Analysis by confocal microscopy of the structure of cambium in the hardwood Kalopanax pictus

An understanding of the morphology and the developmental changes in the sha pes and dimensions of cambial cells requires three-dimensional (3-D) analys is of thick slices of tissue. We devised a simple protocol using confocal l aser scanning microscopy (CLSM), with safranin and acridine orange as fluor escent dyes and glycerol as the clearing and mounting medium, to examine th e 3-D structure of the dormant cambium in Kalopanax pictus, a ring-porous h ardwood. Optical sections and high contrast images provided clear informati on about the shapes and nuclear status of cambial cells. which have previou sly been difficult to determine using conventional microscopy. The axially- oriented cambial cells were found to vary in shape, in particular around th e rays, and were not always typically fusiform. We evaluated the reliabilit y of our method by comparing results with those of a parallel study of the same material by standard analysis of serial sections of epoxy-embedded spe cimens. The images of optical sections obtained by CLSM were of high qualit y and similar to images obtained by conventional light microscopy of semi-t hin mechanical sections. Use of the confocal microscope provided a quick an d easy method for visualization of the structure of the cambium in thick ha nd-cut sections and for studies of the developmental changes in cells from the cambium to the xylem. (C) 2000 Annals of Botany Company.