Micromanipulator-assisted vitreous cryosectioning and sample preparation by high-pressure freezing

Cryo-electron microscopy (cryo-EM) of unfixed, unstained, frozen-hydrated samples conveys the most reliable view of the "live" state of cells and tissues. Advances in sample preparation methods and electron microscope technology over the last decade have made this approach more routine and available to a broader segment of the structural biology community. Many cryo-EM samples are thin enough to be imaged as wholemounts, but most cells and tissues are too thick and must be cryosectioned to obtain samples that are sufficiently thin for successful imaging. Cryosectioning of vitreous material is a challenging and time-consuming task. A number of laboratories have worked hard to develop approaches and technologies for cryosectioning and to carefully characterize the nature of vitreous sections and the artifacts associated with them. Several different cryosectioning methods are in use in cryo-EM laboratories, each of which is effective and routinely yields high-quality structural data. Here, we describe a particular method that utilizes a micromanipulator to aid the cryomicrotomist in controlling vitreous sections as they are being cut and to facilitate transfer of the sections to an EM grid. Each step in the process, from preparing samples by high-pressure freezing to affixing vitreous sections to a grid, is covered in detail, including discussions of cryosectioning hardware, environmental conditions, and sectioning artifacts.