Abstract: Understanding cellular events in three dimensions (3D) is of great importance for the annotation and illustration of biological processes in a contextual way. Imaging techniques based on electron microscopy (EM), such as those derived from scanning electron microscopy (SEM) and transmission electron microscopy (TEM), provide various options to visualize biological samples at scales ranging from cells to macromolecules in situ. Recently, a series of cryogenic techniques has brought EM-based imaging to a new level, enabling specimens to retain their hydrated state throughout the sample preparation and imaging steps, thereby offering a near-native visualization of cellular events. The application of dual-beam focused ion beam (FIB)-SEM to biological samples has enabled high-resolution reconstructions in 3D and streamlined sample preparation workflows for downstream cryo-electron tomography (cryo-ET) imaging. However, applications of these technologies to plant materials are limited due to intrinsic characteristics of plant cells (e.g., non-adhesive growth, large size with a central vacuole, and the presence of cell walls). For the timely application of dual-beam FIB-SEM in three-dimensional subcellular imaging of plant materials, we have recently tested and developed three major workflows with proof-of-concept evidence using developing anthers and in vitro-cultured pollen tubes based on Aquilos 2 Cryo-FIB, including (1) room-temperature FIB-SEM volume imaging, (2) cryo-lamellae preparation from cell suspension culture or high-pressure-frozen organs for cryo-ET imaging, and (3) cryo-FIB-SEM volume imaging, which will facilitate structural studies of plant materials and provide technical guidance for the broader plant cell biology research community.

Key words: 3D imaging, cryo‐ET, cryo‐lift‐out, dual‐beam FIB‐SEM, volume electron microscopy