Cryogenic volume electron microscopy of whole plant protoplasts

Volume electron microscopy (vEM) provides nanometer-scale, three-dimensional imaging of cells, but applying it to plant systems remains challenging. Cell walls, large vacuoles, and tissue thickness complicate sample preparation and cryogenic imaging. Here we report a cryogenic vEM (cryo-vEM) workflow for unstained plant protoplasts that achieves volumetric imaging of whole vitrified sorghum stem protoplasts without chemical fixation, dehydration, resin embedding, 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) slice-and-view acquisition, contrast enhancement and stack alignment, and AI-assisted human-in-the-loop 3D segmentation. Using sorghum stem protoplasts as a demonstration, the workflow captured large-volume frozen-hydrated protoplast ultrastructure, allowing visualization of major organelles, including the nucleus, mitochondria, vacuoles, ER/Golgi-like membranes, lipid bodies, and subcellular features consistent with nuclear-envelope pores. We further quantified organelle volumes and surface areas from the segmented 3D data, highlighting the potential for quantitative cellular ultrastructure analysis. This cryo-vEM workflow provides a platform for near-native structural studies of isolated plant protoplasts.