-based EDS, typically B toU when examining across the K-, L-, and M-edges (seeCalvin [2013] for a discussion of the K-, L-, andM-edges). However, the detection limit of the techniqueis rather poor, generally 0.1 to 1 weight percent for mostelements, which severely limits its field of applicationfor plant-based studies. As indicated earlier, the anal-ysis is surface sensitive, given that the electron beampenetrates only a few micrometers into the sample(SEM) or because ultrathin sections are used (TEM).Accurate quantification for SEM is extremely difficult,and often not attempted, as the method is highly sen-sitive to sample-specific characteristics (bulk composi-tion, density, and so forth) and, hence, calibrationstandards during the analysis are essential (Tylko et al.,2010). However, no commercial biological standardsfor SEM have yet been developed.It is clear that SEM- and TEM-based EDS are useful
approaches where concentrations of the element of in-terest are high and where the risk of elemental redis-tribution upon sample dehydration is low. However,their overall usefulness for visualizing elements in plantsis comparatively low except for hyperaccumulators orplants where elemental concentrations are high (Figs.1 and 2). Some recent examples of studies using SEM- andTEM-EDS include the analyses of contaminants on sur-faces and in longitudinal sections of leaves of Tilia cordata(Mantovani et al., 2018), the distribution of Cd in root
cross sections of Taraxacum ohwianum (Cheng et al.,2019), the distribution of Ni and Co in leaves of Glo-chidion cf. sericeum (van der Ent et al., 2018a), and thedistribution of CuO nanoparticles in the xylem of maize(Wang et al., 2012).
Synchrotron-Based XFM
XFM can be either synchrotron based or laboratorybased (Figs. 1, 3, and 4), with these systems havingseveral important differences. Both synchrotron-basedand laboratory-based systems use x-rays for the inci-dent beam in order to produce fluorescent x-raysfor elemental mapping. Here, we first focus onsynchrotron-based XFM.There are currently approximately 50 synchrotrons in
the world, although not all have XFM beamlines. Themost frequently used beamlines for plant analyses in-clude (but are not limited to) the XFM beamline at theAustralian Synchrotron (Australia; Kopittke et al.,2018), 13IDE at the Advanced Photon Source (UnitedStates; Doolette et al., 2018), the XFM beamline and thehard x-ray nanoprobe beamline at Brookhaven Na-tional Laboratory (United States; Li et al., 2019b), andID21 at the European Synchrotron Radiation Facility(France; Pradas Del Real et al., 2017).Generally, XFM is conducted at ambient temperature
and pressure with no theoretical restrictions on samplesize. As a result, plants can be examined hydrated, andeven in vivo analyses are possible if the entire plant canbe mounted in front of the x-ray beam (Blamey et al.,2018b; Doolette et al., 2018). Nevertheless, care must betaken to ensure that the incident x-ray beam does notresult in artifacts in the specimen during analysis,which can cause localized structural damage and theredistribution of elements. This is particularly impor-tant when examining hydrated samples, with thesebeing especially sensitive to radiation damage. Unfor-tunately, few studies state whether they have explicitly
Figure 2. Freeze-dried cross section ofa root of Conyza cordata examinedusing SEM-EDS showing elemental dis-tributionmaps of O, S, K, Ca, and Cl. Theimages were obtained with an incidentbeam of 15 kV. The specimen was pre-pared by Jolanta Mesjasz-Przybyłowicz.
determined whether sample damage occurs andwhether there is a concomitant redistribution of ele-ments (Jones et al., 2020).
For XFM, given the penetrating nature of the x-rays(both the incident x-rays as well as the fluorescentx-rays), elemental distribution can often be examinedthroughout the entire thickness of plant tissues. How-ever, the depth of analysis varies greatly dependingupon the element of interest, being determined by theenergy of the corresponding fluorescent x-rays (withthese having a lower energy than the incident x-rays).This is best illustrated with the following examples. ForCa, with a K-edge emission line of 3.7 keV, 50% of thefluorescence will be absorbed in a plant sample ap-proximately 70 mm thick and 90% in a sample ap-proximately 200 mm thick. In contrast, for Se, with aK-edge emission line of 11.2 keV, 50% of the fluores-cence will be absorbed in a sample approximately 2,000
mm thick and 90% in a sample approximately 6,500 mmthick. In other words, assuming a root with a thicknessof 1,000 mm, only the Ca in the surface 100 to 200 mmcan be detected (with the Ca in the vascular tissue beinginvisible), while Se will be detected across the entiredepth of the root cylinder. Thus, great care needs to betaken when comparing the distribution of various ele-ments, especially in thicker samples.
