TY - JOUR
AU1 - Kopittke, Peter M.
AU2 - Lombi, Enzo
AU3 - van der Ent, Antony
AU4 - Wang, Peng
AU5 - Laird, Jamie S.
AU6 - Moore, Katie L.
AU7 - Persson, Daniel P.
AU8 - Husted, Søren
AB - Abstract Understanding the distribution of elements in plants is important for researchers across a broad range of fields, including plant molecular biology, agronomy, plant physiology, plant nutrition, and ionomics. However, it is often challenging to evaluate the applicability of the wide range of techniques available, with each having its own strengths and limitations. Here, we compare scanning/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 mass spectroscopy, autoradiography, and confocal microscopy with fluorophores. For these various techniques, we compare their accessibility, their ability to analyze hydrated tissues (without sample preparation) and suitability for in vivo analyses, as well as examining their most important analytical merits, such as resolution, sensitivity, depth of analysis, and the range of elements that can be analyzed. We hope that this information will assist other researchers to select, access, and evaluate the approach that is most useful in their particular research program or application. Visualizing elements in plants is essential for a broad range of studies, including those aiming to improve plant nutrition and crop productivity, improving the nutritional content of edible portions of plants for human nutrition, and reducing concentrations of harmful contaminants in food and the broader environment. Accordingly, gaining a detailed understanding of the distribution and chemical forms of target elements in plants is critical in plant molecular biology, agronomy, plant nutrition, plant physiology, and ionomics. Open in new tabDownload slide Open in new tabDownload slide A variety of approaches can be used to visualize the distribution of elements within plants. These techniques have their own advantages and disadvantages, and for many researchers, selecting the most appropriate technique and evaluating the data from individual techniques can be challenging. For example, these various techniques differ in the range of elements that can be analyzed, their detection limits, ability to be quantitative, their resolving power, and whether specimens can be examined fresh or frozen hydrated (without sample preparation) or whether dehydration (such as freeze-drying) prior to analysis is required. In this review, we aim to compare a suite of techniques that are suitable for mapping the distribution of elements within plants. Specifically, we compare scanning electron microscopy-based energy-dispersive x-ray spectroscopy (SEM-EDS) and transmission electron microscopy-based energy-dispersive x-ray spectroscopy (TEM-EDS), x-ray fluorescence microscopy (XFM), particle-induced x-ray emission (microPIXE), laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS), nanoscale secondary ion mass spectroscopy (NanoSIMS), autoradiography, and confocal microscopy using fluorophores as element-specific labels. For all experimental approaches, it is necessary to consider the methods used for sample preparation. However, this is not discussed in detail here; rather, the reader is referred to other reviews. This review builds upon and complements previous reviews, such as those of van der Ent et al. (2018b), who examined the use of x-ray-based approaches in hyperaccumulator plants, Kopittke et al. (2018), who examined synchrotron-based approaches in plants, Persson et al. (2016a), who examined LA-ICP-MS, Zhao et al. (2014), who discussed the types of research questions enabled by synchrotron-based approaches and mass spectrometry approaches, and Moore et al. (2012a), who examined NanoSIMS and complementary approaches for examining elemental distribution in plants. In this review, we aim to provide a comprehensive comparison of the advantages and disadvantages of the most frequently used techniques and methodologies, thereby enabling readers to select the approach that is most applicable for use in their particular experiment. IMPORTANCE OF VISUALIZING ELEMENTS IN PLANTS The study of the distribution of elements in plants is critical for many research questions. These are briefly considered below before discussing the approaches that can be used to measure elements and their distribution in plants. Functional Characterization in Plant Molecular Biology Studies that link elemental imaging with genetic approaches are critical for characterizing genes that influence elemental homeostasis. Such studies provide opportunities for the analyses of gene × environment interactions in planta, for example, by comparing transporter phenotypes. The approaches described below have been used for functional characterization in molecular biology, with most studies focusing on micronutrients, although some also focused on macronutrients. For example, using a mutant of Arabidopsis (Arabidopsis thaliana) unable to synthesize the metal chelator nicotianamine, it was found with LA-ICP-MS that the mutant accumulated Zn and Mn in the tissues surrounding the vascular cylinder, while Fe was confined to the cortical cell walls in the mutant despite being primarily in the epidermis of the wild type (Persson et al., 2016a). In another recent study, XFM was used to investigate Arabidopsis, finding that METAL TOLERANCE PROTEIN8 (MTP8) determined the distribution of Fe and Mn in seeds (Chu et al., 2017). In this latter study, the use of XFM to image element distribution in vivo avoided potential issues associated with GFP imaging in quiescent seed tissue. In another example, XFM was used by Punshon et al. (2013) to show differences in Ca localization and speciation in a calcium oxalate deficient5 (cod5) mutant of Medicago truncatula. These authors reported that knockout of COD5 prevented biogenic crystal formation by altering Ca distribution and the form of Ca oxalate. Finally, Kim et al. (2006) examined Fe in Arabidopsis seeds, finding that when VACUOLAR IRON TRANSPORTER1 (VIT1) is disrupted, Fe did not accumulate in the provascular strands of the embryo. Improving Plant Nutrition and Productivity Understanding