3D EM analysis of high-pressure frozen/freeze-substituted A. thaliana anthers using SBF-SEM and ET. A–C, Three serial block face EM images depicting the anther cell wall layers, i.e. the epidermis (Ep), endothecium (En), and tapetum (T), and the developing microspores (M) in the anther locule processed as explained in Czymmek et al. (2020). D, Isosurface visualization of anther wall layers and microspores from 64 serial block face SEM images. E, Enlarged view of tapetal cell region highlighted by orange square in (A). Note that the resolution is too low to discern most structural features of cytoplasmic organelles such as the membranes of mitochondria (M), multivesicular endosomes (MVE), and plastids (P). This is due to a tradeoff between the size of the reconstructed area (i.e. low magnification to increase the field of view) and the resolution (i.e. lower resolution due to large pixel size). In this SBF-SEM image dataset, the magnification was chosen to capture multiple cell layers and microspores within the anther resulted in limited subcellular resolution. F, G, Tomographic slice (F) and tomographic model (G) of a portion of cytoplasm from a tapetal cells showing an MVE, a TGN, and a mitochondrion (M). Scale bar = 5 µm in A–D; 100 nm in E–G.