A cryogenic, coincident fluorescence, electron, and ion beam microscope.

Area of Science:

• Structural Biology
• Microscopy Techniques
• Biophysics

Background:

• Cryogenic electron tomography (cryo-ET) with subtomogram averaging enables high-resolution in situ visualization of macromolecular complexes.
• Cryogenic focused ion beam (cryo-FIB) micromachining is crucial for preparing samples for cryo-ET but lacks precise targeting of specific structures.
• Existing fluorescence-guided FIB milling methods involve multiple transfers, risking contamination and compromising targeting accuracy.

Purpose of the Study:

• To develop an improved method for in situ fluorescence microscopy-guided focused ion beam (FIB) fabrication of frozen-hydrated lamellae for cryo-ET.
• To enhance the precision and success rate of cryo-FIB milling for targeting specific cellular structures or proteins.

Main Methods:

• Retrofitting a cryogenic microcooler, custom stage, and inverted fluorescence microscope onto a FIB scanning electron microscope to create a three-beam cryogenic correlative microscope.
• Implementing in situ fluorescence microscopy guidance during every step of the lamella milling process.
• Validation using transmission electron microscope tomogram reconstructions of the milled target regions.

Main Results:

• Demonstrated successful fluorescence microscopy-guided targeting throughout the lamella fabrication process.
• Achieved higher success rates in lamella fabrication due to real-time monitoring during and after milling.
• Validated the method's efficacy through tomogram reconstructions of targeted regions.

Conclusions:

• The developed in situ fluorescence microscopy-guided FIB fabrication method significantly improves the precision and efficiency of cryo-ET sample preparation.
• This approach increases the throughput for producing high-quality lamellae suitable for high-resolution structural analysis of macromolecular complexes.
• The integrated system addresses limitations of standard cryo-FIB milling, enabling more reliable in situ structural determination.