Preprint: Please note that this article has not completed peer review.

Protocol for cryo-focused ion beam lift-out technique

Miroslava Schaffer, Stefan Pfeffer, Julia Mahamid, Stephan Kleindiek, Tim Laugks, Sahradha Albert, Benjamin D. Engel, Andreas Rummel, Andrew Smith, Wolfgang Baumeister, Juergen M. Plitzko
DOI: 10.21203/rs.2.10392/v1

Abstract

Cryo-focused ion beam milling of frozen hydrated cells for the production of thin lamellas in combination with cryo-electron tomography (cryo-ET) has yielded unprecedented insights into the cell interior. This method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, it is only suitable for cells that can be vitrified by plunge freezing (<10 μm). Multicellular organisms and tissues are considerably thicker and high-pressure freezing is required to ensure optimal preservation. Here, we describe a preparation method for extracting lamellas from high pressure frozen samples with a new cryo-gripper tool. This in situ lift-out technique at cryo-temperatures enables cryo-ET to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology.

Keywords
Cryo-electron tomography, cryo-focused ion beam, lift-out, high-pressure freezing, in situ structural biology

Introduction

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Preprint: Please note that this article has not completed peer review.

Protocol for cryo-focused ion beam lift-out technique

Miroslava Schaffer, Stefan Pfeffer, Julia Mahamid, Stephan Kleindiek, Tim Laugks, Sahradha Albert, Benjamin D. Engel, Andreas Rummel, Andrew Smith, Wolfgang Baumeister, Juergen M. Plitzko

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Abstract

Cryo-focused ion beam milling of frozen hydrated cells for the production of thin lamellas in combination with cryo-electron tomography (cryo-ET) has yielded unprecedented insights into the cell interior. This method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, it is only suitable for cells that can be vitrified by plunge freezing (<10 μm). Multicellular organisms and tissues are considerably thicker and high-pressure freezing is required to ensure optimal preservation. Here, we describe a preparation method for extracting lamellas from high pressure frozen samples with a new cryo-gripper tool. This in situ lift-out technique at cryo-temperatures enables cryo-ET to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology.

Introduction

Reagents

Equipment

Procedure

References

Learn more about our company.