Looking deeper: whole-mount confocal imaging of plant tissue for the accurate study of inner tissue layers.

Building on previous work, we further developed a staining procedure that fluorescently labels plant cell walls and, when combined with confocal microscopy, allows visualization of plant cellular organisation in whole mounts to depths exceeding 200 microm. This technique can be combined with beta-glucuronidase histochemical activity assays, allowing the simultaneous study of gene expression. Images taken from stained samples can be used for three-dimensional tissue reconstruction. We used the technique to study Arabidopsis protophloem development. The phloem is one of the innermost tissues of a plant and therefore especially difficult to visualise. Furthermore, in general the technique will improve significantly the speed and accuracy with which any kind of plant tissue organisation can be studied, and it is not limited to the study of Arabidopsis tissues.