This chapter describes the techniques used in rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis. Rapid freezing and freeze-substitution of yeast cells for electron microscopy have been found to result in morphological preservation that is generally superior to that seen after chemical fixation. The accurate preservation of cellular structures depends in part on the speed with which cellular processes are stopped. Rapid freezing results in the almost instantaneous fixation of the cell, which in order of magnitude is faster than the time needed for chemical fixatives to diffuse into a cell and cross-link its components. A potential problem with cryofixation is the formation of ice crystals that distort the structure of the sample. This has been solved largely by employing rapid rates of cooling such that crystalline ice is not formed but rather intracellular water is vitrified. This has been achieved by plunging thin layers of cells into liquid ethane or propane, or by propane jet freezing. After freezing, the cells must be further fixed by freeze-substitution and then embedded in resin in preparation for viewing in the electron microscope. Freeze-substitution involves replacing the frozen water of the cell with an organic solvent at low temperature, thus avoiding the damaging effects of dehydration that occur at ambient temperature.
ASJC Scopus subject areas
- Cell Biology