Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

Area of Science:

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

Background:

• Cryo-electron tomography (cryo-ET) allows imaging of cellular structures in their native state.
• Cryo-focused ion beam (cryo-FIB) milling is crucial for preparing thin lamellas for cryo-ET.
• Targeting specific subcellular structures within limited cryo-FIB lamellas remains challenging.

Purpose of the Study:

• To develop a method for precise targeting of specific cellular structures for cryo-FIB milling.
• To integrate light microscopy with cryo-FIB workflows at cryogenic temperatures.
• To improve the efficiency and accuracy of cryo-ET sample preparation.

Main Methods:

• Development of a cryo-stage for spinning-disk confocal light microscopy at cryogenic temperatures.
• Integration of light microscopy with cryo-FIB for correlative imaging.
• Use of fiducial markers and 3D coordinate transformations for accurate correlation.
• Targeted cryo-FIB milling of vitrified cellular samples, including fluorescently labeled lipid droplets.
• Localization of targeted structures in transmission electron microscopy images.

Main Results:

• Successful development and integration of a cryo-stage for correlative light microscopy and cryo-FIB.
• Demonstrated precise targeting of fluorescently labeled lipid droplets within 300 nm thick lamellas.
• Established a workflow for correlating light microscopy data with cryo-FIB milling and cryo-ET.
• Enabled unambiguous identification and navigation to specific structures within cryo-ET lamellas.

Conclusions:

• The developed correlative approach enhances the precision of cryo-FIB milling for cryo-ET.
• This method facilitates the study of low-abundance and dynamic macromolecular assemblies.
• It significantly improves the ability to acquire cryo-ET data from specifically targeted cellular regions.