AbstractThe topochemical distribution of lignin and cellulose in individual cell wall layers of Populus nigra stem was determined by transmission electron microscopy (TEM) and confocal Raman microscopy. TEM images exhibited the fiber wall as being typically differentiated into three layers: middle lamella (ML), primary wall (P), and secondary wall (S1, S2, and S3). Higher magnification views showed the S2 layer to be differentiated into electron lucent and dense areas in the radial direction. In situ Raman images calculated by integrating over the intensity of characteristic spectral bands enabled visualization of the spatial variation in lignin and cellulose. Raman images acquired by integrating over the spectral band at 1605 cm-1 suggested that higher lignin content was visualized in the cell corner (CC), the compound middle lamella (CML), and the secondary wall of ray parenchyma. Cellulose distribution followed by taking the band regions around 2897 cm-1 into account showed the opposite pattern, with the highest content in fiber secondary wall. The SEM-EDXA provided semi-quantitative results, showing that the lignin content ratio in various cell wall layers was 1.4 (CC):1.1(CML):1(S2).