Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery. Plant cell walls constitute the largest renewable source of biomass on Earth that can supply environmental benefits for the production of fuel, chemicals and materials. They are composed by lignocellulose made from three main polymers (cellulose, hemicelluloses, lignin) assembling as a network whose structural and chemical complexity hampers hydrolysis of cellulose by enzymes and microorganisms 1. In addition, cell walls display high variability depending on genetic and environmental factors, as well as plant tissue and cell types 2–4. Understanding plant cell wall complexity during lignocellulosic bioconversion is therefore important to identify critical features impacting hydrolysis, for optimising pretreatments of biomass 5 and enzyme cocktails 6. Investigation of the dynamics of lignocellulose hydrolysis requires physicochemical characterization and mul-tiscale visualization 7,8. Combined approach using multiple microscopic techniques including scanning electron microscopy, atomic force microscopy, light microscopy, immunocytochemistry and microspectrometry can be used to monitor microstructural and topochemical heterogeneity of plant cell walls and their recalcitrance at tissue, cell and subcellular levels 9–15. In addition, spatial and temporal imaging of enzymes distribution within lignocellulose substrates can be carried out by means of immunoprobe or fluorescent labelled protein 16–18 to study accessibility at the molecular scale 19,20. However real time imaging of cell wall microstructure and enzyme distribution during bioconversion still remains challenging. Enzymatic degradation of cellulose and hemicelluloses involves several types of enzymes, namely glycoside hydrolases that work synergistically 21,22. To overcome the recalcitrance of plant polysaccharides, filamentous fungi and bacteria secrete lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of gly-coside bonds 23–25. In industrial processes, addition of LPMOs to cellulolytic cocktails leads to the reduction of the enzyme loading required for efficient saccharification of lignocellulosic biomass 26. These powerful enzymes are copper-containing enzymes classified within the auxiliary activity (AA) class of the CAZy database (www.cazy. org 27) in AA9-AA11 and AA13 families. AA9 LPMOs are mostly active on cellulose but some have been shown to act on xyloglucan and glucomannan 25,28. Despite their recognized boosting effect on biomass hydrolysis, AA9 LPMOs activity has been essentially investigated on model substrates with only sparse studies focusing on the insoluble fraction of the substrate that show their disruptive action at the surface of cellulosic fibers 29–31. To our