Synchrotron Radiation Techniques

Synchrotron techniques have emerged as a powerful tool to study the speciation and distribution of metal and metalloids in plants exposed to nanomaterials. These techniques are based on the electromagnetic radiation produced when a magnetic field alters the direction of particles moving at nearby the speed of light.

Synchrotron facilities produce high-intensity photons with brilliance that is several orders of magnitude higher than that produced by conventional X-ray sources. High brilliance provides unique capabilities on experiments requiring a high photonflux and a small beam size. The tunability of synchrotron radiation (SR) allows the study of samples with different techniques (e.g., X-rayfluorescence and X-ray absorption spectroscopy). Some specific advantages offered by SR for the study of metals/metalloids in plants are as follows:

(1) Samples can be analyzed with little or no pretreatment.

(2) Sensitivity limit at femtogram level and spatial resolution at micro- and nanoscale levels (Sarret et al.2013).

(3) Potential to identify the chemical forms of the element of interest. (i.e., spe-ciation and coordination environments).

Two SR techniques seem to be more suitable to study the effects of nanoma-terials in plants: X-ray fluorescence and X-ray absorption spectroscopy. Both techniques can be used in tandem to obtain the distribution and speciation of elements of interest. Several reviews have focused on the use of SR techniques in plants (Lombi and Susini2009; Lombi et al.2011; Donner et al.2012; Majumdar et al.2012; Sarret et al. 2013). This section provides a brief description of the SR techniques used to study plants exposed to MNMs and emphasizes results.

3.3.2.1 Micro-X-Ray Fluorescence (μ-XRF)

This technique is based on the emission of characteristic X-rays from atoms excited by the SR. When incident X-rays eject core level electrons from the atom into the continuum, a core-hole vacancy is created. Electrons from higher energy statesfill the core-hole creating photons (fluorescence) of specific energies.

The multi-elemental μ-XRF technique has been predominantly used to create bi-dimensional maps in plants. The high spatial resolution inμ-XRF maps is pro-vided by special optics (e.g., Kirkpatrick-Baez mirrors, Fresnel zone plates) that generate focused beams with sizes that can reach less than 1µm. Due to the high penetration of hard X-rays, thin sections are recommended when analyzing plant tissue, in order to avoid signal originating from different depths (Scheckel et al.

2007; Lombi et al.2011; Hernandez-Viezcas et al.2013; Majumdar et al. 2014).

Multiple elements can be analyzed and mapped simultaneously with the present technique. Metal oxide MNMs have been at the forefront of plant nanotoxicity research due to their unique properties and high production (Kahru and Dubourguier2010; Hendren et al.2011). Nanoparticles of CeO₂, ZnO, TiO₂, and FeO_x are some of the most studied metal oxides MNMs (Piccinno et al. 2012).

Larue et al. (2011) exposed wheat plants to anatase TiO₂NPs (12 nm) in hydro-ponics for seven days. The μ-XRF analysis showed Ti in the parenchyma and vascular cylinder of the roots, suggesting root absorption and translocation of TiO₂ NPs. Servin et al. (2013) cultivated cucumber in soil amended with 750 mg/kg of TiO2NPs and analyzed the fruit with an X-ray beam of 0.3× 0.7 μm²generating a fluorescence map. The synchrotron μ-XRF map showed Ti in the fruit (Fig.3.7).

The transfer of TiO₂NPs to lettuce (Lactuca sativa) leaves was also evaluated with μ-XRF. This spectroscopic technique provided spatial localization of Ti in all let-tuce tissues; however, no phytotoxicity was observed (Larue et al.2014a). In recent studies with plants exposed to nCeO₂(8 nm), SR μ-XRF showed the presence of Ce in the vascular tissue of kidney bean (Phaseolus vulgaris) roots, cucumber leaves, rice roots, and soybean pods (Zhao et al. 2013; Rico et al. 2013;

Hernandez-Viezcas et al. 2013; Majumdar et al.2014). Zhao et al. (2014,2015) used μ-XRF to determine the effects nCeO2 on micro- and macronutrients in cucumber and corn (Zea mays). The authors reported that nCeO₂did not change the nutrient element distribution in cucumber plant and found a reduced Ca translo-cation and elements’ redistribution in kernels of nCeO2 treated corn plants. In plants exposed to ZnO NPs,μ-XRF maps have shown increased Zn concentration

in roots of cowpea (Vigna unguiculata), the root and leaves of mesquite (Prosopis juliflora velutina), and the stem and pods of soybean. Nevertheless, no major signs of toxicity were found in these plant species (Hernandez-Viezcas et al.2011,2013;

Wang et al.2013).

Elemental NPs’ production is smaller when compared to metal oxide NPs;

however, there is a need to study their potential plant nanotoxicity (Piccinno et al.

2012). μ-XRF, in combination with other analytical techniques, was used to demonstrate that Au NPs (3.5–18 nm) were taken up by roots of tobacco plants and subsequently translocated to the aerial plan parts (Sabo-Attwood et al.2012). Larue et al. (2014a) exposed, through the leaves, lettuce plants to several concentrations of Ag NPs and analyzed the leaves with several techniques.μ-XRF showed that the Ag NPs were entrapped in the cuticle, and some of them penetrated the leaves.

3.3.2.2 Synchrotron X-Ray Absorption Spectroscopy (XAS)

XAS is a spectroscopic technique that provides information about the chemistry of the element of interest in a sample. The obtained XAS spectra can provide the oxidation state, interatomic distances, coordination number, and species of the atoms surrounding the analyte. This technique requires a high photonflux and tunability, which makes it almost exclusive to synchrotron facilities (Lombi and Susini2009).

As with XRF, the XAS phenomenon is dominated by the photoelectric effect.

