Author(s): Czymmek K, Sawant A, Goodman K, Pennington J, Pedersen P, Hoon M, Otegui MS. Imaging plant cells by high-pressure freezing and serial block-face scanning electron microscopy. Methods Mol Biol. 2020;2177:69-81. doi: 10.1007/978-1-0716-0767-1_7. PMID 32632806
Journal: Methods In Molecular Biology (Clifton, N.J.), Volume 2177, 2020
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.