Automated cryo-lamella preparation for high-throughput in-situ structural biology.
J Struct Biol
; 210(2): 107488, 2020 05 01.
Article
en En
| MEDLINE
| ID: mdl-32126263
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.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Microscopía por Crioelectrón
/
Microscopía Electrónica de Transmisión
Límite:
Animals
/
Humans
Idioma:
En
Año:
2020
Tipo del documento:
Article