title: Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins.
abstract: While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution.
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: schaffer@biochem.mpg.de.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Schaffer
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Mahamid
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Engel
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Laugks
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: plitzko@biochem.mpg.de.
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
mesh_terms: D002696:Chlamydomonas / Q000648:ultrastructure; D055032:Electron Microscope Tomography / Q000379:methods; D005629:Frozen Sections / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000648:ultrastructure*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D013048:Specimen Handling / Q000379:methods*
chemical_list: D008565:Membrane Proteins
keywords: Cryo-ET; Cryo-FIB; In situ; Sample preparation; Volta phase plate