Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography.
Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples.
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Miroslava
M
Schaffer
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Benjamin D
BD
Engel
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Tim
T
Laugks
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Julia
J
Mahamid
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Jürgen M
JM
Plitzko
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Wolfgang
W
Baumeister