Cryopreparation and electron tomography of yeast cells

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.