Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy

This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View^®) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.