Freeze-fracturing of monolayers (capillary layers) of cell, membranes and viruses: some technical considerations

A novel hinged device for freeze-fracturing of cell monolayer in the Balzers freeze-etch unit is described. It is economical on biological material and enables oriented adsorption of sheet-like membrane fragments. For freeze-fracturing 'by hand' a monolayer is formed on a positively charged piecie of mica (with polylysine) and this is covered with another piece of mica, thin brass plate of filter paper. Such a sandwich is frozen in liquid nitrogen and fractured by means of forceps. Several modifications of this technique as well as practical examples are described. Among possible application are: negative staining of intramembranous protein particles; chemical or physical analyses of single membrane leaflets; identification of protein complexes by immunoelectron microscopy, etc.