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bioRxiv · 10.1101/2022.08.15.503951

ELI trifocal microscope: A precise cryogenic fabrication system to prepare target cryo-lamellae of cells for in situ cryo-ET study

Li, S.·Wang, Z.·Jia, X.·Niu, T.·Zhang, J.·Yin, G.·Zhang, X.·Zhu, Y.·Ji, G.·Sun, F.
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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 in situ cryo-ET structural study. Here, we developed a precise cryogenic fabrication system, the ELI trifocal microscope (ELI-TriScope), by setting up an electron (E) beam, a light (L) beam and an ion (I) beam at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella without sacrificing the throughput. ELI-TriScope was developed starting from a commercial dual-beam scanning electron microscope (SEM) by incorporating a cryo-holder-based transfer system and embedding an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam (FIB) 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 an [~]100% success rate, and discovered new in situ structural features of the human centrosome through a subsequent cryo-ET structural study.

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Li, S., Wang, Z., Jia, X., Niu, T., Zhang, J., Yin, G., Zhang, X., Zhu, Y., Ji, G., Sun, F.. 2022-08-15. ELI trifocal microscope: A precise cryogenic fabrication system to prepare target cryo-lamellae of cells for in situ cryo-ET study. https://doi.org/10.1101/2022.08.15.503951

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