Most synchrotron-based XFM facilities tend to have aresolution on the order of 20 nm to 1 mm (Li et al.,2019b). The time required to conduct analyses alsovaries greatly. For synchrotron-based systems with fastdetector systems, the dwell is now routinely 1 ms orless, meaning that a 1-megapixel image can be collectedin approximately 17 min or less. The elements that canbe examined depend upon a wide range of factors.Often, elements can be accessed from P (2.1 keV) to Ag(25 keV), while higher Z elements can potentially be
Figure 3. Use of synchrotron-basedXFM (Australian Synchrotron) forhigh-throughput screening of plantmutant libraries for Arabidopsis. Theimage in A is an optical micrograph.The images in B to E show the distri-bution of Fe (B), Mn (C), Zn (D), and Se(E) in approximately 6,000 seeds, witheach image having a resolution of ap-proximately 20 megapixels when dis-played at full resolution. The image in Fshows a small portion of a detailed scanfor Fe showing some seeds differing intheir Fe concentration and distribution.The overview scans (B–E) had a 10-mmpixel size with a dwell of 1 ms perpixel, while the detailed scans (a smallportion shown in F) had a 1-mm pixelsize with a dwell of 7 ms per pixel. Intotal, an estimated 40,000 seeds wereexamined, with only approximately6,000 seeds shown here. Note that theanalyses are nondestructive.
examined using the L-edges. The detection limit varieswidely depending upon the facility as well as the ele-ment being analyzed. For elements such as Mn, Fe, andZn, the detection limit is excellent, being on the order ofapproximately 1 mg kg21 or even lower. However, thedetection limit decreases for the lower Z elements, in-cluding P, S, and K, often being approximately 10 to1,000 mg kg21, which is a function of the smaller x-raycross sections (resulting in lower fluorescence yields)and the operation of most XFM beamlines in air, whichabsorbs low-energy x-rays. For synchrotron-basedXFM, analyses are potentially fully quantitative, forexample, using the GeoPIXE software package, whichproduces quantitative self-absorption corrected mapsthat are line overlap resolved and in which the back-ground is subtracted (Ryan, 2000).Given that XFM allows analyses of plants in vivo
with no (or minimal) sample preparation and withgood sensitivity (Fig. 3), this technique is being used toexamine an increasing number of diverse problems.This includes studies where it is imperative to avoidsample processing (such as freeze-drying) or whererepeated measurements of living plants are requiredin vivo. It is especially useful for studies focusing ontrace metals andmetalloids, such asMn, Zn, Fe, Cu, As,and Se, and to a lesser extent the macronutrients P, S, K,and Ca (Fig. 3). Some recent examples of studies usingXFM include analyses of MTP8 (Chu et al., 2017; Erogluet al., 2017; Basiri-Esfahani et al., 2019), kinetic analysesof living leaves of cowpea (Vigna unguiculata) exposedto toxic levels of Mn (Blamey et al., 2018b), analyses ofZn movement following foliar fertilization in wheat(Doolette et al., 2018), and analyses of nanoparticles inplants (Martínez-Criado et al., 2016). A potential new,and yet unexplored, use is for high-throughputscreening of plant mutant libraries. This is especiallyexciting given the nondestructive nature of XFM anal-yses as well as the ability to examine changes in thespatial distribution of elements instead of examiningonly bulk concentrations (Fig. 3). Finally, another
potential advantage of XFM analyses is the potentialto combine imaging with speciation through the useof x-ray absorption near-edge structure spectroscopy(Kopittke et al., 2018), although this is beyond the scopeof our review (see Wang et al. [2015] for an example forexamining the distribution and speciation of Se in riceand wheat tissues).
