Cryogenic 3D electron microscopy captures whole plant cell native ultrastructure

Abstract Volume electron microscopy (vEM) provides nanometer-scale, 3D imaging of cells, but applying it to plant cells has been constrained by rigid cell walls and the need for harsh chemical fixation and staining. Here we report a cryogenic vEM (cryo-vEM) workflow for plant protoplasts that achieves high-resolution volumetric imaging of whole cells at up to 4 nm in a fully hydrated, vitrified state without any chemical fixation or heavy-metal staining. The method integrates optimized protoplast isolation, plunge-freezing vitrification for native-state preservation, automated cryogenic focus ion beam scanning electron microscopy (cryo-FIB-SEM) slicing and imaging, and a computational pipeline for image contrast enhancement, alignment, and machine learning assisted 3D segmentation. Using sorghum stem protoplasts as a demonstration, the workflow reliably captured complete cellular ultrastructure, resolving major organelles of the nucleus, mitochondria, vacuoles, endoplasmic reticulum, lipid bodies, and subcellular features such as membrane contact sites in situ and their native states. We further quantified organelle volumes and surface areas from the segmented 3D data, highlighting the potential for quantitative cellular ultrastructure analysis. This versatile cryo-vEM platform opens the door for in situ structural studies of plant cells, enabling unprecedented insights into native-state organelle architecture and interactions in plant biology. Competing Interest Statement The authors have declared no competing interest.