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

Abstract

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.

Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism

“…To examine whether lobe initiation and subsequent expansion in the wild-type and any1 pavement cells is associated with the reported alignment of the cellulose microfibrils, we investigated the degree of the alignment (the anisotropy degree, hereafter) of cellulose at both neck and lobe regions with high spatial resolution in living cells. We stained cellulose microfibrils with the fluorescent dye Pontamine Fast Scarlet 4B (S4B), which specifically binds to cellulose (Anderson et al, 2010), and used polarized fluorescence microscopy (Mehta et al, 2016;Swaminathan et al, 2017) to examine the anisotropy degree of cellulose microfibrils (Thomas et al, 2017). The anisotropy of S4B's fluorescence polarization reflects the anisotropy degree of cellulose in the periclinal wall of the pavement cells at pixel-by-pixel resolution.…”

Developmental Biology and Induction of Phi Thickenings by Abiotic Stress in Roots of the Brassicaceae

“…Lignification of the phi thickenings, Casparian strip, and xylem could be viewed in whole, cleared roots with conventional lignin stains viewed under bright field, or by imaging blue, lignin autofluorescence from unstained samples resulting from 405 nm (violet) excitation by confocal microscopy (data not shown). However, for high-resolution confocal imaging, cleared roots were double-stained with berberine hemisulfate for lignin (and suberin) [ 3 , 4 , 20 , 21 , 22 ] and for cellulose with pontamine fast scarlet 4B [ 23 , 24 ]. This combination of stains allowed comparison of lignified structures with the location of non-lignified cell walls.…”

Contactless Resonant Cavity Dielectric Spectroscopic Studies of Cellulosic Paper Aging

“…30° to 45° from the normal within the cellulose microfibril structure. This disorientation of cellulose fibers in live plants translates to about the 60° orientation in sheeted paper as we have designated here (Anderson et al 2010;Thomas et al 2017).…”