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  Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

Arnold, J., Mahamid, J., Lucic, V., de Marco, A., Fernandez, J.-J., Laugks, T., et al. (2016). Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy. BIOPHYSICAL JOURNAL, 110(4), 860-869. doi:10.1016/j.bpj.2015.10.053.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-265F-8 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-2660-2
Genre: Journal Article

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 Creators:
Arnold, Jan1, Author              
Mahamid, Julia1, Author              
Lucic, Vladan1, Author              
de Marco, Alex2, Author
Fernandez, Jose-Jesus2, Author
Laugks, Tim1, Author              
Mayer, Tobias2, Author
Hyman, Anthony A.2, Author
Baumeister, Wolfgang1, Author              
Plitzko, Jürgen M.1, Author              
Affiliations:
1Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565142              
2external, ou_persistent22              

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Free keywords: CRYOELECTRON TOMOGRAPHY; ELECTRON TOMOGRAPHY; LIGHT-MICROSCOPY; FLUORESCENCE MICROSCOPY; CELLS; SPECIMENS; PRECISION; MARKERS
 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.

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Language(s): eng - English
 Dates: 2016
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: ISI: 000370763800014
DOI: 10.1016/j.bpj.2015.10.053
 Degree: -

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Title: BIOPHYSICAL JOURNAL
Source Genre: Journal
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Publ. Info: 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA : CELL PRESS
Pages: - Volume / Issue: 110 (4) Sequence Number: - Start / End Page: 860 - 869 Identifier: ISSN: 0006-3495