ELECTRON-MICROSCOPY OF FROZEN BIOLOGICAL OBJECTS - A STUDY USING CRYOSECTIONING AND CRYOSUBSTITUTION

Freezing of bulk biological objects was investigated by X-ray cryodiff raction. Freezing at atmospheric pressure of most microscopic biologic al samples gives rise to large hexagonal crystals and leads to poor st ructural preservation of these specimens. High-pressure freezing induc es the formation of different ices (hexagonal, cubic and a high-pressu re form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ic e crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especia lly using high-pressure freezing. Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy , Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. B ecause samples containing crystalline ices cannot be cryosectioned, th eir structure has to be studied using cryosubstitution and resin embed ding. We show that bacteria, yeast, and ciliate and marine worm elytru m have cellular compartments with an organization that has not been de scribed by classical techniques relying on chemical fixation of the ti ssues. A high-pressure artefact affecting the Paramecium trichocysts i s described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-press ure freezing and thawing steps.