Electron Microscopy Techniques for 3D Plant ER Imaging.

The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex, whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. Here we present two protocols for 3D EM imaging of the ER across a range of scales. For large-scale ER structure analysis, we describe selective ER staining with ZIO that allows for automated or semi-automated ER segmentation. For smaller regions of ER, we describe high-pressure freezing, which enables almost instantaneous fixation of plant tissues but without organelle specific staining. These fixation and staining techniques are suitable for a range of imaging modalities, including serial sections, array tomography, serial block face-scanning electron microscopy (SBF-SEM), or focused ion beam (FIB) SEM.