Abstract
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 (R > 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.
Highlights
Energy-dispersive X-ray microanalysis (EDX) is a technique for analyzing elements at the microscopic level
In the scanning transmission electron microscope (STEM) image, a frame adjacent to the measured region was used for drift correction (Figure 1A,B); this is required, because micrographs at high magnification are distorted by motion of the sample during the scanning and image acquisition
X-rays were detected with the EDX detector at 80 kV with a low current
Summary
Energy-dispersive X-ray microanalysis (EDX) is a technique for analyzing elements at the microscopic level. Vacuolar compartmentalization of Na+ and Cl− could be demonstrated in root cortical cells [22], but the concentrations were apparently lower in vacuoles than in the cell walls [21,22] Those results were somewhat difficult to interpret, because the images compared were not acquired under the same measuring conditions. The use of cryo-EDX to investigate frozen hydrated samples directly with SEM avoids potential drying artifacts that may occur during freeze drying It precludes structure determinations in the elemental images, due to its relatively low resolution. High-spatial resolution of intracellular ion concentrations is a major advantage of TEM-EDX compared to the lower resolution elemental images obtained with cryo-analytical SEM. The proposed TEM-EDX protocol is a feasible method for estimating multiple elemental concentrations within cell compartments
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