element distribution in plants is also important for improving plant mineral nutrition and productivity. As an example, consider the application of foliar fertilizers to improve plant growth in soils containing low levels of plant-available nutrients. The mechanisms by which foliar-applied nutrients move across the leaf surface and are translocated and assimilated remain unclear. To examine this research question, Zn fertilizer was applied to the surface of a leaf of wheat (Triticum aestivum) and changes in leaf Zn concentrations 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 that the Zn had only limited mobility regardless of the form of Zn applied. Similar results were also reported by Tian et al. (2015). In a similar manner, autoradiography has 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 of 65Zn-labeled compounds allowed for time-resolved analyses of Zn distribution in live plants, reporting that 65Zn was translocated throughout the plant (including to the grain, where it is important for human nutrition) following its foliar application. Improving Human Nutrition through Foodstuffs To improve human nutrition through higher quality foods, an understanding of nutrient concentration and distribution within foodstuffs is essential. This is because the nutritive value of foods depends not only upon the total elemental concentration but also its distribution and molecular speciation. Accordingly, biofortification strategies need to consider both the distribution and speciation of nutrients within the foodstuff tissue. As an example of a study aiming to improve human nutrition, grains of buckwheat (Fagopyrum esculentum) were examined using microPIXE (Pongrac et al., 2011). These authors found that the inner 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 it was 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 accumulate in the bran layers (i.e. the aleurone, the tegument, and the pericarp), those elements that are generally more mobile within the phloem (such as K, Mg, P, Fe, Zn, and Cu) tend to accumulate to higher concentrations in the aleurone layer (De Brier et al., 2015). In this regard, Wang et al. (2011) used LA-ICP-MS to examine the stable Zn isotope 70Zn in wheat grain, finding that there are two barriers to Zn transport in wheat grain: between the stem tissue rachis and the grain and between the maternal and filial tissues in the grain. Not only is distribution important in influencing the nutritional value of foods, but it is also necessary to understand how the colocalization of different elements within foods impacts on nutrient availability to humans. For example, colocalization of micronutrients with P (often present as phytate) likely reduces micronutrient availability in the human gut, with such colocalization observed in sweetcorn and maize (Zea mays; Cheah et al., 2019) and wheat (Moore et al., 2012b). Understanding Toxic Elements in Plants and Tolerance Mechanisms Understanding the behavior of toxic elements in plants, their impact on plant growth, their translocation through the plant and accumulation in human foodstuffs, and the mechanisms that plants use to tolerate these toxicants is of critical importance. First, to illustrate the importance of understanding elemental distribution in crop plants, consider the problem of Al toxicity. Soluble concentrations of Al are elevated in the acid soils that constitute approximately 3.95 billion ha of the global ice-free land (Eswaran et al., 1997). Although Al is highly toxic to plant root growth, much remains unknown about how it exerts its toxic effects. In this regard, NanoSIMS has been used to examine Al distribution in root tissues of soybean (Glycine max), finding that Al accumulates almost entirely in the walls of cells in the rhizodermis and outer cortex (Kopittke et al., 2015). These authors reported that this Al in the cell wall of young, elongating roots was toxic and caused a rapid reduction in root elongation. Interestingly, in tea (Camellia sinensis), a known accumulator of Al, much of the Al accumulated in the cell walls of the leaves, representing a potential tolerance mechanism (Tolrà et al., 2011). As another example, the accumulation of As in foods is of interest due to the consumption of this carcinogen by humans. The distribution of As in roots of Arabidopsis was examined using XFM, confirming the localization of a new arsenate reductase (HAC) that limits As accumulation in the tissues (Chao et al., 2014). Another area of major research interest has been in the use of imaging techniques to understand how hyperaccumulating plants are able to tolerate high concentrations of metal(loid)s in their tissues. Ni hyperaccumulator plants (which make up the majority of hyperaccumulator plants known globally) have been the most intensively studied (Reeves et al., 2018). In most species studied to date, Ni is concentrated in the epidermal cell vacuoles of the leaves (Küpper et al., 2001; Bhatia et al., 2004; Kachenko et al., 2008; van der Ent et al., 2017). Hyperaccumulation spans several length scales, from whole plants down to organs, tissues, individual cells, cellular organelles, and transporter molecules, and information at all of these scales is important for understanding the mechanisms associated with hyperaccumulation (van der Ent et al., 2017). X-RAY FLUORESCENCE-BASED APPROACHES FOR VISUALIZATION With x-ray fluorescence-based approaches, elements are detected based upon their characteristic fluorescent x-rays. These fluorescent x-rays are generated by passing 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 different elements (depending on the energy of the incident x-rays, electrons, or protons), which are detected and quantified by a detector to determine elemental concentrations in the specimen. The movement of the specimen through the incident beam in x-y creates a raster map in which each point represents a pixel with concentration data (or relative element intensity) for a range of elements. High-energy x-rays (greater than 15 keV) have great penetrative power and will pass through plant specimens (both sectioned tissues and potentially even through