Achieving micrometer-scale 4D X-ray tomography of living leaf tissue in the laboratory

A methodology for achieving micrometer-scale 4D X-ray lab microscopy of living leaf tissue was developed to overcome challenges associated with delicate tissues, radiation damage, and motion artifacts during in vivo imaging. The study focused on optimizing laboratory-based X-ray micro-computed tomography (microCT) parameters to balance high-resolution imaging with minimized physiological stress and radiation dose quantification. Assessing the dose-safe imaging window required comparing vertical and horizontal leaf mounting setups. Results demonstrated that the horizontal setup provided greater stability, preventing tissue degradation and maintaining sample viability during continuous acquisitions lasting up to 22 h (~ 15,600 Gy). MicroCT clearly resolved microstructures at the cellular level, achieving a spatial resolution down to around 2 voxels. Furthermore, this optimized methodology confirmed the ability to track the spatiotemporal dynamics of applied compounds such as iohexol and aggregated nanoparticles within the leaf tissue. This work establishes that accessible laboratory-based microCT enables in vivo 4D monitoring of anatomical and physiological changes in living plants.