Laboratory-Based XFM
The strengths of XFM are evident from the researchoutput from studies undertaken at synchrotrons.However, the restrictive nature of access to synchrotronXFM facilities is recognized by many users as a limitingfactor in using XFM in their research, and wherelaboratory-based facilities are available, this can over-come that limitation. However, we are not aware ofmany laboratory-based XFM systems, with this being acurrent restriction for their use. The authors are awareof systems used for the investigation of plants at themicroXRF facility at the University of Queensland(Australia; Fig. 4), Washington State University (UnitedStates; Fittschen et al., 2017), and the Maia Mapper atthe Advanced Resource Characterization Facility of theCommonwealth Scientific and Industrial Research Or-ganization in Australia (Ryan et al., 2018). The latter isperhaps the most advanced laboratory XFM system, asit can image up to;80 million pixels over a 500-3 150-mm2 sample area using the Maia detector array.However, it has been developed for drill core sectionsand polished rock slabs, not biological applications. TheUniversity of Queensland microXRF facility has beenspecifically developed for biological applications andhas dual microfocus sources (focusing to 5 and 25 mm),two large silicon drift detectors of 150 mm2, and canscan areas up to 3003 300 mm in air, vacuum, or heliumatmosphere. It also has a cryo-stage (50- 3 50-mm activearea held at 250°C) for analysis of samples in afrozen-hydrated state.
Figure 4. Analysis of a fresh hydratedshoot of the Se hyperaccumulatorNeptunia amplexicaulis, using laboratory-based XFM at the University of Queens-land (Australia). Images show elementalmaps of K, Ca, and Se distribution and amap of the sum of all x-rays (useful forobserving the structure of the sample).
These new laboratory-based XFM facilities do notfully replace synchrotron-based XFM for a number ofspecialized applications but will bridge the gap be-tween what is currently possible in the laboratory en-vironment and the capability of synchrotron facilities.Furthermore, it allows researchers to combine thestrengths of both facilities, for example, by whole-organism mapping at their laboratory followed by in-vestigation of target cells at the synchrotrons, and hencestrengthen the outcome of both platforms.
Laboratory-based XFM systems essentially offer un-limited access (within the institutional constraints ofavailability and financial considerations) as required byexperimental needs. In addition, many laboratory-based systems provide vacuum and helium purgecapabilities that might not be available at synchrotron-based beamlines, thereby offering improved capabilityfor the measurement of light elements such as Al, Si, S,and P.
However, these laboratory-based systems offerworse spatial resolution, often in the range of 5 to 50mm(compared with 20 nm to 1 mm for synchrotron-basedsystems). In addition, laboratory-based systems typi-cally use a concave focused polychromatic x-ray sourcewith Bremsstrahlung background, with this havingimportant differences from a monochromatic, highlyparallel x-ray source in synchrotron-based XFM. Forexample, there is no energy tunability in laboratory-based systems, and hence x-ray absorption spectros-copy is not possible. Finally, the substantially lowerx-ray flux for laboratory-based systems (typically 1,000to 10,000 times less bright) results in longer dwell times(50–100 ms per pixel) compared with synchrotron-based systems (0.5–5 ms per pixel).
MicroPIXE
With microPIXE (Figs. 1 and 5A), an ion beam ofprotons is used as the incident beam, generating fluo-rescent x-rays in the sample.
