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Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging.

Maarten W Tuijtel¹, Sergio Cruz-León², Jan Philipp Kreysing^1,3

¹Department of Molecular Sociology, Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, 60438 Frankfurt am Main, Germany.

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|April 26, 2024

Summary

Cryo-electron tomography (cryo-ET) data quality depends on specimen thickness. Optimal lamella thickness up to 180nm does not impact resolution, with damage limited to 30nm from surfaces.

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Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

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Published on: July 12, 2022

Area of Science:

Cellular and Molecular Imaging
Structural Biology
Biophysics

Background:

Cryo-electron tomography (cryo-ET) enables in situ structural analysis of biomolecules.
High-quality cryo-ET data relies on optimal specimen thickness.
Cryo-focused ion beam (cryo-FIB) milling thins specimens but can induce damage.

Purpose of the Study:

Systematically investigate the impact of lamella thickness on cryo-ET resolution.
Quantify ion-induced structural damage from cryo-FIB milling.
Determine optimal lamella thickness for high-resolution cryo-ET imaging.

Main Methods:

Cryo-focused ion beam (cryo-FIB) milling of cellular specimens.
Systematic variation of lamella thickness.
Cryo-electron tomography data acquisition and subtomogram averaging.
Analysis of resolution as a function of lamella thickness and distance from surfaces.

Main Results:

Ion-induced damage is confined to within 30 nanometers of each lamella surface.
Lamella thicknesses up to approximately 180 nanometers do not negatively affect resolution.
Resolution is not compromised by using thicker lamellae.

Conclusions:

Cryo-FIB milling damage is localized and does not preclude high-resolution imaging with thicker lamellae.
Optimal lamella thickness can be chosen to encompass cellular features of interest.
Thicker lamellae expand the applicability of cryo-ET to larger cellular complexes and structures.