Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water

Biological membranes are often poorly visible with the electron microscope after high-pressure freezing and freeze-substitution. The water content of the sample and of the substitution medium is one factor among others that strongly influences membrane visibility. In order to investigate this effect, high-pressure frozen yeast cells, rat-pancreas tissue and arthropod tissue were freeze-substituted with and without adding water to the substitution medium. The visibility of the biological membranes was generally improved if the substitution medium contained 1-5% water. The effect was especially pronounced in yeast cells, where membrane visibility was poor after freeze-substitution with water-free medium but good after addition of 5% water to the substitution medium.