For microPIXE, the resolution is generally in therange of 1 to 3 mm, or occasionally slightly better. PIXEexcites the K-lines of virtually all elements, and hence itis generally possible tomeasure elements in the range of1.5 to 60 keV (corresponding to Al toW), with this beingconsiderably wider than that achieved using XFM.Moreover, very light elements in the range of 0.05 to 1.3keV (corresponding to Li to Mg) can be analyzed withparticle-induced g-ray emission. As such, PIXE opens
Figure 5. A, PIXE elemental map of an intact Noccaea caerulescensseed (pixel size 2 mm, dwell 5 ms per pixel). The southern France ac-cessions (St. Laurent de Minier/Ganges) have the ability to hyper-accumulate Cd with up to 900 mg kg21 Cd in the seeds. B, Stele of amature barley root examined using LA-ICP-MS (193-nm excimer laser
[Analyte G2; Teledyne Photon Machines] equipped with a Cobalt cell[Teledyne Photon Machines], with a pixel size of 2 mm) showing 24Mgdistribution. C, LA-ICP-MS images from the inner tissues of a maturebarley root with a pixel size of 5 mm, showing 24Mg, 67Zn, 66Zn, and alight micrograph (the red square indicating the area analyzed using LA-ICP-MS). The root was first starved for Zn and then exposed to 67Znstable isotope (94.3% enriched) for 2 h. The natural 66Zn/67Zn isotopicratio is 6.8 (66Zn, 27.9%; 67Zn, 4.1%). The 67Zn image shows how theZn (added as 67Zn) is taken up and transported radially toward the stele.
up imaging of metals such as Ag and Cd and the met-alloids Sb, Te, and I, which are very difficult to measurewith synchrotron XFM. The detection limit usingmicroPIXE is excellent, typically in the range of lowermg kg21, with analyses also generally being fullyquantitative with the use of techniques (such as Ruth-erford backscattering spectrometry) to determine sam-ple matrix composition. The new PIXE Maia facility atthe University of Melbourne (Australia) combines thebenefits of PIXE with those of the revolutionary Maiadetector array able to process 4 to 9 million events s21
(measured on a plant specimen; Laird et al., 2019) or upto ;900 times greater than that typically used in theprevious single detector system (Laird et al., 2013).Based on this discussion, it is clear that microPIXE is
useful for examining elements in a wide range of sys-tems. Given that it can analyze a wider range of ele-ments than many other approaches (such as XFM), it isespecially valuable for examining light elements (suchas Na, Mg, or Al) as well as heavier elements (such asCd or the rare earth elements, as shown in Fig. 5A),which often cannot be analyzed with other approaches,all with a good detection limit (Figs. 1 and 5A). Somerecent studies using microPIXE include for the analysisof Zn and Cd in a hyperaccumulator, Sedum plumbi-zincicola (Hu et al., 2015), and for the study of Nihyperaccumulation in Phyllanthus balgooyi (Mesjasz-Przybylowicz et al., 2016).
MASS SPECTROMETRY-BASED APPROACHESFOR VISUALIZATION
For mass spectrometry-based approaches, smallportions of the sample are progressively removedduring scanning for analysis. Because analysis is bymass spectrometry, not only are these generally highlysensitive techniques, but they also allow isotopic anal-yses. However, given that small portions of the sam-ple are removed for analysis during scanning, thesemass spectrometry-based approaches are considereddestructive.
LA-ICP-MS
LA-ICP-MS uses a focused laser to ablate the surfaceof the sample (Figs. 1 and 5, B and C). These ablatedparticles are then transported to an ICP-MS device in astream of He gas for both elemental and isotopicanalyses.LA-ICP-MS offers excellent detection limits (less than
1 mg kg21) and a very wide range of elements (Li to U).Furthermore, multielement analyses are routine, andstable isotope analyses are also possible, as illustratedin Figure 5C. In addition, LA-ICP-MS offers a modestresolution (approximately 1 mm), being similar tomicroPIXE and SEM-based EDS. A wide range of ele-ments can be examined using LA-ICP-MS, from Li to U.Sensitivity is greater than for x-ray-based approaches,with the detection limit being sub-mg kg21 for many
