Good Ultrastructural Preservation of Human Tissues and Cultured Cells by Glutaraldehyde Fixation, Sandwich Freezing, and Freeze-Substitution

Sandwich freezing (freezing the specimen rapidly with liquid propane by placing it between two copper disks) and freeze-substitution of living yeast cells has been used for observing exquisite close-to-native ultrastructure of cells. Glutaraldehyde fixation, sandwich freezing, and freeze substitution of bacteria and other microorganisms also yield close-to-native ultrastructure. Here, we have used glutaraldehyde fixation, sandwich freezing, and freeze substitution to observe human cells and tissues. We obtained clear and natural cell images of tissues sliced to 0.2 mm thickness. This is a remarkable result because, in the past, tissues as thick as 0.2 mm could only be frozen by high-pressure freezing. The present study has shown that it is possible to observe clear and natural cell structures in animal and human tissues anytime because glutaraldehyde-fixed tissues can be stored at 4 degrees C for several months before freezing, and a sandwich-freezing device will soon become commercially available. Also, natural ultrastructure of cultured cells in suspension was found to be observed more clearly by glutaraldehyde-fixation, sandwich freezing, and freeze-substitution than sandwich freezing and freeze substitution of living cells. The present method should be used as a standard method to observe the close-to-native ultrastructure of animal and human tissues.