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

Abstract

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.

Keywords: confocal microscopy; gene expression; plant development; three-dimensional imaging; vasculature; whole-mount.

Figure 1

CLSM images of mPS-PI stained samples. (A–D) Cellular layers of an Arabidopsis leaf: (A) Adaxial epidermis, (B) Palisade cells, (C) Vasculature, (D) Spongy mesophyll. (E) 3D reconstruction of an Arabidopsis cotyledon using the OsiriX software. A virtual slice through the cotyledon can be seen in the bottom section. (F) Sieve plate within a vertical section through an Arabidopsis stem. Scale bars: 10 µm.