While X-ray computed tomography (CT) is commonly used to study static plant structures, micrometer-scale 3D studies of physiological processes in plants are scarce and sub-cellular plant physiology remains mainly a domain of 2D surface or indirect characterization methods. We have developed a methodology to visualize at deep sub-micrometer scale the 3D microstructure of living plant tissue with the aim to capture 3D nutrition uptake in plants by deploying nanoparticles as carriers. Nano-holo-tomography in projection geometry is a suitable method for in-vivo imaging in the sense that high X-ray energies (short wavelengths) are accessible (29 keV compared to a traditional sub 15 keV TXM setups) enabling the optimization of dose-to-contrast ratio. This aspect together with the option of continuous change in magnification were the essential prerequisites that allowed us this first demonstration of capturing the 3D microstructure of plants at the nanoscale in physiologically relevant scenarios. We imaged living barley plants with 50 nm pixel size using holo-tomography at ID16B beamline at the ESRF to visualize liquid distribution and thereby follow nanoparticle pathways in plant leaves. For this study leaves were cut into 5x10 mm segments directly from a live barley plant, which were then sealed in a pipette tip with added water to maintain humidity to optimize image quality. We observed nanoparticle clustering in the sub-stomatal cavity and reported nanoparticle-based fertilizers in the cell wall matrix. In addition to the tomography experiments, we have supplemented our findings with quantitative results obtained through SAXS, XRF, TXM, and XANES measurements. These analyses offer insights into the uptake, aggregation, dissolution, and bioavailability of the administered nano fertilizer material as it enters the plant. Conclusion In this study, we show a feasibility study of live plant tissue imaging down to 50 nm pixel size in 3D, thereby visualizing nanoparticle aggregation processes in the cell wall matrix. This gives perspectives in discovering the pathways of nanoparticles in living plants and thereby contributing to the challenge of sustainable agriculture by efficient nutrition delivery without degrading the soil.
Emil Visby Kristensen (Wed,) studied this question.