Cryo-electron microscopy of vitreous sections

Full text: For the last two decades, cryo-electron microscopy (cryo-em) of thin layers of vitrified biological suspensions has considerably extended applications in electron microscopy. Biomacromolecules or their assemblies can be observed in their fully hydrated native state, without any or few microscopy related preparation artefacts. Only electron beam damage still limits resolution, thus leaving room for specialists of image processing, capable of extracting the very last bit of information created by a limited number of electrons. They're skills and programs have been very good when applied to thin specimens but this method does not apply readily to bulk specimens. However over the last 20 years, cryo-em of vitreous bulk material and sections has also been under development. In principle, it is the dream method of structural cell biology. It consists in vitrifying a sample of tissue by rapid cooling, cutting into ultra-thin sections and cryo-em observation with all details perfectly preserved. Practically the technical problems are considerable. First of all, vitrification, which is relatively easy for sub-micron sample, must be extended to macroscopic dimensions. For this purpose, freezing under high pressure has proved very effective. Cutting a piece of vitreous material into < 100 nm sections is the second major challenge. Unlike resin, vitreous water does not behave well under the knife. A compromise must be found between fracturing the brittle material or plastic deformation when it is more viscous. Here the recent development of an oscillating knife is promising. Finally, to become fluent with the various manipulations and adjustments leading to optimal observations requires time and experience. Copyright (2002) Australian Society for Electron Microscopy Inc