Wood cell walls are naturally fluorescent due to the presence of lignin. Autofluorescence offers a more specific method for localising lignin than staining and can potentially be used to assess cell wall modification resulting from a range of biological, chemical and physical treatments. In order to optimise conditions for imaging lignin by autofluorescence and to evaluate possible differences in fluorescence between softwood and hardwood lignin, wood sections of radiata pine and poplar were examined by confocal laser scanning microscopy to measure fluorescence spectra in a range of mounting media. Glycerol/buffer mixtures at three different pH values were compared with immersion oil and thiodiethanol using both UV and visible excitation. Glycerol/buffer at pH9 produced the strongest lignin fluorescence at visible excitation indicating that this is the optimal mounting medium for imaging and spectroscopy. For UV excitation, thiodiethanol gave increased brightness relative to glycerol. Poplar lignin was four times brighter than pine lignin with excitation at 488 nm at pH9, and showed characteristic differences in spectral emission under these conditions. This characteristic fluorescence was localised to the inner secondary wall of fibres, expressed as a gradient from the outer S2 region increasing towards the lumen, as visualised by spectral unmixing. Comparison with sapwood from other hardwood species suggests that this fluorescence emission is characteristic of poplar and willow (Salicaceae). Members of the Salicaceae family are known to contain characteristic syringyl p-hydroxybenzoate lignin and it is likely that this special lignin type is responsible for the characteristic pH dependant fluorescence of poplar fibre walls.
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