PT  - JOURNAL ARTICLE
AU  - Tobias Zachs
AU  - João M. Medeiros
AU  - Andreas Schertel
AU  - Gregor L. Weiss
AU  - Jannik Hugener
AU  - Martin Pilhofer
TI  - Fully automated, sequential focused ion beam milling for cryo-electron tomography
AID  - 10.1101/797514
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 797514
4099  - http://biorxiv.org/content/early/2019/10/08/797514.short
4100  - http://biorxiv.org/content/early/2019/10/08/797514.full
AB  - Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, with the unique ability to determine structures of macromolecular complexes in their cellular context. A major 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.