physiologically relevant elements (Persson et al.,2016a). Analyses are potentially fully quantitative, al-though this is not without substantial difficulties. Spe-cifically, the laser beam interaction with the sample willvary according to the sample properties, resulting inchanges in the amount of analyte (sample) removed perpulse. For example, portions of the plant tissues that areheavily lignified will ablate less material than softerparts of the plant tissue, making full quantificationchallenging. Thus, analyses of plant tissues are gener-ally considered to be semiquantitative.Although LA-ICP-MS analyses are generally con-
ducted at ambient temperature and pressure in an inertAr atmosphere, extreme care must be taken to avoidsample desiccation due to the exposure of samples tothe dry stream of Ar gas when examining hydratedtissues, with this leading to experimental artifacts. As aresult, most analyses utilizing LA-ICP-MS examinedehydrated samples and, hence, in vivo analyses arechallenging. Nevertheless, it is indeed possible to ana-lyze fresh (hydrated) tissues and living plants in somesituations (Salt et al., 2008; Klug et al., 2011), and pro-tocols for sample preparations that produce intact, drysamples with unaltered ion distribution within tissueare available (Persson et al., 2016a).LA-ICP-MS is therefore particularly useful in studies
where high sensitivity is required with access to anextremely broad range of elements with good sensi-tivity (Figs. 1 and 5, B and C). It is a surface-sensitivetechnique that offers advantages compared with XFMor microPIXE in some situations, but it can be disad-vantageous in others. The use of isotopic analyses isalso a potentially useful advantage for examining theflux and distribution of exogenously applied isotopes.Recent studies to use LA-ICP-MS for the study of plantsinclude for the imaging of Fe, Zn, and Mn in roots ofArabidopsis (Persson et al., 2016a), Mn in roots andgrain of barley (Hordeum vulgare; Long et al., 2018; Chenet al., 2019), Ca, Na, and K in stems and leaves of to-bacco (Nicotiana tabacum; Thyssen et al., 2017), Zn, S,and P analyses of biofortified wheat grains (Perssonet al., 2016b), Cd, Pb, Cu, and Zn in roots of pea(Pisum sativum; Hanc et al., 2016), for mapping thedistribution of pollutants in leaves of sweet basil (Oci-mum basilicum; Ko et al., 2018), and for examining nu-trient distribution in nodes of rice mutants (Yamaji andMa, 2019).
NanoSIMS
In NanoSIMS (Figs. 1 and 6), ions are used as theincident (primary) beam, and these ions collide with thesample surface and cause atoms, ions, and moleculesfrom the sample surface to be ejected into the vacuum(sputtering). The ionized particles (secondary ions) arethen collected and transported to a mass spectrometerfor analysis. For NanoSIMS, the sputtering depth isapproximately 5 to 20 nm (Hoppe et al., 2013), making ita very surface-sensitive technique.
Currently, there are considerably fewer NanoSIMSfacilities in the world than there are synchrotrons. As aresult, accessing a NanoSIMS device for an experimentcan potentially be difficult for many researchers. Nev-ertheless, the facilities most commonly used for inves-tigations of plant tissues include those at the Universityof Manchester (England) and the University of WesternAustralia (Australia).
NanoSIMS operates in an ultra-high vacuum, mean-ing that samplesmust first be dehydrated before analysis.Furthermore,NanoSIMS requires aflat surface, andhenceit is typically only possible to examine sectioned tissues(Fig. 6). As for other techniques where sample dehydra-tion is required prior to analysis, extreme care must betaken to ensure that the method used for sample pro-cessing does not cause experimental artifacts through re-distribution of the elements of interest.
NanoSIMS offers an excellent lateral resolution, withanalyses routinely conducted at resolutions as low as100 nm (Fig. 6). Using this technique, it is possible toanalyze a very wide range of elements of relevance toplant studies, from H to U. The sensitivity is also verygood, with the detection limit being in the low mg kg21 range. The sensitivity for any given element dependsupon the primary beam selected, with either an O2
beam or a Cs1 beam available. The negatively chargedprimary beam (i.e. O2) tends to favor the production ofpositively charged secondary ions, while the positivelycharged primary beam (i.e. Cs1) tends to favor theproduction of negatively charged secondary ions. As aresult, for elements such as Na, Mg, Al, K, Ca, Mn, Fe,and Zn, the O2 beam is generally preferred (Nuñezet al., 2017). In contrast, for elements such as Si, P, S,Cl, As, and Se, the Cs1 beam is generally preferred.
