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Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

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Bieber,  Anna
Max Planck Institute of Biochemistry, Max Planck Society;

Capitanio,  Cristina
Max Planck Institute of Biochemistry, Max Planck Society;

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Wilfling,  Florian
Research Group Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, Max Planck Society;
Max Planck Institute of Biochemistry, Max Planck Society;

Plitzko,  Jürgen
Max Planck Institute of Biochemistry, Max Planck Society;

Erdmann,  Philipp S.
Max Planck Institute of Biochemistry, Max Planck Society;
Fondazione Human Technopole;

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Citation

Bieber, A., Capitanio, C., Wilfling, F., Plitzko, J., & Erdmann, P. S. (2021). Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography. Journal of Visualized Experiments, (176): e62886. doi:10.3791/62886.


Cite as: https://hdl.handle.net/21.11116/0000-0009-7553-6
Abstract
Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).