Towards high-throughput in-situ structural biology

Datum

03.02.2020

Zeit

11:00 - 12:00

Sprecher

Alex de Marco

Zugehörigkeit

Monash University, Dept. of Biochemistry and Molecular Biology, ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, Australia

Sprache

en

Hauptthema

Biologie

Host

Gaia Pigino

Beschreibung

Cryo-transmission electron tomography (cryoET) in association with cryo-focused ion beam (cryoFIB) 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 cryoET imaging. CryoFIB 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 throughout the process. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that it is possible to increase the throughput and achieve a rate of 5 lamellae/hour without the need to supervise the preparation. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryoET analyses. This method in association with the integration of a cryo-light microscope inside the cryoFIB and with the optimization of the ion source leads to the possibility to prepare efficiently targeted lamellae in tissue.

Letztmalig verändert: 04.02.2020, 00:06:26