Artefact-aware Brillouin microscopy framework for root mechanical phenotyping.

Plant roots continuously sense and respond to the mechanical signals of their growth environment: root cap function, mucilage secretion, and hormonal regulation jointly adapt root tip mechanics, shaping whether roots penetrate compacted layers or reorient around obstacles. Most current measurements of root mechanical properties require removal of the root from the surrounding medium, triggering mechano-adaptive responses and confounding follow-up observations. Here we present an artefact-aware Brillouin microscopy framework that combines RoPods, a 3D-printed growing and imaging chamber with colocalised Raman microscopy, to enable repeated in situ measurements of root mechanical properties, while retaining growth-induced stresses. As a proof-of-concept application, we show that roots adapt their mechanical properties to different Gelrite concentrations, and characterise methodological biases arising from chamber geometry and sample handling.