In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

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 micros...

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Bibliographic Details
Published in:Scientific reports 2019-12, Vol.9 (1), p.19133-10, Article 19133
Main Authors: Fuest, Marie, Schaffer, Miroslava, Nocera, Giovanni Marco, Galilea-Kleinsteuber, Rodrigo I., Messling, Jan-Erik, Heymann, Michael, Plitzko, Jürgen M., Burg, Thomas P.
Format: Article
Language:English
Subjects:
101/28
101/62
631/1647/328/1259
639/166
Electron microscopy
Freezing
Humanities and Social Sciences
Ion beams
Larvae
Microfluidics
Microscopy
multidisciplinary
Preservation
Science
Science (multidisciplinary)
Sectioning
Structure-function relationships
Tomography
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Summary: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
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-019-55413-2