Synchrotron-Based X-Ray Fluorescence Microscopy as a Technique for Imaging of Elements in Plants

Abstract

Understanding the distribution of elements within plant tissues is important across a range of fields in plant science. In this review, we examine synchrotron-based x-ray fluorescence microscopy (XFM) as an elemental imaging technique in plant sciences, considering both its historical and current uses as well as discussing emerging approaches. XFM offers several unique capabilities of interest to plant scientists, including in vivo analyses at room temperature and pressure, good detection limits (approximately 1-100 mg kg^-1), and excellent resolution (down to 50 nm). This has permitted its use in a range of studies, including for functional characterization in molecular biology, examining the distribution of nutrients in food products, understanding the movement of foliar fertilizers, investigating the behavior of engineered nanoparticles, elucidating the toxic effects of metal(loid)s in agronomic plant species, and studying the unique properties of hyperaccumulating plants. We anticipate that continuing technological advances at XFM beamlines also will provide new opportunities moving into the future, such as for high-throughput screening in molecular biology, the use of exotic metal tags for protein localization, and enabling time-resolved, in vivo analyses of living plants. By examining current and potential future applications, we hope to encourage further XFM studies in plant sciences by highlighting the versatility of this approach.

Figure 1.

Distribution of nutrients in plant tissues examined using XFM. A, Tricolor image showing Fe, Mn, and Zn distribution in a longitudinal section (70 µm thick) of a barley grain. The original scan was 2,320 × 880 pixels (approximately 2 megapixels) with a pixel transit time of approximately 0.6 ms, resulting in a scan duration of approximately 20 min (Lombi et al., 2011c). B, Image from XFM analyses of a scan of a living seedling of Noccaea caerulescens (a hyperaccumulator) showing Zn (green), K (red), and Ca (blue). Data are from A. van der Ent (unpublished). C and D, Images from XFM analyses of scans of a leaflet of P. vittata (an As hyperaccumulator) showing K (green), As (red), and Ca (blue). Data are from R.V. Tappero (unpublished). E, An initial attempt using XFM to rapidly scan whole and intact wheat seeds (cv Shield). The transit per 20-μm pixel was 15 mm s⁻¹ velocity, with the entire scan duration being approximately 8 min. Data are from E. Lombi (unpublished).

Figure 2.

Distribution of nutrients in leaf tissues of sunflower examined using XFM following foliar fertilization with Zn. A and B, Distribution of Zn (A) and Ca (B) in an intact, hydrated leaf of sunflower to which two 5-μL droplets of ZnSO₄ (1,000 mg L⁻¹ Zn) had been applied on the adaxial surface toward the tip. Note that the highest concentrations of foliar-absorbed Zn accumulated in the trichomes (A), as also can be seen from the high Ca concentrations in these regions (B). C, The concentrations shown for the transect correspond to the dotted white line in A. D, Cross section of a sunflower leaf to which a droplet of ZnSO₄ had been applied on the adaxial surface. The entire adaxial area shown was from beneath the droplet.

Figure 3.

Mn accumulating in an excised (hydrated) trifoliate leaf of cowpea exposed to 30 µm Mn in nutrient solution. A, Image of the leaf. B and C, Distribution of Mn. D, Tricolor image showing Mn, Ca, and Zn. The area scanned in C is indicated by the red rectangle in B, and the area scanned in D is indicated by the white rectangle in C. In D, note how Mn (red) initially accumulates in the cell wall (white arrow), with the green (Ca) circular structures corresponding to vacuoles. For more details and full experimental procedures, see Blamey et al. (2018a).

Figure 4.

XFM images showing the distribution of Mn in a portion of a cowpea leaf exposed to 30 μm Mn. The area shown here represents only approximately 1.5% of the 8.5-megapixel image obtained for each time interval, which included portions of leaves exposed to both 0.2 and 30 μm Mn. The total area analyzed (i.e. 8.5 megapixels) at each time interval was 120 mm², as indicated by the white rectangle in Supplemental Figure S2B. See Blamey et al. (2018b).