Routine determination of ice thickness for cryo-EM grids

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Cryo-electron microscopy (cryo-EM) is a powerful technique for determining near-atomic resolution structures.
• High-quality data collection in cryo-EM is often limited by sample preparation, specifically the thickness of vitreous ice.
• Optimizing ice thickness is crucial for obtaining high-resolution cryo-EM data.

Purpose of the Study:

• To present two novel methods for measuring vitreous ice thickness during cryo-EM data collection.
• To integrate these methods into the Leginon/Appion workflow for seamless implementation.
• To demonstrate the utility of routine ice thickness monitoring for improving cryo-EM data quality.

Main Methods:

• Developed two methods to measure ice thickness: one using an energy filter, the other using scattering outside the objective aperture.
• Implemented these methods within the Leginon/Appion data collection workflow.
• Ensured methods can be applied to potentially every image without interrupting data collection.

Main Results:

• The presented methods allow for real-time measurement of ice thickness during cryo-EM data collection.
• These techniques can be integrated into existing workflows, minimizing disruption.
• Examples from test cases demonstrate the practical application and effectiveness of the methods.

Conclusions:

• Routine monitoring of ice thickness is essential for optimizing cryo-EM sample preparation.
• These methods facilitate improved data collection strategies.
• Implementing ice thickness measurement aids in enhancing overall cryo-EM data processing and structural determination.