Pontamine fast scarlet 4B: A new fluorescent dye for visualising cell wall organisation in radiata pine tracheids

Using confocal microscopy, tracheid wall organisation was investigated with pontamine fast scarlet 4B (P4B), a cellulose-specific stain that fluoresced red following green excitation. P4B fluorescence was present throughout unlignified walls (cambium, ray cells, resin canals and parenchyma cells) and in two concentric bands around opposite and compression wood tracheids. Scanning electron micrographs demonstrated these bands as the S1 and S3 layers of normal wood, and the S1 and inner S2 layers of compression wood. Fluorescence was also strongly dependent on the polarisation of the excitation light, a phenomenon referred to as bifluorescence. Compared to two other cell wall stains, Congo red and calcofluor white, P4B showed a higher specificity for the S1 and S3 layers and increased bifluorescence. These results suggest that P4B is an ideal tool with which to investigate the orientation of cellulose microfibrils in the S1 and S3 layers of the tracheid wall.