Lignocellulosic biomass offers advantages over corn starch for the production of biofuels, from economic and environmental standpoints. However, due to the chemical resistance of lignin and complex structure of the plant cell wall, energy-intensive and polluting pretreatments of the biomass are necessary for its fractionation. Recently, ionic liquids have attracted considerable interest for their ability to dissolve biomass at high temperatures. In this study, the swelling of poplar wood cells during ionic liquid (1-ethyl-3-methylimidazolium acetate) pretreatment was monitored with time-lapse series of autofluorescence images. After a 3 h ionic liquid pretreatment at room temperature, the secondary cell walls were noticeably swollen, indicated by an increase of the cell wall areas ranging from 60% to 100%. After the ionic liquid pretreatment, a rinsing with de-ionized water resulted in an immediate reduction of the secondary cell wall swelling and an increase of the cell total area (cell wall and lumen). The swelling during ionic liquid pretreatment and contraction upon rinsing was used to incorporate gold particles into the wood cell wall. The particles remained in the cell wall even after water rinsing, as observed by surface-enhanced Raman spectroscopy. This particle incorporation process enables the development of new sensing, imaging capabilities and pretreatments.