ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study.
Nat Methods
; 20(2): 276-283, 2023 02.
Article
in En
| MEDLINE
| ID: mdl-36646897
ABSTRACT
Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent cryo-ET. Here we describe a precise cryogenic fabrication system, ELI-TriScope, which sets electron (E), light (L) and ion (I) beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. ELI-TriScope uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embed an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome with a success rate of ~91% and discovered new in situ structural features of the human centrosome by cryo-ET.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Electrons
/
Electron Microscope Tomography
Limits:
Humans
Language:
En
Journal:
Nat Methods
Journal subject:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Year:
2023
Document type:
Article
Affiliation country:
China