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The protocol includes sample preparation, the use of a custom-designed incubation chamber to keep the sample in optimal conditions and instructions for image acquisition. Using the natural autofluorescence of the lignin contained in the cell walls, confocal image stacks of samples of raw and hot water pre-treated poplar sections can be acquired at regular intervals during 24 hours of enzymatic hydrolysis. The acquisition parameters have been optimised to determine a compromise between image resolution and reduction of sample photobleaching. The acquired 3D images can then be processed to extract precise quantitative information on the deconstruction of the cell wall.
This protocol is an important first step towards elucidating the structural parameters underlying the recalcitrance of plant walls by allowing the acquisition of high-resolution images in time and space. This work was carried out as part of Aya Zoghlami's PhD thesis in collaboration with the PICT platform of the URCA.

Read: Zoghlami A, Refahi Y, Terryn C, Paës G (2020) Three-Dimensional Imaging of Plant Cell Wall Deconstruction Using Fluorescence Confocal Microscopy. Sustainable Chemistry 1, 45-85. https://doi.org/10.3390/suschem1020007

Contact: Dr Gabriel Paës, gabriel.paes@inrae.fr