The main advantages of NanoSIMS are the excellentdetection limit and spatial resolution aswell as thewiderange of elements that can be analyzed. As a result, thisapproach is particularly suited to examining the sub-cellular distribution of elements within cross sections ofplant tissues (Figs. 1 and 6). Isotopic analyses are also
possible using this approach (for an example in plants, seeMoore et al. [2016]). Some recent studies utilizing Nano-SIMS include the studyof Fewith amyloplasts in pea seeds(Moore et al., 2018), detoxification of Mn by Si in leaves ofsoybean and sunflower (Helianthus annuus; Blamey et al.,2018a), determining the mechanisms by which foliar-applied Zn fertilizer moves across the leaf surface (Liet al., 2019a), as well as examining the distribution of Alin roots of soybean (Kopittke et al., 2015) with the mass ofAl being too low to examine using XFM (Fig. 6).
AUTORADIOGRAPHY
Autoradiography (Figs. 1 and 7A) is the oldest of thetechniques discussed here, having been used for plantssince the 1920s (Hevesy, 1923). In autoradiography,radioactive isotopes are supplied to a plant, which aretaken up and redistributed throughout the plant tis-sues. To then examine their distribution in the plant, animage is obtained of the decay emissions from thevarious plant tissues (Fig. 7A). These decay emissionscan be detected using an x-ray film or, more recently,using digital autoradiography.
Comparedwith some other approaches, such as XFMorNanoSIMS, access to autoradiography facilities is likelynot too difficult. This approach also has a range of otheradvantages, including being able to examine hydratedplant tissues, including for in vivo analyses. Furthermore,autoradiography can be used to examine large samples oreven entire plants. Another major advantage is the abilityto separate background isotopes of an element (i.e. thosenatively present in the plant tissues) from the radioisotopeof the same element added exogenously. The resolutionachievable with autoradiography varies between ap-proximately 25 and 1,000mm (Fig. 1) and depends upon arange of factors (Zhang et al., 2008).
The greatest challenges for using autoradiographyare the highly restrictive and complicated health andsafety regulations in many jurisdictions for working
Figure 6. NanoSIMS analyses of aportion of a transverse cross section ofsoybean root exposed to 30 mM Al for0.5 h with an radio frequency plasmaO2 source. The images for Al (left) andNa (right) were obtained with a pixelsize of 0.3 mm and a dwell of 60 ms perpixel. For more information on plantgrowth and analyses, see Kopittke et al.(2015).
with radioisotopes. In addition to this limitation, it isonly possible to examine a single element at a time(Solon et al., 2010). Furthermore, it is only possible touse this technique for elements where a suitable radi-oisotope exists. For studies of plants, these elementsinclude 26Al, 32P, 33P, 35S, 45Ca, 51Cr, 54Mn, 55Fe, 59Ni,67Cu, 65Zn, 73As, and 109Cd (Kanno et al., 2012).The main uses of autoradiography are for in vivo
studies or studies in which sample processing needs tobe avoided. It also offers excellent detection limits andallows the separation of background elements from thoseadded exogenously as radioisotopes, this being criticalwhenonly aportion of the total element is of interest (Figs.
1 and 7A). Recent studies to use autoradiography to ex-amine elemental distribution in plants include the studyof 65Zn applied as foliar fertilizers in wheat (Read et al.,2019) and tracking 64Cu-labeled nanoparticles in lettuce(Lactuca sativa; Davis et al., 2017).
LASER CONFOCAL MICROSCOPYWITH FLUOROPHORES
The final approach considered here is the use of laserconfocal microscopy with element-selective fluorophores(Figs. 1 and 7B).
Figure 7. A, Autoradiography of trea-ted wheat leaves onto which 65Zn-labeled foliar fertilizers had beenapplied as 65ZnCl2, 65ZnEDTA, 65ZnO-NPs (nanoparticles), and 65ZnO-MPs(microparticles; 750 mg L21). The dig-ital photograph (left) shows the leavesonto which the Zn was applied. A totalof 10 droplets were applied onto eachleaf before being washed from the leafsurface. For more information, seeRead et al. (2019). B, Zinpyr-1 (fluores-cent indicator for Zn21; green color)stained and autofluorescence of a leafsection of N. caerulescens obtained us-ing confocal fluorescence microscopy.
