RT Journal Article SR Electronic T1 Automated cryo-lamella preparation for high-throughput in-situ structural biology JF bioRxiv FD Cold Spring Harbor Laboratory SP 797506 DO 10.1101/797506 A1 Genevieve Buckley A1 Gediminas Gervinskas A1 Cyntia Taveneau A1 Hari Venugopal A1 James C. Whisstock A1 Alex de Marco YR 2019 UL http://biorxiv.org/content/early/2019/10/08/797506.abstract AB Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.