Recent Advances in Gas Injection System-Free Cryo-FIB Lift-Out Transfer for Cryo-Electron Tomography of Multicellular Organisms and Tissues

Abstract: Cryo-electron tomography (cryo-ET) enables visualization of protein complexes within their native cellular environment at molecular resolution. Most cells and all tissues, however, are too thick to be imaged directly by transmission electron microscopy (TEM). Overcoming this limitation requires the production thin biological sections called lamellae. The procedure obtain lamellae cells, either seeded or grown on grids, cryo-focused ion beam (cryo-FIB) milling samples. This method faces an additional challenge when dealing with tissues multicellular organisms, as these samples must high-pressure frozen, which embeds sample in a layer ice. Nonetheless, can still prepared from such extracting small volume transferring it receiver grid for lamella preparation, process lift-out. Here, we describe available workflows produce lift-out cryogenic conditions recent developments gas injection system (GIS)-free approaches transfer. These advances expand applications cryo-ET, enabling investigation whole organisms situ