Graphene Oxide-Supported Microwell Grids for Preparing Cryo-EM Samples with Controlled Ice Thickness

Area of Science:

• Structural Biology
• Materials Science
• Biophysics

Background:

• Cryo-electron microscopy (cryo-EM) is crucial for 3D protein structure determination and studying radiation/air-sensitive materials.
• Current cryo-EM sample preparation methods lack precise control over ice thickness, hindering efficiency and throughput.
• Controlling nanoscale vitreous ice thickness is vital for high signal-to-noise ratios in cryo-EM imaging of low-contrast specimens.

Purpose of the Study:

• To develop an advanced sample preparation device for cryo-EM.
• To enable precise control over vitreous ice thickness for improved sample quality.
• To enhance cryo-EM imaging of diverse nanoscale samples, including biological and inorganic materials.

Main Methods:

• Development of a silicon-chip-based device with micropatterned holes and a graphene oxide (GO) window on silicon nitride (SiₓNᵧ).
• Utilizing precisely regulated hole depths to control vitreous ice thickness.
• Employing GO window's biomolecule affinity to concentrate samples.

Main Results:

• The device enables accurate regulation of vitreous ice thickness.
• Graphene oxide window facilitates higher sample molecule concentration.
• Enhanced cryo-EM imaging was demonstrated for various samples, including viral particles, protein complexes, and inorganic materials.

Conclusions:

• The developed micropatterned chip with a GO window significantly improves cryo-EM sample preparation.
• Precise ice thickness control leads to higher quality cryo-EM data.
• This technology advances high-throughput structural analysis of sensitive nanoscale specimens.