Advanced cryo-tomography workflow developments - correlative microscopy, milling automation and cryo-lift-out

Area of Science:

• Cellular and Molecular Biology
• Microscopy and Imaging Technologies
• Structural Biology

Background:

• Cryo-electron tomography (cryo-ET) provides high-resolution 3D structural information of biomolecules within their cellular context.
• Native-state imaging is crucial for understanding cellular processes without artifacts from chemical fixation.
• Focused ion beam (FIB) technology has improved cellular sample preparation for cryo-ET.

Purpose of the Study:

• To introduce recent advancements in the cryo-electron tomography workflow.
• To highlight improvements in cryo-correlative light and electron microscopy (cryo-CLEM) for cellular targeting.
• To showcase progress in cryo-FIB automation and cryo-lift-out technology for sample preparation.

Main Methods:

• Cryo-focused ion beam (cryo-FIB) milling to prepare thin cryo-lamellae (200-300 nm) from vitrified cells.
• Cryo-correlative light and electron microscopy (cryo-CLEM) for precise targeting of cellular regions of interest.
• Automated cryo-FIB software for streamlined lamella preparation.
• Cryo-lift-out technology for processing high-pressure frozen bulk samples.

Main Results:

• Developed integrated cryo-CLEM and cryo-FIB interfaces for improved workflow efficiency.
• Achieved significant automation in cryo-FIB sample preparation, reducing preparation time.
• Demonstrated successful 3D imaging of bulk-frozen samples using cryo-FIB and cryo-lift-out techniques.

Conclusions:

• Recent developments enhance the cryo-ET workflow, enabling more efficient and detailed cellular structural analysis.
• The integrated approach of cryo-CLEM, cryo-FIB, and cryo-ET provides unprecedented insights into cellular architecture and function.
• Further advancements in automation and sample preparation will continue to push the boundaries of cryo-ET applications.