Lignocellulose biomass can be transformed into sustainable chemicals, materials and energy but its natural recalcitrance requires the use of pretreatment to enhance subsequent catalytic steps. Dilute acid pretreatment is one of the most common and efficient ones, however its impact has not yet been investigated simultaneously at nano- and cellular-scales. Poplar samples have been pretreated by dilute acid at different controlled severities, then characterized by combined structural and spectral techniques (scanning electron microscopy, confocal microscopy, autofluorescence, fluorescence lifetime, Raman). Results show that pretreatment favours lignin depolymerization until severity of 2.4-2.5 while at severity of 2.7 lignin seems to repolymerize as revealed by broadening of autofluorescence spectrum and strong decrease in fluorescence lifetime. Importantly, both nanoscale and cellular-scale markers can predict hydrolysis yield of pretreated samples, highlighting some connections in the multiscale recalcitrance of lignocellulose.