Abstract text: Water-conducting xylem vessels display strikingly ordered secondary cell walls (SCWs). Protoxylem vessels (PXVs) typically form periodic banded or spiral thickenings, whereas metaxylem vessels (MXVs) develop continuously thickened SCWs interspersed with pits. Intermediate reticulate patterns also occur in older vessels or under abiotic stress. Despite recent progress, the mechanisms underlying the formation and periodic deposition of these SCW patterns remain incompletely understood.

Most studies of xylem SCW architecture rely on maximum-intensity projections or single optical sections of confocal z-stacks to visualize these patterns. While informative, such approaches capture only a limited view of the vessel wall and restrict quantitative analysis of the full three-dimensional structure.

To overcome these limitations, we developed an image-analysis pipeline for Arabidopsis thaliana that combines optimized ClearSee clearing, chemical labeling of cellulose and lignin, and confocal z-stack acquisition. Automated segmentation identifies vessel geometry in three-dimensional datasets, enabling the vessel wall to be computationally extracted and “unrolled” into two-dimensional radial projections that reveal the entire SCW surface.

This approach enables quantitative measurements of vessel diameter, SCW band and gap thickness, and spiral orientation, and detects characteristic SCW defects in xylem mutants. The workflow is provided as an open-source Fiji plugin supporting both single-sample and batch analyses.