Pontamine fast scarlet 4B bifluorescence and measurements of cellulose microfibril angles

Summary Pontamine fast scarlet 4B is a red paper and textiles dye that has recently been introduced as a fluorescent probe for plant cell walls. Pontamine exhibits bifluorescence, or fluorescence dependent on the polarization of the excitation light: Because cellulose is aligned within the cell wall, pontamine‐labelled cell walls exhibit variable fluorescence as the excitation polarization is modulated. Thus, bifluorescence measurements require polarized excitation that can be directly or indirectly modulated. In our confocal microscopy observations of various cellulose samples labelled with pontamine, we modulated excitation polarization either through sample rotation or by the confocal's scanfield rotation function. This variably rotated laser polarizations on Leica confocal microscopes, but not those from other makers. Beginning with samples with directly observable microfibril orientations, such as purified bacterial cellulose, the velamen of orchid roots and the inner S2 layer of radiata pine compression wood, we demonstrate that modelling the variations in pontamine fluorescence with a sine curve can be used to measure the known microfibril angles. We then measured average local microfibril angles in radiata pine samples, and showed similar microfibril angles in compression and normal (opposite) wood. Significantly, bifluorescence measurements might also be used to understand the degree of local cellulose alignment within the cell wall, as opposed to variations in the overall cellulose angle. Lay description The physical properties of materials derived from plant cell walls, including timber, textiles and paper, depend to a large degree on how the cellulose is organised and oriented in the plant cells that make up these materials. Measurements of cellulose orientations in plant cells are, however, difficult and time consuming to make. In this study, we have investigated the properties of a fluorescent dye, pontamine fast scarlet 4B, that binds specifically to cellulose. As this dye only will absorb light, and thus fluoresce, when it is oriented parallel to the polarisation of light, any variations that occur in fluorescence when the polarisation of the excitation light is changed will reveal structural information about the orientation of cellulose within the cell wall. We modulated the relative polarisation of the excitation light in two ways, either by physically rotating the sample through a plane polarised laser (part of a confocal microscope), o