Visualisation of mycorrhizal fungal structures and quantification of theirsurface area and volume using laser scanning confocal microscopy

A method has been developed for the visualisation and three-dimensional (3D ) measurement of mycorrhizal fungal structures inside plant roots. Sections of Allium porrum L. roots colonised by Glomus sp. 'City Beach' (WUM 16) an d Lilium sp. roots colonised by Scutellospora calospora (Nicol. & Gerd.) Wa lker & Sanders (WUM 12(2)) were stained with acid fuchsin. This allowed flu orescence from the fungal structures to be observed under a laser scanning confocal microscope (LSCM) without interference from the plant cells. A ser ies of horizontal optical sections were collected from a Glomus sp. arbuscu le and from a hyphal coil of S. calospora. These data were used to produce extended focus images. Axial distortion in microscopic visualisation 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 mu m was used for the z-interval between the xy-slices. A series of bin ary xy-images from each structure was rendered into a 3D graphical model fo r viewing. The volume and surface area of the structures were estimated usi ng computerised 3D measurement and also by stereological integration of bin ary xy-images. With both structures, the surface area estimates varied grea tly between the two measuring systems, whereas differences in volume estima tes were small. Computerised 3D measurement was considered more accurate th an stereological integration of confocal binary images.