In this paper, we present the use of multiplex click/bioorthogonal
chemistry combined with super-resolution Airyscan microscopy to track
biomolecules in living systems with a focus on studying lignin formation
in plant cell walls. While laser scanning confocal microscopy (LSCM)
provided insights into the tissue-scale dynamics of lignin formation
and distribution in our previous reports, its limited resolution precluded
an in-depth analysis of lignin composition at the unique cell wall
or substructure level. To overcome this limitation, we explored the
use of Airyscan microscopy, which, among the super-resolution techniques
available, offers an optimal balance between performance, cost, accessibility,
and ease of implementation. Our study demonstrates that a triple labeling
strategy using copper-catalyzed azide–alkyne cycloaddition
(CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC),
and inverse electronic-demand Diels–Alder cycloaddition (IEDDA)
to label modified lignin metabolic precursors can be combined with
Airyscan microscopy to reveal the zones of active lignification at
the single cell level with improved sensitivity and resolution. This
approach enables insights into the lignin composition in wall substructures,
such as pits or in wall layers that are otherwise not distinguishable
by classical LSCM. Our work emphasizes the importance of studying
lignin formation in plant cell walls and demonstrates the potential
of combining bioorthogonal chemistry and super-resolution microscopy
techniques for studying biomolecules in living systems.