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Background <p>Cryo-Electron Microscopy (CryoEM) has revolutionized structural biology by enabling high-resolution visualization of macromolecules in near-native states. However, challenges such as particle interaction with the air-water interface (AWI), preferential orientation, and beam-induced motion limit the resolution and reproducibility of this technique. Graphene oxide (GO) has emerged as a promising support material to address these challenges.</p> Methods <p>Here, we present an optimized method for preparing GO-supported grids using the Vitrobot Mark IV, a widely used cryoEM sample preparation device. This modified drop-cast protocol employs polyethyleneimine (PEI) to enhance GO adhesion and ensures reproducibility through precise control of environmental and blotting parameters using the Vitrobot Mark IV.</p> Results <p>Grids prepared using this approach demonstrated an average GO coverage of 55%, with monolayer and bilayer coverage accounting for 40%. CryoEM reconstructions of β-galactosidase and apoferritin achieved resolutions of 2.3 Å and 1.9 Å, respectively, demonstrating the feasibility of this method for high-resolution structure determination.</p> Discussion <p>This Vitrobot-based method provides a simple and reproducible approach for preparing GO-supported grids without specialized equipment. Controlled environmental settings and PEI-enhanced GO adhesion ensure consistent grid quality and particle distribution. The protocol supports high-resolution reconstructions and offers a practical foundation for future applications, including functionalization and studies of challenging samples.</p>

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Ready to GO: a reliable and reproducible method for preparing graphene oxide grids using the Vitrobot

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年7月6日

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  • Hok Sau Kwong,
  • Alessandro Grinzato,
  • Gian Luca Freiherr von Scholley,
  • Marc-Andre Hograindleur,
  • Eaazhisai Kandiah

摘要

Background

Cryo-Electron Microscopy (CryoEM) has revolutionized structural biology by enabling high-resolution visualization of macromolecules in near-native states. However, challenges such as particle interaction with the air-water interface (AWI), preferential orientation, and beam-induced motion limit the resolution and reproducibility of this technique. Graphene oxide (GO) has emerged as a promising support material to address these challenges.

Methods

Here, we present an optimized method for preparing GO-supported grids using the Vitrobot Mark IV, a widely used cryoEM sample preparation device. This modified drop-cast protocol employs polyethyleneimine (PEI) to enhance GO adhesion and ensures reproducibility through precise control of environmental and blotting parameters using the Vitrobot Mark IV.

Results

Grids prepared using this approach demonstrated an average GO coverage of 55%, with monolayer and bilayer coverage accounting for 40%. CryoEM reconstructions of β-galactosidase and apoferritin achieved resolutions of 2.3 Å and 1.9 Å, respectively, demonstrating the feasibility of this method for high-resolution structure determination.

Discussion

This Vitrobot-based method provides a simple and reproducible approach for preparing GO-supported grids without specialized equipment. Controlled environmental settings and PEI-enhanced GO adhesion ensure consistent grid quality and particle distribution. The protocol supports high-resolution reconstructions and offers a practical foundation for future applications, including functionalization and studies of challenging samples.