Cryofixation of epithelial cells grown on sapphire coverslips by impact freezing

Abstract

Rapid cryofixation of cells cultured on coverslips without the use of chemical fixatives has proved advantageous for the immunolocalization of antigens by electron microscopy. Here, we demonstrate the application of sapphire-attached tissue culture cells (PtK2 epithelial cells and mouse myoblasts) to metal-mirror impact freezing. The potential of the Leica EM-CPC cryoworkstation for routine freezing and for safe transfer of the cryofrozen samples into a sapphire disc magazine for freeze-substitution (SD-FS unit) has been exploited. Subsequently, the SD-FS unit has been tested for its use in methanol freeze-substitution and low temperature embedding for immunoelectron microscopy. The structural preservation of Lowicryl HM20-embedded cells has been assessed as being free of damage by large ice crystals.