entire, intact plant tissues), whereas electrons and protons will only penetrate 5 to 50 µm into a specimen. In principle, the incident x-ray beam does not destroy the sample, hence the method is typically considered nondestructive. However, as x-rays are ionizing radiation, depending on the energy and dwell on the sample, damage might occur due to the formation of free radicals, which are highly reactive and damaging to the tissue being analyzed. Furthermore, the incident x-ray beams do not generate heat in the specimen, in contrast to electron and proton beams, which consist of particles and have a far greater potential to damage the specimen during scanning. Obtaining sufficient element sensitivity while keeping dwell low enough not to cause beam-induced damage can 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 in order to produce the characteristic fluorescent x-rays. These approaches are the most commonly used methods for examining elemental distribution in plant tissues. Figure 1. Open in new tabDownload slide Comparison of seven broad techniques used for examining element distribution in plants. All values are indicative of typical systems. Figure 1. Open in new tabDownload slide Comparison of seven broad techniques used for examining element distribution in plants. All values are indicative of typical systems. Figure 2. Open in new tabDownload slide Freeze-dried cross section of a root of Conyza cordata examined using SEM-EDS showing elemental distribution maps of O, S, K, Ca, and Cl. The images were obtained with an incident beam of 15 kV. The specimen was prepared by Jolanta Mesjasz-Przybyłowicz. Figure 2. Open in new tabDownload slide Freeze-dried cross section of a root of Conyza cordata examined using SEM-EDS showing elemental distribution maps of O, S, K, Ca, and Cl. The images were obtained with an incident beam of 15 kV. The specimen was prepared by Jolanta Mesjasz-Przybyłowicz. Given that most SEM- and TEM-based EDS systems operate under a high vacuum, the plant tissue specimen must be totally dehydrated (and coated with carbon to make it conductive for electrons) prior to analysis (Fig. 2). However, where a cryo-SEM system is available, it is possible to examine frozen plant tissue specimens in the hydrated state. Appropriate specimen preparation for cryo-SEM and cryotransfer remains extremely challenging technically, and detection limits are poorer than for dehydrated specimens. In addition, it is also increasingly possible to analyze living plants using environmental SEM, although there are issues with sample size restrictions and beam damage (Danilatos, 1981; McGregor and Donald, 2010). In the majority of studies, specimen dehydration (typically by freeze-drying or lyophilization) is required, and this has the potential to cause artifacts due to elemental redistribution (see van der Ent et al. [2018b] for a full discussion of considerations). When imaging a specimen, SEM can typically achieve a resolution of approximately 1 to 50 nm. However, when examining elemental composition using 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 µm and worsening with increasing accelerating voltage. For TEM-based EDS, the resolution is better than for SEM-based EDS because the use of TEM requires the plant tissues to be cut as ultrathin sections (approximately 60–100 nm in thickness), thereby greatly reducing problems associated with the depth of penetration. Thus, for TEM-based EDS, it is possible to achieve a resolution of approximately 100 nm. A comparatively large range of elements can be detected use SEM- and TEM-based EDS, typically B to U when examining across the K-, L-, and M-edges (see Calvin [2013] for a discussion of the K-, L-, and M-edges). However, the detection limit of the technique is rather poor, generally 0.1 to 1 weight percent for most elements, which severely limits its field of application for plant-based studies. As indicated earlier, the analysis is surface sensitive, given that the electron beam penetrates 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 sensitive to sample-specific characteristics (bulk composition, density, and so forth) and, hence, calibration standards during the analysis are essential (Tylko et al., 2010). However, no commercial biological standards for SEM have yet been developed. It is clear that SEM- and TEM-based EDS are useful approaches where concentrations of the element of interest are high and where the risk of elemental redistribution upon sample dehydration is low. However, their overall usefulness for visualizing elements in plants is comparatively low except for hyperaccumulators or plants where elemental concentrations are high (Figs. 1 and 2). Some recent examples of studies using SEM- and TEM-EDS include the analyses of contaminants on surfaces 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 Glochidion cf. sericeum (van der Ent et al., 2018a), and the distribution of CuO nanoparticles in the xylem of maize (Wang et al., 2012). Synchrotron-Based XFM XFM can be either synchrotron based or laboratory based (Figs. 1, 3, and 4), with these systems having several important differences. Both synchrotron-based and laboratory-based systems use x-rays for the incident beam in order to produce fluorescent x-rays for elemental mapping. Here, we first focus on synchrotron-based XFM. Figure 3. Open in new tabDownload slide Use of synchrotron-based XFM (Australian Synchrotron) for high-throughput screening of plant mutant libraries for Arabidopsis. The image in A is an optical micrograph. The images in B to E show the distribution of Fe (B), Mn (C), Zn (D), and Se (E) in approximately 6,000 seeds, with each image having a resolution of approximately 20 megapixels when displayed at full resolution. The image in F shows a small portion of a detailed scan for Fe showing some seeds differing in their Fe concentration and distribution. The overview scans (B–E) had a 10-µm pixel size with a dwell of 1 ms per pixel, while the detailed scans (a small portion shown in F) had a 1-µm pixel size with a dwell of 7 ms per pixel. In total, an estimated 40,000 seeds were examined, with only approximately 6,000 