Fig. 3.7 aTricolorμ-XRF images of the cross sections of cucumber fruit treated with nTiO2, b Ti temperature map, c μ-XANES spectra, spots of interest (3–4) were chosen from image (a).

(Adopted from Servin et al. (2013). Copyright @ 2013 American Chemical Society)

In XAS measurements, incident photons progressively increase their energy while impacting the sample, starting at 50 eV below the binding energy of the analyte and finishing a few hundreds to over a thousand eV above it. The XAS spectrum indicates the energy absorption of the element due to the ejection of the photoelectron to the continuum. Also, oscillations caused by the interferences of the photoelectron with neighboring atoms are indicated in the spectrum. The XAS spectrum is divided into two parts, X-ray absorption near edge structure (XANES) and extended X-ray absorptionfine structure (EXAFS). XANES encompasses the region approximately 50 eV below and above the absorption edge. This portion of the spectrum provides information about the oxidation state, local symmetry, and molecular species, when compared to model (pure) compounds. The EXAFS part of the spectrum extends from 50 to 1000 eV above the absorption edge and contains information about the coordination environment of the analyte. Detailed explana-tions about the XAS principles can be found in excellent reference reviews (Fendorf et al.1994; Bertsch and Hunter2001; Lombi and Susini2009; Sarret et al.2013).

The incident beam that excites the elements of interest in the sample can have different sizes depending on the optics used: focused beam on the order of micrometer or submicrometer, and“bulk” beams on the order of millimeters.

Lopez-Moreno et al. (2010a,b) investigated the speciation of Ce in hydropon-ically grown soybean, corn, cucumber, alfalfa, and tomato plants exposed to CeO₂ NPs. Root samples were freeze-dried and homogenized with mortar and pestle, loaded into aluminum sample holders, covered with Kaptonfilm, and analyzed by XAS. By comparing the obtained XAS spectra with the spectra of model com-pounds, authors reported that CeO₂NPs were absorbed and stored within the roots with no modification. In another study, ZnO NPs were exposed to tumbleweed (Salsola tragus), mesquite, and palo verde (Parkinsonia florida). XAS studies showed no presence of ZnO NPs within the roots tissues and Zn was present as Zn (II) (Lopez-Moreno et al. 2010a; De La Rosa et al. 2011). Wang et al. (2013) performed XAS on cowpea grown in soil amended with ZnO NPs and corroborated that ZnO NPs are not stored within tissues. Further experiments concluded that ZnO NPs is not stable in the soil.

Several studies have shown that µ-XRF and µ-XAS can be used together to provide complementary information about the uptake, distribution, and speciation of NPs within plant tissues. After a µ-XRF map is created, specific areas in the image can be analyzed byµ-XAS to determine the oxidation state of the analyte.

The distribution and speciation of rare earth nano-oxides (nLa₂O₃, nYb₂O_3, and nCeO₂) were studied withµ-XRF and µ-XAS in cucumber tissues. A small portion of nLa₂O₃ and nYb₂O₃ was present in the root cells as LaPO₄ and YbPO₄, respectively, whereas a portion of the nCeO₂was biotransformed into CePO₄in the root and Ce(CH₃COO)₃ in the shoot (Ma et al. 2011; Zhang et al. 2012a, b).

Similarly, Cui et al. (2014) found that 6 % of nCeO₂biotransformed into Ce(III) carboxylates in the root of lettuce exposed to nCeO₂. Hernandez-Viezcas et al.

(2013) usedµ-XRF to localize Ce in the soybean pod and by using µ-XAS, they

found that most of the Ce remained as Ce(IV) in the form of CeO2NPs. Servin et al.

(2012) also usedµ-XRF and µ-XAS to study cucumber plants exposed to nTiO2

(anatase 82 %, rutile 18 %) (Fig.3.7). The results showed that TiO2NPs can be absorbed and translocated to the aerial parts in cucumber. Interestingly, the anatase phase remained in the root, while the rutile phase was found in the aerial parts of the plant.

3.3.2.3 Synchrotron-Based X-Ray Microscopy and Tomography

Synchrotron-based X-ray microscopy measures the absorbance above and below the edge energy of the analyte of interest, and the differences are used to generate 2D images. The samples can be rotated, reanalyzed, and the resulting images used to reconstruct 3D images. This technique can achieve resolutions as high as 20– 40 nm. A disadvantage of plant analysis is the need for high concentrations in the samples. Fluorescence tomography, on the other hand, uses afluorescence detector to produce 2D images; the sample is then rotated and reanalyzed to render a 3D image. This type of tomography has a higher sensitivity than contrast absorption measurements (Lombi et al. 2011). Patty et al. (2009) used transmission X-ray microscopy (TXM) to create absorption contrast transmission 2D and 3D images of cordgrass (Spartina foliosa) root exposed to mercury (Fig.3.8). The results of the study show Hg NPs in the root tissue. Ma et al. (2011) and Zhang et al. (2012a) also used TXM to study rare earth nano-oxides, nLa₂O₃, nYb₂O₃, and nCeO₂ in cucumber tissues (Zhang et al.2012c). Their studies suggest that the NPs distribute inside the cucumber tissues at varying degrees. Later on, Hernandez-Viezcas et al.

(2013) employed TXM to investigate the behavior of ZnO NPs in the soybean; the technique showed clusters of Zn in the pod tissue but no presence of ZnO NPs.

Synchrotron radiation techniques have proven to be a powerful tool to study the interactions of MNM and plants. With the challenges of nanotechnology, life sci-ences, and engineering, synchrotron facilities tend to provide beamlines capable of micro- and nanoscale analysis. Nevertheless, XAS can only provide a certain amount of information, and complementary techniques should be used to improve the data collection. Table3.3summarizes recent studies on the use of SR to assess of the effects of NMs in plants.