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          Institute: MPI für molekulare Zellbiologie und Genetik     Collection: MPI-CBG Publications 2016 (archival)     Display Documents



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ID: 732391.0, MPI für molekulare Zellbiologie und Genetik / MPI-CBG Publications 2016 (archival)
Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.
Authors:Arnold, Jan; Mahamid, J.; Lucic, Vladan; Marco, Alex de; Fernandez, Jose-Jesus; Laugks, Tim; Mayer, Tobias; Hyman, Anthony; Baumeister, W.; Plitzko, Jürgen M
Date of Publication (YYYY-MM-DD):2016
Title of Journal:Biophysical Journal
Volume:110
Issue / Number:4
Start Page:860
End Page:869
Copyright:not available
Audience:Experts Only
Intended Educational Use:No
Abstract / Description: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.
External Publication Status:published
Document Type:Article
Version Comment:Automatic journal name synchronization
Communicated by:Thüm
Affiliations:MPI für molekulare Zellbiologie und Genetik
Identifiers:LOCALID:6465
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