seeds shown here. Note that the analyses are nondestructive. Figure 3. Open in new tabDownload slide Use of synchrotron-based XFM (Australian Synchrotron) for high-throughput screening of plant mutant libraries for Arabidopsis. The image in A is an optical micrograph. The images in B to E show the distribution of Fe (B), Mn (C), Zn (D), and Se (E) in approximately 6,000 seeds, with each image having a resolution of approximately 20 megapixels when displayed at full resolution. The image in F shows a small portion of a detailed scan for Fe showing some seeds differing in their Fe concentration and distribution. The overview scans (B–E) had a 10-µm pixel size with a dwell of 1 ms per pixel, while the detailed scans (a small portion shown in F) had a 1-µm pixel size with a dwell of 7 ms per pixel. In total, an estimated 40,000 seeds were examined, with only approximately 6,000 seeds shown here. Note that the analyses are nondestructive. Figure 4. Open in new tabDownload slide Analysis of a fresh hydrated shoot of the Se hyperaccumulator Neptunia amplexicaulis, using laboratory-based XFM at the University of Queensland (Australia). Images show elemental maps of K, Ca, and Se distribution and a map of the sum of all x-rays (useful for observing the structure of the sample). Figure 4. Open in new tabDownload slide Analysis of a fresh hydrated shoot of the Se hyperaccumulator Neptunia amplexicaulis, using laboratory-based XFM at the University of Queensland (Australia). Images show elemental maps of K, Ca, and Se distribution and a map of the sum of all x-rays (useful for observing the structure of the sample). There are currently approximately 50 synchrotrons in the world, although not all have XFM beamlines. The most frequently used beamlines for plant analyses include (but are not limited to) the XFM beamline at the Australian Synchrotron (Australia; Kopittke et al., 2018), 13IDE at the Advanced Photon Source (United States; Doolette et al., 2018), the XFM beamline and the hard x-ray nanoprobe beamline at Brookhaven National Laboratory (United States; Li et al., 2019b), and ID21 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 sample size. As a result, plants can be examined hydrated, and even in vivo analyses are possible if the entire plant can be mounted in front of the x-ray beam (Blamey et al., 2018b; Doolette et al., 2018). Nevertheless, care must be taken to ensure that the incident x-ray beam does not result in artifacts in the specimen during analysis, which can cause localized structural damage and the redistribution of elements. This is particularly important when examining hydrated samples, with these being especially sensitive to radiation damage. Unfortunately, few studies state whether they have explicitly determined whether sample damage occurs and whether there is a concomitant redistribution of elements (Jones et al., 2020). For XFM, given the penetrating nature of the x-rays (both the incident x-rays as well as the fluorescent x-rays), elemental distribution can often be examined throughout the entire thickness of plant tissues. However, the depth of analysis varies greatly depending upon the element of interest, being determined by the energy of the corresponding fluorescent x-rays (with these having a lower energy than the incident x-rays). This is best illustrated with the following examples. For Ca, with a K-edge emission line of 3.7 keV, 50% of the fluorescence will be absorbed in a plant sample approximately 70 µm thick and 90% in a sample approximately 200 µm thick. In contrast, for Se, with a K-edge emission line of 11.2 keV, 50% of the fluorescence will be absorbed in a sample approximately 2,000 µm thick and 90% in a sample approximately 6,500 µm thick. In other words, assuming a root with a thickness of 1,000 µm, only the Ca in the surface 100 to 200 µm can be detected (with the Ca in the vascular tissue being invisible), while Se will be detected across the entire depth of the root cylinder. Thus, great care needs to be taken when comparing the distribution of various elements, especially in thicker samples. Most synchrotron-based XFM facilities tend to have a resolution on the order of 20 nm to 1 µm (Li et al., 2019b). The time required to conduct analyses also varies greatly. For synchrotron-based systems with fast detector systems, the dwell is now routinely 1 ms or less, meaning that a 1-megapixel image can be collected in approximately 17 min or less. The elements that can be 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 examined using the L-edges. The detection limit varies widely depending upon the facility as well as the element being analyzed. For elements such as Mn, Fe, and Zn, the detection limit is excellent, being on the order of approximately 1 mg kg−1 or even lower. However, the detection limit decreases for the lower Z elements, including P, S, and K, often being approximately 10 to 1,000 mg kg−1, which is a function of the smaller x-ray cross sections (resulting in lower fluorescence yields) and the operation of most XFM beamlines in air, which absorbs low-energy x-rays. For synchrotron-based XFM, analyses are potentially fully quantitative, for example, using the GeoPIXE software package, which produces quantitative self-absorption corrected maps that are line overlap resolved and in which the background is subtracted (Ryan, 2000). Given that XFM allows analyses of plants in vivo with no (or minimal) sample preparation and with good sensitivity (Fig. 3), this technique is being used to examine an increasing number of diverse problems. This includes studies where it is imperative to avoid sample processing (such as freeze-drying) or where repeated measurements of living plants are required in vivo. It is especially useful for studies focusing on trace metals and metalloids, such as Mn, 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 using XFM include analyses of MTP8 (Chu et al., 2017; Eroglu et al., 2017; Basiri-Esfahani et al., 2019), kinetic analyses of living leaves of cowpea (Vigna unguiculata) exposed to toxic levels of Mn (Blamey et al., 2018b), analyses of Zn movement following foliar fertilization in