Methods to Visualize Elements in Plants

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.

Figure 1.

Comparison of seven broad techniques used for examining element distribution in plants. All values are indicative of typical systems.

Figure 2.

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 3.

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.

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 5.

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 6.

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 7.

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.