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  In situ microfluidic cryofixation for cryo Focused Ion Beam milling and cryo electron tomography.

Fuest, M., Schaffer, M., Nocera, G. M., Galilea-Kleinsteuber, R. I., Messling, J. E., Heymann, M., et al. (2019). In situ microfluidic cryofixation for cryo Focused Ion Beam milling and cryo electron tomography. Scientific Reports, 9: 19133. doi:10.1038/s41598-019-55413-2.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0005-6E79-9 Version Permalink: http://hdl.handle.net/21.11116/0000-0005-6E7D-5
Genre: Journal Article

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 Creators:
Fuest, M.1, Author              
Schaffer, M., Author
Nocera, G. M.1, Author              
Galilea-Kleinsteuber, R. I.1, Author              
Messling, J. E., Author
Heymann, M., Author
Plitzko, J. M., Author
Burg, T. P.1, Author              
Affiliations:
1Research Group of Biological Micro- and Nanotechnology, MPI for Biophysical Chemistry, Max Planck Society, ou_578602              

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 Abstract: We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

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Language(s): eng - English
 Dates: 2019-12-13
 Publication Status: Published online
 Pages: -
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 Rev. Method: Peer
 Identifiers: DOI: 10.1038/s41598-019-55413-2
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Title: Scientific Reports
Source Genre: Journal
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Publ. Info: London, UK : Nature Publishing Group
Pages: 10 Volume / Issue: 9 Sequence Number: 19133 Start / End Page: - Identifier: ISSN: 2045-2322
CoNE: https://pure.mpg.de/cone/journals/resource/2045-2322