wheat (Doolette et al., 2018), and analyses of nanoparticles in plants (Martínez-Criado et al., 2016). A potential new, and yet unexplored, use is for high-throughput screening of plant mutant libraries. This is especially exciting given the nondestructive nature of XFM analyses as well as the ability to examine changes in the spatial distribution of elements instead of examining only bulk concentrations (Fig. 3). Finally, another potential advantage of XFM analyses is the potential to combine imaging with speciation through the use of x-ray absorption near-edge structure spectroscopy (Kopittke et al., 2018), although this is beyond the scope of our review (see Wang et al. [2015] for an example for examining the distribution and speciation of Se in rice and wheat tissues). Laboratory-Based XFM The strengths of XFM are evident from the research output from studies undertaken at synchrotrons. However, the restrictive nature of access to synchrotron XFM facilities is recognized by many users as a limiting factor in using XFM in their research, and where laboratory-based facilities are available, this can overcome that limitation. However, we are not aware of many laboratory-based XFM systems, with this being a current restriction for their use. The authors are aware of systems used for the investigation of plants at the microXRF facility at the University of Queensland (Australia; Fig. 4), Washington State University (United States; Fittschen et al., 2017), and the Maia Mapper at the Advanced Resource Characterization Facility of the Commonwealth Scientific and Industrial Research Organization in Australia (Ryan et al., 2018). The latter is perhaps the most advanced laboratory XFM system, as it can image up to ∼80 million pixels over a 500- × 150-mm2 sample area using the Maia detector array. However, it has been developed for drill core sections and polished rock slabs, not biological applications. The University of Queensland microXRF facility has been specifically developed for biological applications and has dual microfocus sources (focusing to 5 and 25 μm), two large silicon drift detectors of 150 mm2, and can scan areas up to 300 × 300 mm in air, vacuum, or helium atmosphere. It also has a cryo-stage (50- × 50-mm active area held at −50°C) for analysis of samples in a frozen-hydrated state. These new laboratory-based XFM facilities do not fully replace synchrotron-based XFM for a number of specialized applications but will bridge the gap between what is currently possible in the laboratory environment and the capability of synchrotron facilities. Furthermore, it allows researchers to combine the strengths of both facilities, for example, by whole-organism mapping at their laboratory followed by investigation of target cells at the synchrotrons, and hence strengthen the outcome of both platforms. Laboratory-based XFM systems essentially offer unlimited access (within the institutional constraints of availability and financial considerations) as required by experimental needs. In addition, many laboratory-based systems provide vacuum and helium purge capabilities that might not be available at synchrotron-based beamlines, thereby offering improved capability for the measurement of light elements such as Al, Si, S, and P. However, these laboratory-based systems offer worse spatial resolution, often in the range of 5 to 50 µm (compared with 20 nm to 1 µm for synchrotron-based systems). In addition, laboratory-based systems typically use a concave focused polychromatic x-ray source with Bremsstrahlung background, with this having important differences from a monochromatic, highly parallel x-ray source in synchrotron-based XFM. For example, there is no energy tunability in laboratory-based systems, and hence x-ray absorption spectroscopy is not possible. Finally, the substantially lower x-ray flux for laboratory-based systems (typically 1,000 to 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 of protons is used as the incident beam, generating fluorescent x-rays in the sample. Figure 5. Open in new tabDownload slide A, PIXE elemental map of an intact Noccaea caerulescens seed (pixel size 2 µm, dwell 5 ms per pixel). The southern France accessions (St. Laurent de Minier/Ganges) have the ability to hyperaccumulate Cd with up to 900 mg kg−1 Cd in the seeds. B, Stele of a mature 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 µm) showing 24Mg distribution. C, LA-ICP-MS images from the inner tissues of a mature barley root with a pixel size of 5 µm, showing 24Mg, 67Zn, 66Zn, and a light micrograph (the red square indicating the area analyzed using LA-ICP-MS). The root was first starved for Zn and then exposed to 67Zn stable isotope (94.3% enriched) for 2 h. The natural 66Zn/67Zn isotopic ratio is 6.8 (66Zn, 27.9%; 67Zn, 4.1%). The 67Zn image shows how the Zn (added as 67Zn) is taken up and transported radially toward the stele. Figure 5. Open in new tabDownload slide A, PIXE elemental map of an intact Noccaea caerulescens seed (pixel size 2 µm, dwell 5 ms per pixel). The southern France accessions (St. Laurent de Minier/Ganges) have the ability to hyperaccumulate Cd with up to 900 mg kg−1 Cd in the seeds. B, Stele of a mature 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 µm) showing 24Mg distribution. C, LA-ICP-MS images from the inner tissues of a mature barley root with a pixel size of 5 µm, showing 24Mg, 67Zn, 66Zn, and a light micrograph (the red square indicating the area analyzed using LA-ICP-MS). The root was first starved for Zn and then exposed to 67Zn stable isotope (94.3% enriched) for 2 h. The natural 66Zn/67Zn isotopic ratio is 6.8 (66Zn, 27.9%; 67Zn, 4.1%). The 67Zn image shows how the Zn (added as 67Zn) is taken up and transported radially toward the stele. For microPIXE, the resolution is generally in the range of 1 to 3 µm, or occasionally slightly better. PIXE excites the K-lines of virtually all elements, and hence it is generally possible to measure elements in the range of 1.5 to 60 keV (corresponding to Al to W), with this being considerably wider than that achieved using XFM. Moreover, very light elements in the range of 0.05 to 1.3 keV (corresponding to Li to Mg) can be analyzed with particle-induced γ-ray emission. As such, PIXE opens up imaging of metals such as Ag and Cd and the metalloids Sb, Te, and I, which are very difficult to measure with synchrotron XFM. The detection limit using microPIXE is excellent, typically in the range of lower mg kg−1, with analyses also generally being fully quantitative with the use of techniques (such as Rutherford backscattering spectrometry) to determine sample matrix composition. The new PIXE Maia facility at the University of Melbourne (Australia) combines the benefits of PIXE with those of the revolutionary Maia detector array able to process 4 to 9 million events s−1 (measured on a plant specimen; Laird et al., 2019) or up to ∼900 times greater than that typically used in the previous 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 systems. Given that it can analyze a wider range of elements than many other approaches (such as XFM), it is especially valuable for examining light elements (such as Na, Mg, or Al) as well as heavier elements (such as Cd 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). Some recent studies using microPIXE include for the analysis of Zn and Cd in a hyperaccumulator, Sedum plumbizincicola (Hu et al., 2015), and for the study of Ni hyperaccumulation in Phyllanthus balgooyi (Mesjasz-Przybylowicz et al., 2016). MASS SPECTROMETRY-BASED APPROACHES FOR VISUALIZATION For mass spectrometry-based approaches, small portions of the sample are progressively removed during scanning for analysis. Because analysis is by mass spectrometry, not only are these generally highly sensitive techniques, but they also allow isotopic analyses. However, given that small portions of the sample are removed for analysis during scanning, these mass spectrometry-based approaches are considered destructive. LA-ICP-MS LA-ICP-MS uses a focused laser to ablate the surface of the sample (Figs. 1 and 5, B and C). These ablated particles are then transported to an ICP-MS device in a stream of He gas for both elemental and isotopic analyses. LA-ICP-MS offers excellent detection limits (less than 1 mg kg−1) and a very wide range of elements (Li to U). Furthermore, multielement analyses are routine, and stable isotope analyses are also possible, as illustrated in Figure 5C. In addition, LA-ICP-MS offers a modest resolution (approximately 1 µm), being similar to microPIXE and SEM-based EDS. A wide range of elements 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 kg−1 for many physiologically relevant elements (Persson et al., 2016a). Analyses are potentially fully quantitative, although this is not without substantial difficulties. Specifically, the laser beam interaction with the sample will vary according to the sample properties, resulting in changes in the amount of analyte (sample) removed per pulse. For example, portions of the plant tissues that are heavily lignified will ablate less material than softer parts of the plant tissue, making full quantification challenging. Thus, analyses of plant tissues are generally considered to be semiquantitative. Although LA-ICP-MS analyses are generally conducted at ambient temperature and pressure in an inert Ar atmosphere, extreme care must be taken to avoid sample desiccation due to the exposure of samples to the dry stream of Ar gas when examining hydrated tissues, with this leading to experimental artifacts. As a result, most analyses utilizing LA-ICP-MS examine dehydrated samples and, hence, in vivo analyses are challenging. Nevertheless, it is indeed possible to analyze fresh (hydrated) tissues and living plants in some situations (Salt et al., 2008; Klug et al., 2011), and protocols for sample preparations that produce intact, dry samples with unaltered ion distribution within tissue are available (Persson et al., 2016a). LA-ICP-MS is therefore particularly useful in studies where high sensitivity is required with access to an extremely broad range of elements with good sensitivity (Figs. 1 and 5, B and C). It is a surface-sensitive technique that offers advantages compared with XFM or microPIXE in some situations, but it can be disadvantageous in others. The use of isotopic analyses is also a potentially useful advantage for examining the flux and distribution of exogenously applied isotopes. Recent studies to use LA-ICP-MS for the study of plants include for the imaging of Fe, Zn, and Mn in roots of Arabidopsis (Persson et al., 2016a), Mn in roots and grain of barley (Hordeum vulgare; Long et al., 2018; Chen et al., 2019), Ca, Na, and K in stems and leaves of tobacco (Nicotiana tabacum; Thyssen et al., 2017), Zn, S, and P analyses of biofortified wheat grains (Persson et al., 2016b), Cd, Pb, Cu, and Zn in roots of pea (Pisum sativum; Hanć et al., 2016), for mapping the distribution of pollutants in leaves of sweet basil (Ocimum basilicum; Ko et al., 2018), and for examining nutrient distribution in nodes of rice mutants (Yamaji and Ma, 2019). NanoSIMS In NanoSIMS (Figs. 1 and 6), ions are used as the incident (primary) beam, and these ions collide with the sample surface and cause atoms, ions, and molecules from the sample surface to be ejected into the vacuum (sputtering). The ionized particles (secondary ions) are then collected and transported to a mass spectrometer for analysis. For NanoSIMS, the sputtering depth is approximately 5 to 20 nm (Hoppe et al., 2013), making it a very surface-sensitive technique. Figure 6. Open in new tabDownload slide NanoSIMS analyses of a portion of a transverse cross section of soybean root exposed to 30 µm Al for 0.5 h with an radio frequency plasma O− source. The images for Al (left) and Na (right) were obtained with a pixel size of 0.3 µm and a dwell of 60 ms per pixel. For more information on plant growth and analyses, see Kopittke et al. (2015). Figure 6. Open in new tabDownload slide NanoSIMS analyses of a portion of a transverse cross section of soybean root exposed to 30 µm Al for 0.5 h with an radio frequency plasma O− source. The images for Al (left) and Na (right) were obtained with a pixel size of 0.3 µm and a dwell of 60 ms per pixel. For more information on