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

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), or by rotating the polarisation of the laser with the confocal optics and not moving the sample. Using cellulose from bacteria and plant cell walls in which the orientation of cellulose could be directly measured, we show that the variations in pontamine fluorescence could be fitted to a sine wave equation, and this equation would give a direct measurement of the cellulose angle. We then applied this method to cell walls where the cellulose was not directly visible. Our experiments 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.
Source: Journal of Microscopy - Category: Laboratory Medicine Authors: Tags: Original Article Source Type: research