In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method

Area of Science:

• Structural Biology
• Cryo-Electron Microscopy
• Biophysics

Background:

• Cryo-focused ion beam (FIB) milling is crucial for in situ cellular cryo-electron tomography (cryo-ET).
• Conventional cryo-FIB milling of thin specimens faces limitations in throughput and sample distribution.
• Thicker specimens are challenging to prepare for high-resolution cryo-ET analysis.

Purpose of the Study:

• To present the
• Waffle Method
• protocol for vitrifying thick specimens for cryo-FIB milling.
• To describe a semi-automated milling procedure for generating cryo-lamellae from diverse biological samples.
• To enhance the throughput and efficiency of cryo-ET sample preparation.

Main Methods:

• Vitrification of thick specimens using the
• Waffle Method
• protocol.
• Semi-automated cryo-FIB milling using Thermo Fisher Scientific Aquilos 2 and AutoTEM Cryo software.
• Generation of cryo-lamellae with target thicknesses of 100-200 nm.

Main Results:

• Successful generation of cryo-lamellae from various samples including microsporidia spores, yeast, bacteria, mammalian cells, and purified protein complexes.
• Achieved throughput of 2-3 cryo-lamellae per 8-hour working day with dimensions of ~12 μm width and 15-20 μm length.
• Potential for higher throughput (up to 16 cryo-lamellae in 24 h) with low-contamination cryo-FIB/SEMs.

Conclusions:

• The
• Waffle Method
• protocol combined with semi-automated cryo-FIB milling significantly improves cryo-ET sample preparation efficiency.
• This method is versatile, applicable to a wide range of biological specimens.
• It offers a robust solution for obtaining high-quality cryo-lamellae for structural analysis.