Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Cryo-electron microscopy (cryo-EM) achieves near-atomic resolution for isolated macromolecular complexes.
• Cryo-electron tomography (cryo-ET) offers in-situ structural insights but is limited by sample thickness.
• Thicker biological samples, like intact cells, are challenging for high-resolution cryo-ET.

Purpose of the Study:

• To describe a protocol for preparing thin lamellae from intact frozen cells using cryo-focused-ion-beam (cryo-FIB) milling.
• To enable high-resolution cryo-ET of biomolecules within their native cellular environment.
• To integrate fluorescence microscopy for targeted lamella preparation and cryo-ET imaging.

Main Methods:

• Utilized dual-beam microscopes equipped with cryo-focused-ion-beam (cryo-FIB) and scanning electron microscopy (SEM) on a cryo-stage.
• Fabricated 100-250-nm-thin lamellae from frozen-hydrated cells.
• Integrated fluorescence microscopy for precise targeting of regions of interest before milling.

Main Results:

• Successfully prepared thin lamellae suitable for high-resolution cryo-ET from intact cells.
• Demonstrated the feasibility of targeted lamella preparation using fluorescence microscopy guidance.
• Provided a protocol adaptable to various dual-beam microscopes with cryo-capabilities.

Conclusions:

• Cryo-FIB milling is a crucial technique for preparing samples for in-situ structural biology via cryo-ET.
• The described protocol facilitates the study of biomolecular structures in their native cellular context.
• This method significantly expands the applicability of cryo-ET to a wider range of biological samples.