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

Area of Science:

• Cellular and Molecular Imaging
• Biophysics
• Microscopy Techniques

Background:

• Cryo-focused ion beam (cryo-FIB) milling is crucial for preparing cryo-lamellae of frozen specimens for in situ cryo-electron tomography (cryo-ET).
• Limited targeting precision remains a significant bottleneck, hindering the broader application of cryo-FIB milling.
• Accurate localization of specific cellular structures is essential for high-resolution cryo-ET studies.

Purpose of the Study:

• To develop an advanced cryo-correlative light and electron microscopy (cryo-CLEM) system for improved cryo-FIB targeting.
• To enhance the precision of identifying and targeting regions of interest for cryo-lamella fabrication.
• To facilitate more efficient and accurate workflows for in situ cryo-electron tomography.

Main Methods:

• Integration of a 3D structured illumination fluorescence microscopy (SIM) system with an upgraded high-vacuum stage.
• Development of cryo-CLEM software (3D-View) for precise correlation and targeting.
• Utilized the HOPE-SIM system for cryo-lamella preparation of specific cellular components and viral structures.

Main Results:

• Achieved a correlation precision of 110 nm for targeting regions of interest, sufficient for cryo-lamella fabrication.
• Successfully prepared cryo-lamellae targeting mitochondria and centrosomes in HeLa cells.
• Demonstrated successful targeting of the herpesvirus assembly compartment in infected BHK-21 cells.

Conclusions:

• The developed HOPE-SIM system significantly enhances targeting precision in cryo-FIB milling.
• This cryo-CLEM approach offers high potency for future in situ cryo-ET workflows.
• The system enables accurate cryo-lamella preparation for studying complex cellular and viral structures at high resolution.