The last two decades have seen exponential growth in cryo-electron microscopy, resulting in exciting advances, new hypotheses, and a better understanding of the ultrastructure of living and inert matter. Using an accessible, step-by-step format, this handbook presents the most sophisticated methods used in research laboratories. It describes the basic principles of cryo-fixation, cryo-ultramicrotomy, embedding at low temperatures and freeze-fracture. The book also explores applications such as electron crystallography, 2D/3D structure analysis, cryo-electron tomography, and techniques for protein localization. Each chapter covers a specific method, including detailed protocols, summary tables, and hundreds of illustrations and diagrams to clarify the techniques.