Given that many researchers are likely able to accesslaser confocal microscopy without substantial diffi-culty, this approach is one of the easier ones in terms offacility access, as most research institutions will havelaser confocal microscopes, especially in medical fac-ulties where they are routinely used. This approach isalso nondestructive and can be used on hydratedsamples, including for in vivo analyses of living plants.The maximum resolution is similar to some otherapproaches, being approximately 1 mm.
All the approaches considered above have analyzedelemental composition directly. However, laser confo-cal microscopy relies on the binding of ion-selectivefluorophores to the element of interest for their subse-quent detection using excitation by specific wave-lengths emitted by lasers (Fig. 7B). This in itselfrepresents a potential limitation of this technique, asfluorophores will generally only bind to free ions notalready bound strongly to other ligands in the plant.Thus, the proportion of the total pool identified usingthe fluorophore can be uncertain. In addition, issueswith penetration into the plant tissue are largely un-known and are hard to quantify. The range of elementsthat can be investigated using laser confocal micros-copy is entirely dependent on the commercial avail-ability of fluorophores with specific affinities forelements of interest. These include, but are not limitedto, Zn (Zinpyr-1, FluoZin, TSQ), Ni/Co (NewportGreen), Cu (Phen Green), and Pb/Cd (LeadmiumGreen).
The use of laser confocal microscopy with element-selective fluorophores is particularly suited to caseswhere in vivo analyses are required for an element forwhich a suitable fluorophore exists, with an excellentdetection limit (Figs. 1 and 7B). However, questions stillremain regarding the binding of the fluorophores andtheir penetration into the plant tissue. Recent studiesusing confocal microscopy with fluorophores includeimaging the distribution Ni21 with the dye NewportGreen in Alyssum murale (Agrawal et al., 2013) andAlyssum lesbiacum (Ingle et al., 2008), the use of Zinpyr-1 for imaging the distribution of Zn21 in Noccaea caer-ulescens (Kozhevnikova et al., 2017; Dinh et al., 2018)and Arabidopsis (Sinclair et al., 2007), and the use ofLeadmium Green for imaging Zn21 and Cd21 in Sedumalfredii and Picris divaricata (Lu et al., 2008; Hu et al.,2012).
CONCLUDING REMARKS
Understanding the distribution of elements withinplant tissues is critical for a range of research programswithin plant science, including for functional charac-terization in molecular biology, improving plant nu-trition and productivity, improving human nutrition,and understanding toxic elements in plants and toler-ance mechanisms. For analyzing plants, a range oftechniques are suitable, but it can often be confusing asto which approach is best given their range of advan-tages and limitations. It is clear from this review thatthere is no single technique that is best. Rather, eachtechnique has its own strengths and weaknesses. Bycomparing the accessibility, ability to analyze hydratedtissues (without sample preparation) and conductin vivo analyses, as well as comparing the resolu-tion, sensitivity, depth of analysis, and range of ele-ments that can be analyzed for the seven describedapproaches, we hope that this information will assistother researchers to select and access the approach thatis most useful in their particular research program. Inaddition, it will be helpful to use correlative approachesin which the same sample is examined with multipletechniques to exploit the advantages listed here. Ofcentral importance in the future will be the analyses ofliving plants (including in vivo analyses) with minimalsample preparation at excellent resolution and withgood detection limits across the wide range of physio-logically relevant elements, this requiring a strongcorrelative approach (see Outstanding Questions). Theuse of such correlative approaches will enable impor-tant research questions to be answered within the fieldof plant science.
ACKNOWLEDGMENTS
The autoradiography images were collected during a study undertaken atAustralian Nuclear Science and Technology Organization, and the authorsacknowledge TomCresswell and Nick Howell (Australian Nuclear Science andTechnology Organization) for collecting and preparing the images. Thea Readand Casey Doolette (University of South Australia) are also acknowledged for
their contributions to the autoradiography study. We also thank Dr. CiprianStremtan (Teledyne Advanced Chemistry Systems) as well as Dr. Joke Belzaand Dr. Thibaut Van Acker (University of Ghent) for technical assistance withthe LA-ICP-MS analyses.
Received October 24, 2019; accepted January 16, 2020; published January 23,2020.
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