Quantitative X-ray Elemental Imaging in Plant Materials at the Subcellular Level with a Transmission Electron Microscope: Applications and Limitations.

Energy-dispersive X-ray microanalysis (EDX) is a technique for determining the distribution of elements in various materials. Here, we report a protocol for high-spatial-resolution X-ray elemental imaging and quantification in plant tissues at subcellular levels with a scanning transmission electron microscope (STEM). Calibration standards were established by producing agar blocks loaded with increasing KCl or NaCl concentrations. TEM-EDX images showed that the salts were evenly distributed in the agar matrix, but tended to aggregate at high concentrations. The mean intensities of K⁺, Cl, and Na⁺ derived from elemental images were linearly correlated to the concentrations of these elements in the agar, over the entire concentration range tested ( > 0.916). We applied this method to plant root tissues. X-ray images were acquired at an actual resolution of 50 nm ´ 50 nm to 100 nm ´ 100 nm. We found that cell walls exhibited higher elemental concentrations than vacuoles. Plants exposed to salt stress showed dramatic accumulation of Na⁺ and Cl in the transport tissues, and reached levels similar to those applied in the external solution (300 mM). The advantage of TEM-EDX mapping was the high-spatial-resolution achieved for imaging elemental distributions in a particular area with simultaneous quantitative analyses of multiple target elements.