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.
International Workshop of 3D Molecular Imaging by Cryo-Electron Microscopy, Third K. H. Kuo Summer School of Electron Microscopy and Crystallography in 2010.
International Workshop of Advanced Image Processing of Cryo-Electron Microscopy, 2013
Get acquainted with Cryo-Electron Microscopy: First Chinese Workshop for Structural Biologists, 2015
International Workshop of Advanced Image Processing of Cryo-Electron Microscopy, 2015
Instutions
Instutions
Institute of Biophysics, Chinese Academy of Sciences
The Scripps Research Institute
Max Planck Institute of Biochemistry
Database
Database
National Center for Biotechnology Information(NCBI)
Protein Data Bank
The Electron Microscopy Data Bank
ExPASy Proteomics Server
Pfam
3D EM
3DEM
Tools and Softwars
Tools and Softwars
CCP4
CCP-EM
MOLE 2.0 (characterization of channels and pores in protein complex)