plant growth and analyses, see Kopittke et al. (2015). Currently, there are considerably fewer NanoSIMS facilities in the world than there are synchrotrons. As a result, accessing a NanoSIMS device for an experiment can potentially be difficult for many researchers. Nevertheless, the facilities most commonly used for investigations of plant tissues include those at the University of Manchester (England) and the University of Western Australia (Australia). NanoSIMS operates in an ultra-high vacuum, meaning that samples must first be dehydrated before analysis. Furthermore, NanoSIMS requires a flat surface, and hence it is typically only possible to examine sectioned tissues (Fig. 6). As for other techniques where sample dehydration is required prior to analysis, extreme care must be taken to ensure that the method used for sample processing does not cause experimental artifacts through redistribution of the elements of interest. NanoSIMS offers an excellent lateral resolution, with analyses routinely conducted at resolutions as low as 100 nm (Fig. 6). Using this technique, it is possible to analyze a very wide range of elements of relevance to plant studies, from H to U. The sensitivity is also very good, with the detection limit being in the low mg kg−1 range. The sensitivity for any given element depends upon the primary beam selected, with either an O− beam or a Cs+ beam available. The negatively charged primary beam (i.e. O−) tends to favor the production of positively charged secondary ions, while the positively charged primary beam (i.e. Cs+) tends to favor the production of negatively charged secondary ions. As a result, for elements such as Na, Mg, Al, K, Ca, Mn, Fe, and Zn, the O− beam is generally preferred (Nuñez et al., 2017). In contrast, for elements such as Si, P, S, Cl, As, and Se, the Cs+ beam is generally preferred. The main advantages of NanoSIMS are the excellent detection limit and spatial resolution as well as the wide range of elements that can be analyzed. As a result, this approach is particularly suited to examining the subcellular distribution of elements within cross sections of plant tissues (Figs. 1 and 6). Isotopic analyses are also possible using this approach (for an example in plants, see Moore et al. [2016]). Some recent studies utilizing NanoSIMS include the study of Fe with amyloplasts in pea seeds (Moore et al., 2018), detoxification of Mn by Si in leaves of soybean and sunflower (Helianthus annuus; Blamey et al., 2018a), determining the mechanisms by which foliar-applied Zn fertilizer moves across the leaf surface (Li et al., 2019a), as well as examining the distribution of Al in roots of soybean (Kopittke et al., 2015) with the mass of Al being too low to examine using XFM (Fig. 6). AUTORADIOGRAPHY Autoradiography (Figs. 1 and 7A) is the oldest of the techniques discussed here, having been used for plants since the 1920s (Hevesy, 1923). In autoradiography, radioactive isotopes are supplied to a plant, which are taken up and redistributed throughout the plant tissues. To then examine their distribution in the plant, an image is obtained of the decay emissions from the various plant tissues (Fig. 7A). These decay emissions can be detected using an x-ray film or, more recently, using digital autoradiography. Figure 7. Open in new tabDownload slide A, Autoradiography of treated wheat leaves onto which 65Zn-labeled foliar fertilizers had been applied as 65ZnCl2, 65ZnEDTA, 65ZnO-NPs (nanoparticles), and 65ZnO-MPs (microparticles; 750 mg L−1). The digital photograph (left) shows the leaves onto which the Zn was applied. A total of 10 droplets were applied onto each leaf before being washed from the leaf surface. For more information, see Read et al. (2019). B, Zinpyr-1 (fluorescent indicator for Zn2+; green color) stained and autofluorescence of a leaf section of N. caerulescens obtained using confocal fluorescence microscopy. Figure 7. Open in new tabDownload slide A, Autoradiography of treated wheat leaves onto which 65Zn-labeled foliar fertilizers had been applied as 65ZnCl2, 65ZnEDTA, 65ZnO-NPs (nanoparticles), and 65ZnO-MPs (microparticles; 750 mg L−1). The digital photograph (left) shows the leaves onto which the Zn was applied. A total of 10 droplets were applied onto each leaf before being washed from the leaf surface. For more information, see Read et al. (2019). B, Zinpyr-1 (fluorescent indicator for Zn2+; green color) stained and autofluorescence of a leaf section of N. caerulescens obtained using confocal fluorescence microscopy. Compared with some other approaches, such as XFM or NanoSIMS, access to autoradiography facilities is likely not too difficult. This approach also has a range of other advantages, including being able to examine hydrated plant tissues, including for in vivo analyses. Furthermore, autoradiography can be used to examine large samples or even entire plants. Another major advantage is the ability to separate background isotopes of an element (i.e. those natively present in the plant tissues) from the radioisotope of the same element added exogenously. The resolution achievable with autoradiography varies between approximately 25 and 1,000 µm (Fig. 1) and depends upon a range of factors (Zhang et al., 2008). The greatest challenges for using autoradiography are the highly restrictive and complicated health and safety regulations in many jurisdictions for working with radioisotopes. In addition to this limitation, it is only possible to examine a single element at a time (Solon et al., 2010). Furthermore, it is only possible to use this technique for elements where a suitable radioisotope exists. For studies of plants, these elements include 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 to be avoided. It also offers excellent detection limits and allows the separation of background elements from those added exogenously as radioisotopes, this being critical when only a portion of the total element is of interest (Figs. 1 and 7A). Recent studies to use autoradiography to examine elemental distribution in plants include the study of 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 MICROSCOPY WITH