Cryopreparation and Electron Tomography of Yeast Cells
- Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347
- ↵1Correspondence: eileen.otoole{at}colorado.edu; mark.winey{at}colorado.edu
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.
