High-Resolution Imaging of the Rhizosphere Using Resin-Embedded Cross-Sectional Polishing and Scanning Electron Microscopy for Biological Samples (bioCP-SEM)

Abstract

Understanding microbial colonization in plant-associated environments requires high-resolution imaging of intact plant-microbe interfaces under natural soil conditions. However, conventional electron microscopy is limited by sample distortion and poor compatibility with heterogeneous substrates. Here, we present bioCP-SEM, a cross-sectional polishing scanning electron microscopy technique optimized for biological specimens inhabiting unsterilized soil. This method integrates resin embedding, diamond band saw sectioning, and mechanical polishing to enable high-quality imaging across large, complex samples. Using bioCP-SEM, we visualized bacterial clusters in a model plant root, and multilayered biofilm-like structures and fungal structures, including arbuscule-like forms, in a wild weed species. We further applied this method to intact moss collected from an urban environment, capturing spatially resolved interactions among biofilms, spores, protozoa, and annelids. Notably, microbial cells and subcellular structures, such as cilia, were preserved in situ. These results demonstrate the versatility of bioCP-SEM for mapping microbial architecture across diverse biological and environmental contexts.