title: Cryopreparation and Electron Tomography of Yeast Cells.
abstract: Three-dimensional imaging of cells using electron tomography enables analysis of cell structure at unprecedented resolution. The preparation of cells for tomography using rapid freezing followed by freeze-substitution is an essential first step to ensure the optimal preservation of the cell structure for 3D studies. This protocol outlines a method for obtaining well-preserved cells using high-pressure freezing followed by freeze-substitution. We have found that this method is particularly well suited for electron tomography studies and has the added bonus of preserving antigenicity for immuno-electron microscopy. The steps involved in imaging cells and performing tomographic analysis of cellular structures are also outlined.
authors.affiliation: Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 eileen.otoole@colorado.edu mark.winey@colorado.edu.
authors.forename: Eileen T
authors.initials: ET
authors.lastname: O'Toole
authors.affiliation: Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Giddings
authors.affiliation: Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 eileen.otoole@colorado.edu mark.winey@colorado.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Winey
mesh_terms: D055032:Electron Microscope Tomography / Q000379:methods*; D017110:Freeze Substitution; D005615:Freezing; D056229:Fungal Structures / Q000648:ultrastructure; D006874:Hydrostatic Pressure; D021621:Imaging, Three-Dimensional; D011309:Preservation, Biological; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*