Waffle Method: A general and flexible approach for improving throughput in FIB-milling

Area of Science:

• Structural Biology
• Cryo-Electron Microscopy
• Biophysics

Background:

• Cryo-electron tomography (cryo-ET) provides high-resolution in-situ structural information.
• Conventional cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) workflows face challenges with thick specimens, preferred orientation, and low throughput.

Purpose of the Study:

• To introduce a novel method, the 'Waffle Method,' to overcome limitations in cryo-FIB/SEM workflows.
• To demonstrate the Waffle Method's effectiveness in improving sample preparation for cryo-ET.

Main Methods:

• The Waffle Method leverages high-pressure freezing for sample preparation.
• Cryo-ET was applied to analyze microsporidian spores and other cellular/single particle samples.
• A novel stress-relief gap for waffled lamellae was developed.

Main Results:

• The Waffle Method successfully addressed challenges including preferred orientation and vitrification issues.
• High-resolution structural information of the polar tube in microsporidian spores was obtained in multiple orientations.
• The method was validated across diverse sample types and cryo-FIB-milling hardware.

Conclusions:

• The Waffle Method offers a broadly applicable approach to enhance cryo-ET sample preparation.
• It provides advantages similar to cryo-liftout directly on the specimen grid.
• This method simplifies obtaining high-resolution structural data from challenging biological samples.