Frozen in time: A new method using cryo-scanning electron microscopy to visualize root-fungal interactions: Methods

• A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. • Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80°C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. • Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. • This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.