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 shapes and dimensions of cambial cells requires three-dimensional (3-D) analysis of thick slices of tissue. We devised a simple protocol using confocal laser scanning microscopy (CLSM), with safranin and acridine orange as fluorescent dyes and glycerol as the clearing and mounting medium, to examine the 3-D structure of the dormant cambium in Kalopanax pictus, a ring-porous hardwood. Optical sections and high contrast images provided clear information about the shapes and nuclear status of cambial cells, which have previously been difficult to determine using conventional microscopy. The axially-oriented cambial cells were found to vary in shape, in particular around the rays, and were not always typically fusiform. We evaluated the reliability 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 specimens. The images of optical sections obtained by CLSM were of high quality and similar to images obtained by conventional light microscopy of semi-thin mechanical sections. Use of the confocal microscope provided a quick and easy method for visualization of the structure of the cambium in thick hand-cut sections and for studies of the developmental changes in cells from the cambium to the xylem.