Automated cryo-lamella preparation for high-throughput in-situ structural biology

Genevieve Buckley, Gediminas Gervinskas, Cyntia Taveneau, Hariprasad Venugopal, James C. Whisstock, Alex de Marco

Research output: Contribution to journalArticleResearchpeer-review

9 Citations (Scopus)

Abstract

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 typical 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, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our 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.

Original languageEnglish
Article number107488
Number of pages8
JournalJournal of Structural Biology
Volume210
Issue number2
DOIs
Publication statusPublished - 1 May 2020

Keywords

  • Automation
  • cryo-EM
  • Cryo-FIB
  • Cryo-lamella
  • In situ structural biology

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