3D MicroCT Imaging of Medicago sativa Root Nodules

The symbiotic relationship between the legume Medicago sativa and the soil bacteria Sinorhizobium meliloti results in the formation of nitrogen-fixing root nodules. Traditional destructive methods, including paraffin sectioning, vibratome sectioning, and cryosectioning, have been applied to visualize how bacteria occupy the nodule, making it extremely difficult to obtain reliable three-dimensional information. These approaches are often combined with fluorescent labeling or staining, which can introduce additional stress affecting plant growth and nodule formation. MicroCT has emerged as a relatively quick, easy, and robust tool for plant biology that can non-destructively visualize plant histological features in three dimensions (3D), thereby avoiding destructive artifacts during sample preparation and ensuring high-fidelity 3D reconstruction. While microCT has been applied to legume root nodules, a detailed established protocol that documents the process from plant harvest and sample preparation to scanning and software visualization is lacking. In this study, we show a step-by-step microCT workflow using Medicago sativa as a model. The protocol includes nodule excision from roots, fixation, contrast enhancement, mounting, scanning, and three-dimensional reconstruction. Critical parameters affecting elements such as image quality, tissue preservation, and contrast are highlighted. Using this approach, it is possible to visualize the overall tissue organization, bacteroid-infected cells, and vascular bundles in three dimensions without physically sectioning the nodules. The pipeline described here provides a reproducible method for non-destructive, high-resolution imaging of native root nodules and is likely adaptable to other legume species, offering researchers a practical tool for studying nodule structure and bacterial organization within nodules in 3D.