Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. The conversion of cell wall biomass to sugar for fermentation to ethanol thus requires chemical or physical pretreatments to disrupt the recalcitrant plant cell walls and to make the cellulose accessible to cellulolytic enzymes. Within the frame of a French national project on the production of 2G ethanol, we have developed a multimodal approach to investigate lignocellulose (LC) traits related to enzymatic saccharification. Besides classical wet chemistry characterization, multiscale analysis of LC was implemented using 2D NMR and non-invasive methods, such as fluorescence and infrared spectroscopy. Using representative biomass feedstocks (poplar, miscanthus and wheat straw) which were pretreated at different severity levels during a steam explosion step, we have shown that easily-measured spectral features showed strong correlation with enzymatic saccharification (1-2). Complementary, evaluation of plant cell wall accessibility through a combination of innovating fluorescence microscopy techniques using small probes (FRAP, FRET) brought further insights into the impact of physicochemical pretreatments on LC samples (3-4). 1- Auxenfans et al. Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment. Biotechnology for Biofuels 2017, 10, 36. 2- Auxenfans et al. Seeing biomass recalcitrance through fluorescence. Scientific Reports 2017, 7, 8838. 3- Paës et al. Exploring accessibility of pretreated poplar cell walls by measuring dynamics of fluorescent probes. Biotechnology for Biofuels 2017, 10, 15. 4- Chabbert B et al. Fluorescence techniques can reveal cell wall organization and predict saccharification in pretreated wood biomass. Industrial Crops and Products 2018, 123, 84-92.