title: Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.
abstract: The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Arnold
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany. Electronic address: mahamid@biochem.mpg.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Mahamid
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Vladan
authors.initials: V
authors.lastname: Lucic
authors.affiliation: FEI Company, Graefelfing, Germany.
authors.forename: Alex
authors.initials: A
authors.lastname: de Marco
authors.affiliation: Centro Nacional de Biotecnologia, Madrid, Spain.
authors.forename: Jose-Jesus
authors.initials: JJ
authors.lastname: Fernandez
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Laugks
authors.affiliation: FEI Company, Graefelfing, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Mayer
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Hyman
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.affiliation: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany. Electronic address: plitzko@biochem.mpg.de.
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
mesh_terms: D055032:Electron Microscope Tomography / Q000295:instrumentation / Q000379:methods*; D057918:Fiducial Markers; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence