Preparation and Cryo-FIB micromachining of <em>Saccharomyces cerevisiae</em> for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to strong interaction of an electron with matter, high-resolution cryo-ET studies are limited specimens a thickness less than 200 nm, which restricts applicability peripheral regions cell. A complex workflow comprises preparation thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during last decade enable acquisition from interior larger cells. We present protocol for lamellae sample vitrified plunge freezing utilizing Saccharomyces cerevisiae as prototypical example eukaryotic cell wide utilization and molecular biology research. describe protocols vitrification S. into isolated patches few cells or continuous monolayer TEM grid lamella cryo-FIB these two samples.