Integrating scanning X-ray scattering and fluorescence for multi-scale analysis of seed structure supported by machine learning tools

Abstract Background Understanding the structure of plant seeds cultivated for human consumption and food manufacturing is vital to provide sustainable products as well as to investigate early growth stages. This includes structural variation between different plant species, varieties and cultivars depending on genetic setup, as well as structural modifications upon germination, aging and storing or seed treatment during processing. For plant seeds as multi-component biological materials, structural characterization must extend across multiple length scales, from molecular organization to cellular architecture. Results We apply scanning Small- and Wide-Angle X-ray Scattering (SWAXS) and X-ray Fluorescence (XRF) on yellow pea seeds to combine local structural information on the molecular scale with imaging of cellular structures on the micrometer scale, enabling a comprehensive analysis of hierarchical organization. To identify and characterize heterogeneous regions within the pea seeds, we implement a fitting-free, data-driven segmentation and analysis workflow based on machine learning tools. This approach allows for classification of structurally distinct domains and enables quantitative comparison across samples without relying on predefined models. Furthermore, we incorporate multi-modal analysis by combining structural imaging with complementary elemental information obtained from XRF. The integration of compositional and structural data provides deeper insight into structure-composition relationships. Conclusions This multi-scale, multi-modal approach opens new possibilities for investigating hierarchical structures and their development under diverse conditions and enables systematic comparison between different species or seeds at different developmental stages or exposed to different processing steps. The approach is broadly applicable to various kinds of samples and other hierarchically organized biological materials, which makes it a valuable technique for plant science as well as plant-based food science.