FLUOROPHORES The final approach considered here is the use of laser confocal microscopy with element-selective fluorophores (Figs. 1 and 7B). Given that many researchers are likely able to access laser confocal microscopy without substantial difficulty, this approach is one of the easier ones in terms of facility access, as most research institutions will have laser confocal microscopes, especially in medical faculties where they are routinely used. This approach is also nondestructive and can be used on hydrated samples, including for in vivo analyses of living plants. The maximum resolution is similar to some other approaches, being approximately 1 µm. All the approaches considered above have analyzed elemental composition directly. However, laser confocal microscopy relies on the binding of ion-selective fluorophores to the element of interest for their subsequent detection using excitation by specific wavelengths emitted by lasers (Fig. 7B). This in itself represents a potential limitation of this technique, as fluorophores will generally only bind to free ions not already bound strongly to other ligands in the plant. Thus, the proportion of the total pool identified using the fluorophore can be uncertain. In addition, issues with penetration into the plant tissue are largely unknown and are hard to quantify. The range of elements that can be investigated using laser confocal microscopy is entirely dependent on the commercial availability of fluorophores with specific affinities for elements of interest. These include, but are not limited to, Zn (Zinpyr-1, FluoZin, TSQ), Ni/Co (Newport Green), Cu (Phen Green), and Pb/Cd (Leadmium Green). The use of laser confocal microscopy with element-selective fluorophores is particularly suited to cases where in vivo analyses are required for an element for which a suitable fluorophore exists, with an excellent detection limit (Figs. 1 and 7B). However, questions still remain regarding the binding of the fluorophores and their penetration into the plant tissue. Recent studies using confocal microscopy with fluorophores include imaging the distribution Ni2+ with the dye Newport Green in Alyssum murale (Agrawal et al., 2013) and Alyssum lesbiacum (Ingle et al., 2008), the use of Zinpyr-1 for imaging the distribution of Zn2+ in Noccaea caerulescens (Kozhevnikova et al., 2017; Dinh et al., 2018) and Arabidopsis (Sinclair et al., 2007), and the use of Leadmium Green for imaging Zn2+ and Cd2+ in Sedum alfredii and Picris divaricata (Lu et al., 2008; Hu et al., 2012). Open in new tabDownload slide Open in new tabDownload slide CONCLUDING REMARKS Understanding the distribution of elements within plant tissues is critical for a range of research programs within plant science, including for functional characterization in molecular biology, improving plant nutrition and productivity, improving human nutrition, and understanding toxic elements in plants and tolerance mechanisms. For analyzing plants, a range of techniques are suitable, but it can often be confusing as to which approach is best given their range of advantages and limitations. It is clear from this review that there is no single technique that is best. Rather, each technique has its own strengths and weaknesses. By comparing the accessibility, ability to analyze hydrated tissues (without sample preparation) and conduct in vivo analyses, as well as comparing the resolution, sensitivity, depth of analysis, and range of elements that can be analyzed for the seven described approaches, we hope that this information will assist other researchers to select and access the approach that is most useful in their particular research program. In addition, it will be helpful to use correlative approaches in which the same sample is examined with multiple techniques to exploit the advantages listed here. Of central importance in the future will be the analyses of living plants (including in vivo analyses) with minimal sample preparation at excellent resolution and with good detection limits across the wide range of physiologically relevant elements, this requiring a strong correlative approach (see Outstanding Questions). The use of such correlative approaches will enable important research questions to be answered within the field of plant science. ACKNOWLEDGMENTS The autoradiography images were collected during a study undertaken at Australian Nuclear Science and Technology Organization, and the authors acknowledge Tom Cresswell and Nick Howell (Australian Nuclear Science and Technology Organization) for collecting and preparing the images. Thea Read and Casey Doolette (University of South Australia) are also acknowledged for their contributions to the autoradiography study. 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The laboratory XFM instrument was funded through the University of Queensland Major Equipment and Infrastructure Advanced Micro-X-Ray Fluorescence Facility for Biological, Medical, Materials Science, and Geochemistry (grant no. UQMEI1835893). A Housing Grant (to P.M.K.) was provided by the National Bank (Denmark). Parts of this research were undertaken on the XFM beamline at the Australian Synchrotron, part of the Australian Nuclear Science and Technology Organisation. [OPEN] Articles can be viewed without a subscription. 3 Senior author A.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. and J.S.L. wrote the microPIXE section; D.P.P. and S.H. wrote the section on LA-ICP-MS; K.L.M. wrote the section on NanoSIMS; E.L. wrote the section on autoradiography; P.M.K. and A.v.d.E. wrote the section on laser confocal microscopy; P.M.K. coordinated the overall drafting of the article; all authors edited and approved the final version of the article. www.plantphysiol.org/cgi/doi/10.1104/pp.19.01306 © 2020 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2020. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - Methods to Visualize Elements in Plants
JF - Plant Physiology
DO - 10.1104/pp.19.01306
DA - 2020-04-06
UR - https://www.deepdyve.com/lp/oxford-university-press/methods-to-visualize-elements-in-plants-U4ZkOICu60
SP - 1869
EP - 1882
VL - 182
IS - 4
DP - DeepDyve
ER -