ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study
Shuoguo Li, Ziyan Wang, Xing Jia, Tongxin Niu, Jianguo Zhang, Guoliang Yin, Xiaoyun Zhang, Yun Zhu, Gang Ji & Fei Sun
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.
附件下载：
ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study, Nat Methods, 16 Jan 2023
Nat Methods, 16 January, 2023, DOI：https://doi.org/10.1038/s41592-022-01748-0
ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study
Shuoguo Li, Ziyan Wang, Xing Jia, Tongxin Niu, Jianguo Zhang, Guoliang Yin, Xiaoyun Zhang, Yun Zhu, Gang Ji & Fei Sun
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.
文章链接：https://www.nature.com/articles/s41592-022-01748-0
相关报道：http://www.ibp.cas.cn/kyjz/zxdt/202301/t20230113_6599317.html