The plant cell wall is a complex composite material made of polysaccharides, polyphenols, proteins, and minerals. In this work, a multimodal imaging approach was taken, using Raman and Fourier‐transform infrared (FTIR) microspectroscopy along with fluorescence imaging, scanning electron microscopy (SEM), and elemental mapping by energy dispersive X‐ray spectroscopy (EDX). We characterized the chemical composition of sorghum leaf cross‐sections extracted from fresh tissue as well as after paraffin embedding. The complementary vibrational information of Raman and FTIR spectra related a silica deposition to a specific organic composition in the epidermis, specifically with respect to lignin. Moreover, the data enable in situ correlation of autofluorescence with a specific lignin structure. Our results showed that lignin 5–5’ linkages that produce biphenyl structures are important determinants of the cell wall fluorescence properties. The reported multimodal approach will help to clarify the process of biosilica formation and related questions regarding cell wall biochemistry.