Cryogenic Preparations of Biological Specimens for Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become a method of choice for in-cell structural biology studies by integrating the non-invasive power of fluorescence light microscopy, artifact-free thinning of intact cells, and bridging to the atomic resolution achieved by single-particle cryo-electron microscopy (cryo-EM) of biochemically purified macromolecules. While the latter has reached a level of maturity and standardization for specimen preparation, cryo-ET needs to meet a multitude of requirements posed by its highly heterogenous sample types. Cryo-ET is compatible with samples prepared by both plunge-freezing and high-pressure freezing. Still, little is known about the vitrification process of complex and heterogenous cells during cryogenic specimen preparation. Thus, we first describe the physics of vitrification with emphasis on biological specimens prepared on EM-compatible supports. We then provide a comprehensive account of the current state-of-the-art cryo-ET sample preparation techniques for specimens ranging from in-vitro reconstitutions, single cells, to 3D cell cultures, and whole multicellular organisms. We point out specific considerations that facilitate a wide range of downstream applications that are described in the following chapters. Finally, we discuss aspects that demand technological improvements and current strategies to fill these gaps.