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bioRxiv · 10.1101/2022.09.16.508243

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Li, S.·Jia, X.·Niu, T.·Zhang, X.·Qi, C.·Xu, W.·Deng, H.·Sun, F.·Ji, G.
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Abstract

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we developed a new cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 150 nm enough for the subsequent cryo-lamella fabrication. We successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

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Li, S., Jia, X., Niu, T., Zhang, X., Qi, C., Xu, W., Deng, H., Sun, F., Ji, G.. 2022-09-16. HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography. https://doi.org/10.1101/2022.09.16.508243

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