Thomas, JimmyIngerfeld, ManfredNair, HemaChauhan, Shakti SinghCollings, David A.2025-12-312025-12-310043-7719ORCID:/0000-0003-3863-5979/work/162946651https://hdl.handle.net/1885/733797813Using 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.From the School of Biological Sciences at the University of Canterbury, the authors thank Alan Woods for the design and preparation of the rotatable microscope stage and Neil Andrews for assistance with scanning electron microscopy. We thank John Walker (School of Forestry, University of Canterbury) for his continued encouragement for this research, and his comments. And we also thank Eric Chung (Leica Microsystems, Australia) for discussions on polarised light and confocal microscopy, and Jonathan Harrington (Scion Ltd, Rotorua, New Zealand) for discussions. JT gratefully acknowledges funding for his PhD scholarship generously provided by Scion, while HN thanks the Foundation for Research Science and Technology, New Zealand (Grant No. UOCX0801) for the financial support during this work.17en201310.1007/s00226-012-0483-x84872329844