Fully automated, sequential focused ion beam milling for cryo-electron tomography.

Tobias Zachs,Gregor L Weiss,João Medeiros,Andreas Schertel,Joao Matos,Martin Pilhofer,Jannik Hugener

doi:10.7554/elife.52286

Tobias Zachs, Gregor L Weiss + Show 5 more

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https://doi.org/10.7554/elife.52286

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Abstract

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

Highlights

Cryo-electron tomography is a powerful imaging technique at the interface of cell biology and structural biology, due to its capabilities for imaging cells in a near-native state and determining structures of macromolecular machines in their cellular context (Beck and Baumeister, 2016; Koning et al, 2018; Kooger et al, 2018; Oikonomou and Jensen, 2017; Plitzko et al, 2017)
Biological cryo-focused ion beam (cryoFIB) milling is an emerging sample thinning technique, which uses a gallium ion beam to ablate segments of the sample in order to generate thin lamellae that can be imaged by Cryo-electron tomography (cryoET) (Marko et al, 2007; Rigort et al, 2010)
Complementary to efforts from the de Marco and Raunser/Plitzko labs (Buckley et al, 2020; Tacke et al, 2020), here we report an automated sequential FIB milling method aimed at preparing lamellae for subsequent cryoET imaging

Summary

Introduction

Cryo-electron tomography (cryoET) is a powerful imaging technique at the interface of cell biology and structural biology, due to its capabilities for imaging cells in a near-native state and determining structures of macromolecular machines in their cellular context (Beck and Baumeister, 2016; Koning et al, 2018; Kooger et al, 2018; Oikonomou and Jensen, 2017; Plitzko et al, 2017). CryoET is restricted to samples that are well below 800 nm in thickness, and requires sample thinning techniques for specimens like mammalian cells, C. elegans, yeast, cyanobacteria and biofilms. Biological cryoFIB milling is an emerging sample thinning technique, which uses a gallium ion beam to ablate segments of the sample in order to generate thin lamellae that can be imaged by cryoET (Marko et al, 2007; Rigort et al, 2010). CryoFIB milling for cryoET is at an early stage of technical maturation and the available techniques are highly manual procedures with relatively low throughput

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