Lignin is one of the most prevalent biopolymers on the planet and a major 
component of lignocellulosic biomass. This phenolic polymer plays a vital 
structural and protective role in the development and life of higher plants. 
Although the intricate mechanisms regulating lignification processes in vivo 
strongly impact the industrial valorization of many plant-derived products, the 
scientific community still has a long way to go to decipher them. In a simple 
three-step workflow, the dual labeling protocol presented herein enables 
bioimaging studies of actively lignifying zones of plant tissues. The first step 
consists in the metabolic incorporation of two independent chemical reporters, 
surrogates of the two native monolignols that give rise to lignin H- and 
G-units. After incorporation into growing lignin polymers, each reporter is then 
specifically labeled with its own fluorescent probe via a sequential combination 
of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin 
autofluorescence, this approach leads to the generation of three-color 
localization maps of lignin within plant cell walls by confocal fluorescence 
microscopy and provides precise spatial information on the presence or absence 
of active lignification machinery at the scale of plant tissues, cells and 
different cell wall layers.