Imaging nutrient distributions in plant tissue using time-of-flight secondary ion mass spectrometry and scanning electron microscopy.

Abstract

A new approach to trace the transport routes of macronutrients in plants at the level of cells and tissues and to measure their elemental distributions was developed for investigating the dynamics and structure-function relationships of transport processes. Stem samples from Phaseolus vulgaris were used as a test system. Shock freezing and cryo-preparation were combined in a cryogenic chain with cryo-time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS) for element and isotope-specific imaging. Cryo-scanning electron microscopy (cryo-SEM) was integrated into the cryogenic workflow to assess the quality of structural preservation. We evaluated the capability of these techniques to monitor transport pathways and processes in xylem and associated tissues using supplementary sodium (Na) and tracers for potassium (K), rubidium (Rb), and (41)K added to the transpiration stream. Cryo-ToF-SIMS imaging produced detailed mappings of water, K, calcium, magnesium, the K tracers, and Na without quantification. Lateral resolutions ranged from 10 microm in survey mappings and at high mass resolution to approximately 1 microm in high lateral resolution imaging in reduced areas and at lower mass resolution. The tracers Rb and (41)K, as well as Na, were imaged with high sensitivity in xylem vessels and surrounding tissues. The isotope signature of the stable isotope tracer was utilized for relative quantification of the (41)K tracer as a fraction of total K at the single pixel level. Cryo-SEM confirmed that tissue structures had been preserved with subcellular detail throughout all procedures. Overlays of cryo-ToF-SIMS images onto the corresponding SEM images allowed detailed correlation of nutrient images with subcellular structures.