Lab-based X-ray Microscopy for in situ 3D Visualization of Mycorrhizal Fungal Structures Associated with Roots

Arbuscular mycorrhizal fungi (AMF) have inhabited soil for millions of years and have formed beneficial relationships in the roots of many plant species. In general terms the fungus extends the reach of host roots and provides water and nutrients to the plant in exchange for fatty acids and photosynthetic sugars. Specific in vitro research into the complex interaction between roots and fungal structures is extensive, detailing the exchange of water [1], phosphate [2], and fatty acids [3]. Microscopic observation of this nutrient exchange using advanced imaging systems has been done in vitro using fluorescent protein reporters [4] and quantum dots [5]. Unfortunately direct imaging of this relationship in situ is lacking as it occurs underground, or in test systems that are too large or opaque for conventional light, laser, and electron microscopy instruments. We are developing a system for growing plants in cylinders with growth media that are supportive of the root-AMF interaction and amenable to in situ 3D volume imaging using a lab-based X-ray microscope (XRM). Various chemical contrast agents have been used successfully for XRM imaging in plant biology by imparting contrast to relevant structures to increase X-ray attenuation [6,7]. For this project we tested phosphotungstic acid, Lugol’s iodine, osmium tetroxide (OsO4), and gold nanoparticles conjugated to various lectins as potential contrast agents for in situ visualization of AMF structures. Results illustrating our progress in this research will be presented [8]. 3D volume renderings from XRM scans of carrot root colonized in vitro by Rhizophagus irregularis. Spores (s) and hyphae (arrows) were contrasted by incubation with (A) a lectin conjugated to 40nm gold particles, or (B) by fixation with OsO4. Scale bars: A = 50µm, B = 80µm. 3D volume rendering from XRM scan of Brachypodium distachyon root growing in sand-clay medium and colonized by Rhizophagus irregularis, contrasted with OsO4. An ORS Dragonfly Deep Learning model was used to segment the root (A, cyan) and mycorrhizal structures (A,B yellow), making it possible to quantitatively measure fungal biomass in situ. Scale bar = 700µm.