Closer to the native state: Sandwich freezing and freeze‐substitution of microorganisms, cultured cells, and animal and plant tissues for electron microscopy

Since the invention of the electron microscope, morphologists have developed numerous techniques to visualise the native ultrastructure of biological cells and tissues. Although conventional chemical fixation followed by room temperature dehydration remains widely used, it often fails to preserve native structures, leading to distortion and leakage of cellular components. In contrast, cryofixation combined with freeze substitution offers a superior alternative, producing micrographs that more accurately reflect native ultrastructure. We have developed a 'sandwich freezing method' for rapid cryofixation, in which small biological specimens are placed between two copper disks, rapidly frozen by plunging into melting propane cooled with liquid nitrogen, then freeze-substituted in acetone containing osmium tetroxide at low temperature, and finally embedded in epoxy resin. This technique yields exquisitely preserved, near-native ultrastructure in living microorganisms and plant tissues. For cultured cells and animal tissues, prefixation with glutaraldehyde prior to sandwich freezing results in even more native-like preservation compared to specimens frozen without fixation. We propose that glutaraldehyde prefixation prevents the immediate distortion and ultrastructural changes that occur upon removal of cells and tissues from their culture medium or host organism. Importantly, glutaraldehyde rapidly immobilises cellular components without altering cellular architecture. We recommend the routine application of the sandwich freezing-freeze substitution technique to achieve ultrastructural preservation that is as close to the native state as possible in electron microscopy.