Lignins display spatial and compositional changes at the nm-level between cell wall layers and cell types. These spatial modifications of lignins are essential to support the different functions of lignified tissues such as the reversible deformation of cells, their impermeable intercellular cohesion or their structural mechanical reinforcement (Pesquet et al., 2025). To date, lignins are mainly analyzed using wet chemical methods that consider plants as uniform bulk, thus averaging out any lignin spatial specificity. The main chemical differences of lignin units include their aromatic substitution and aliphatic functions, which are generally not simultaneously quantified. Recent breakthroughs in spatial chemical imaging of lignins, using Raman or absorbance microspectroscopy, have circumvented previous averaging due to bulk analyses but can only resolve part of the lignin chemistry (Blaschek et al., 2024). To extend our repertoire of spatial chemical imaging methods for lignins, we herein established the use of lignin autofluorescence microspectroscopy for quantitative and qualitative measurements. We validated our excitation/emission spectral scanning on model synthetic lignins and plant cross-sections genetically engineered both to affect lignin aromatic and aliphatic chemistries. Our novel “beyond green chemistry” method, only using ethanol and water as solvents for imaging, presented nm resolution levels for all previously reported aromatic and aliphatic chemistries of lignin units. Usable on wet samples of both plant biopsies and plant-derived products, our novel spatial imaging method can be used to determine lignin concentration, unit composition and the homogeneity of its spatial distribution. We further emphasize the advantage of tunable lasers in characterizing lignin autofluorescence chemotypes by use of excitation spectroscopy rather than emission-based spectra. This investigation identifies key excitation/emission regions in single and multiphoton imaging to differentiate spatially lignin unit chemistry and determine their spatial density.