Czymmek, K., A. Sawant, K. Goodman, J. Pennington, P. Pedersen, M. Hoon, and M. Otegui. Imaging Plant Cells by High-Pressure Freezing and Serial Block-Face Scanning Electron Microscopy. Methods Mol Biol, 2020, p. PubMed Text.
Abstract
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.
Keywords: High-pressure freezing; Serial block-face electron microscopy; Three-dimensional electron microscopy.