Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution

Freezing of bulk biological objects was investigated by X-ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure 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 ice 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, especially 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. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high-pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps.