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Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy. 2020

Kirk Czymmek, and Abhilash Sawant, and Kaija Goodman, and Janice Pennington, and Pal Pedersen, and Mrinalini Hoon, and Marisa S Otegui
Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center, St. Louis, MO, USA.

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.

UI MeSH Term Description Entries
D008855 Microscopy, Electron, Scanning Microscopy in which the object is examined directly by an electron beam scanning the specimen point-by-point. The image is constructed by detecting the products of specimen interactions that are projected above the plane of the sample, such as backscattered electrons. Although SCANNING TRANSMISSION ELECTRON MICROSCOPY also scans the specimen point by point with the electron beam, the image is constructed by detecting the electrons, or their interaction products that are transmitted through the sample plane, so that is a form of TRANSMISSION ELECTRON MICROSCOPY. Scanning Electron Microscopy,Electron Scanning Microscopy,Electron Microscopies, Scanning,Electron Microscopy, Scanning,Electron Scanning Microscopies,Microscopies, Electron Scanning,Microscopies, Scanning Electron,Microscopy, Electron Scanning,Microscopy, Scanning Electron,Scanning Electron Microscopies,Scanning Microscopies, Electron,Scanning Microscopy, Electron
D008867 Microtomy The technique of using a microtome to cut thin or ultrathin sections of tissues embedded in a supporting substance. The microtome is an instrument that hold a steel, glass or diamond knife in clamps at an angle to the blocks of prepared tissues, which it cuts in sections of equal thickness. Thin Sectioning,Ultramicrotomy,Sectioning, Thin,Sectionings, Thin,Thin Sectionings
D017110 Freeze Substitution A modification of the freeze-drying method in which the ice within the frozen tissue is replaced by alcohol or other solvent at a very low temperature. Substitution, Freeze
D017360 Arabidopsis A plant genus of the family BRASSICACEAE that contains ARABIDOPSIS PROTEINS and MADS DOMAIN PROTEINS. The species A. thaliana is used for experiments in classical plant genetics as well as molecular genetic studies in plant physiology, biochemistry, and development. Arabidopsis thaliana,Cress, Mouse-ear,A. thaliana,A. thalianas,Arabidopses,Arabidopsis thalianas,Cress, Mouse ear,Cresses, Mouse-ear,Mouse-ear Cress,Mouse-ear Cresses,thaliana, A.,thaliana, Arabidopsis,thalianas, A.

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