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

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 have developed a 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 110 nm enough for the subsequent cryo-lamella fabrication. We have 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. A cryo-correlative light and electron microscopy (cryo-CLEM) system with 3D structured illumination fluorescence microscopy and software efficiently targets regions of interest for cryo-focused ion beam milling (cryo-FIB) in cryo-ET studies.

Automated summary

A cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM has been developed to efficiently target cryo-FIB for cryo-ET studies by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage. With the 3D super resolution of cryo-SIM and cryo-CLEM software 3D-View, the correlation precision of the targeting region of interest can reach 110 nm, sufficient for subsequent cryo-lamella fabrication. The HOPE-SIM system has been successfully utilized to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells, and herpesvirus assembly compartments of infected BHK-21 cells, demonstrating its high potential for future in situ cryo-ET workflows.