High-pressure freezing of epithelial cells on sapphire coverslips.

Rapid freezing of cell monolayers at ambient pressure is limited regarding the thickness of ice crystal damage-free freezing. The specific freezing conditions of the cells under investigation are decisive for the success of such methods. Improved reproducibility of results could be expected by cryoimmobilization at high pressure because this achieves a greater thickness of adequate freezing. In a novel approach, we tested the suitability of sapphire discs as cell substrata for high-pressure freezing. Frozen samples on sapphire were subjected to freeze-substitution while in the same flat sample holders as used for high-pressure freezing. We obtained cells that displayed an excellent preservation of fine structure. Because sapphire is a tissue culture substratum suitable for light microscopy, its use in combination with high-pressure freezing could become a powerful tool in correlative studies of cell dynamics at light and electron microscopic levels.