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

ELife
T. ZachsMartin Pilhofer

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.

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Citations

Oct 7, 2020·FEBS Letters·Martin Turk, Wolfgang Baumeister
Nov 4, 2020·Molecular Microbiology·Trishant R UmrekarMorgan Beeby
Jan 9, 2021·Quarterly Reviews of Biophysics·Deryck J Mills
Sep 19, 2020·Histochemistry and Cell Biology·Jan Philipp SchneiderChristoph Wrede
Aug 2, 2020·Current Opinion in Structural Biology·Xiaojie Zhang, Julia Mahamid
Feb 3, 2021·Viruses·Georg Wolff, Montserrat Bárcena
Mar 3, 2021·ACS Nano·Carlos Sanchez-CanoWolfgang J Parak
Aug 20, 2020·Structure·Gong-Her WuWah Chiu
Sep 29, 2020·Journal of Structural Biology·Florian FäßlerFlorian K M Schur
Aug 14, 2020·Cell·Reika WatanabeElizabeth Villa
Jun 22, 2021·Annual Review of Biochemistry·Xuelang MuTamir Gonen
Jun 25, 2021·Quarterly Reviews of Biophysics·David J DeRosier
Aug 14, 2021·Journal of Molecular Biology·Felix J B Bäuerlein, Wolfgang Baumeister
Sep 2, 2021·IScience·Ryan K Hylton, Matthew T Swulius
Sep 9, 2021·Biochemical Society Transactions·Zhuan Qin, Peijun Zhang
Nov 8, 2021·Journal of Structural Biology·Georgi DimchevFlorian Km Schur

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Methods Mentioned

BETA
imaging technique
transmission electron microscopy

Software Mentioned

PEET
IMOD
cryoET
SerialEM
SmartFIB
ACE600
SmartSEM

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