Visualisation of the mycorrhizal fungal structures and quantification of their surface area and volume using laser scanning confocal microscopy

A method has been developed for the visualization and three-dimensional (3D) measurement of mycorrhizal fungal structures inside plant roots. Sections of Allium porrum L. roots colonized by Glomus sp. 'City Beach' (WUM 16) and Lilium sp. roots colonized by Scutellospora calospora (Nicol. and Gerd.) Walker and Sanders (WUM 12(2)) were stained with acid fuchsin. This allowed fluorescence from the fungal structures to be observed under a laser scanning confocal microscope (LSCM) without interference from the plant cells. A series of horizontal optical sections were collected from a Glomus sp. arbuscule and from a hyphal coil of S. calospora. These data were used to produce extended focus images. Axial distortion in microscopic visualization due to the refractive index mismatch between the immersion and mounting media was quantified using vertical scanning of the hyphae. A correction factor of 0.71 μm was used for the z-interval between the xy-slices. A series of binary xy-images from each structure was rendered into a 3D graphical model for viewing. The volume and surface area of the structures were estimated using computerized 3D measurement and also by stereological integration of binary xy-images. With both structures, the surface area estimates varied greatly between the two measuring systems, whereas differences in volume estimates were small. Computerized 3D measurement was considered more accurate than stereological integration of confocal binary images