Visualizing the internal structural heterogeneity of Paeonia lactiflora starch granules by synchrotron soft X-ray microscopy

The traditional assessment of harvest timing for Paeonia lactiflora roots relies on the empirical “sufficient powderiness” phenotype, which lacks objective biomarkers. This protocol describes a synchrotron-based soft X-ray microscopy method at the oxygen K-edge for label-free, high-resolution visualization of oxygen distribution patterns within starch granules. • Dual-energy imaging (530 eV and 545 eV) combined with resin embedding and 500 nm sectioning enables visualization of the characteristic oxygen-deficient core and oxygen-enriched periphery. • ImageJ-based gray-value analysis provides relative quantitative comparison of oxygen density between optimal and non-optimal harvest stages. • The method offers a reproducible, objective tool for harvest-time evaluation and starch ultrastructure studies in medicinal plants.