Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Area of Science:

• Electron microscopy
• Structural biology
• Materials science

Background:

• Beam-induced specimen charging is a major challenge in electron microscopy, particularly for imaging tilted 2D crystals.
• This charging artifact leads to image shift, causing loss of diffraction data and reducing the quality of biological macromolecule images.
• Current preparation methods using single carbon support films yield low success rates for tilted specimens.

Purpose of the Study:

• To investigate the efficacy of the carbon sandwich preparation method in mitigating specimen charging during electron microscopy.
• To assess the impact of this technique on the quality of images obtained from tilted 2D crystals.
• To determine if the carbon sandwich method can improve the yield of analyzable data for electron crystallography.

Main Methods:

• Utilized 2D crystals of aquaporin-4 as a model system.
• Compared the conventional single carbon support film technique with the novel carbon sandwich preparation method.
• Acquired electron microscopy images of tilted specimens prepared using both methods.

Main Results:

• The carbon sandwich preparation method resulted in a higher proportion of images exhibiting sharp diffraction spots perpendicular to the tilt axis.
• This technique effectively reduced image shift caused by beam-induced specimen charging.
• Compared to single carbon films, the carbon sandwich method significantly improved the quality and yield of images from tilted specimens.

Conclusions:

• The carbon sandwich preparation technique is a reproducible method that overcomes severe specimen charging issues in electron microscopy.
• This approach enhances image quality and data acquisition for tilted specimens.
• The method has the potential to accelerate structure determination of biological macromolecules using electron crystallography.