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Peer-reviewedCC BY-NC-ND 4.0Nature Methods

Control of ice thickness in cryo-EM via confinement

  • Liming Zheng1
  • Jiling Song2
  • Xiaole Zhao1
  • Jiacong Cao3
  • Jie Xu2
  • Zhengni Wang2
  • Chenhui Zhang4
  • Weiyu Sun1,5
  • Buhang Chen6
  • Xiaoyin Gao1
  • Haonan Liu7
  • Junhao Yang2
7 more authors
  • Yu Xu8,9
  • Luzhao Sun6
  • Zhaohe Dai3
  • Xiaoding Wei3
  • Nan Liu4
  • Hailin Peng1,5
  • Hong-Wei Wang2
9 affiliations
  1. 1Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
  2. 2Ministry of Education Key Laboratory of Protein Sciences, Beijing Frontier Research Center for Biological Structures, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China
  3. 3State Key Laboratory for Turbulence and Complex System, School of Mechanics and Engineering Science, Peking University, Beijing, China
  4. 4School of Biological Sciences, The University of Hong Kong, Hong Kong, China
  5. 5Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
  6. 6Beijing Graphene Institute (BGI), Beijing, China
  7. 7Key Laboratory for the Physics and Chemistry of Nanodevices and Center for Carbon-based Electronics, School of Electronics, Peking University, Beijing, China
  8. 8Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, China
  9. 9Institute of Bio-Architecture and Bio-Interactions, Shenzhen Medical Academy of Research and Translation, Shenzhen, China
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Abstract

Achieving thin ice with optimal thickness is a prerequisite for successful cryo-EM analysis of embedded macromolecules. Despite decades of effort, precise control of ice thickness in cryo-EM remains a major challenge. Here we reveal that the intrinsic instability of sub-100-nm liquid film, a previously overlooked determinant before vitrification, plays a critical role in controlling ice thickness. We found that well-designed graphene reservoirs can confine liquid film to an optimal thickness and overcome the limitations posed by liquid film instability, achieving precise control of ice thickness with robust reproducibility and large-area uniformity. These graphene reservoirs demonstrate superior liquid retention capabilities than conventional techniques, enabling tunable ice thickness through reservoir depth modulation. Moreover, this approach yields uniform confined ice in graphene sandwiches with controllable thickness and high efficiency, facilitating cryo-EM imaging of a broad range of macromolecule particles with high contrast, reduced motion and improved orientations for more robust high-resolution reconstruction.

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Cite

BibTeXRIS
BibTeX
@article{zheng2026control,
  title = {{Control of ice thickness in cryo-EM via confinement}},
  author = {Liming Zheng and Jiling Song and Xiaole Zhao and Jiacong Cao and Jie Xu and Zhengni Wang and Chenhui Zhang and Weiyu Sun and Buhang Chen and Xiaoyin Gao and Haonan Liu and Junhao Yang and Yu Xu and Luzhao Sun and Zhaohe Dai and Xiaoding Wei and Nan Liu and Hailin Peng and Hong-Wei Wang},
  journal = {Nature Methods},
  year = {2026},
  month = sep,
  doi = {10.1038/s41592-026-03244-1},
  url = {https://doi.org/10.1038/s41592-026-03244-1},
  note = {CC BY-NC-ND 4.0},
}
RIS
TY  - JOUR
TI  - Control of ice thickness in cryo-EM via confinement
AU  - Liming Zheng
AU  - Jiling Song
AU  - Xiaole Zhao
AU  - Jiacong Cao
AU  - Jie Xu
AU  - Zhengni Wang
AU  - Chenhui Zhang
AU  - Weiyu Sun
AU  - Buhang Chen
AU  - Xiaoyin Gao
AU  - Haonan Liu
AU  - Junhao Yang
AU  - Yu Xu
AU  - Luzhao Sun
AU  - Zhaohe Dai
AU  - Xiaoding Wei
AU  - Nan Liu
AU  - Hailin Peng
AU  - Hong-Wei Wang
PY  - 2026
DA  - 2026/09/25
JO  - Nature Methods
DO  - 10.1038/s41592-026-03244-1
UR  - https://doi.org/10.1038/s41592-026-03244-1
AB  - Achieving thin ice with optimal thickness is a prerequisite for successful cryo-EM analysis of embedded macromolecules. Despite decades of effort, precise control of ice thickness in cryo-EM remains a major challenge. Here we reveal that the intrinsic instability of sub-100-nm liquid film, a previously overlooked determinant before vitrification, plays a critical role in controlling ice thickness. We found that well-designed graphene reservoirs can confine liquid film to an optimal thickness and overcome the limitations posed by liquid film instability, achieving precise control of ice thickness with robust reproducibility and large-area uniformity. These graphene reservoirs demonstrate superior liquid retention capabilities than conventional techniques, enabling tunable ice thickness through reservoir depth modulation. Moreover, this approach yields uniform confined ice in graphene sandwiches with controllable thickness and high efficiency, facilitating cryo-EM imaging of a broad range of macromolecule particles with high contrast, reduced motion and improved orientations for more robust high-resolution reconstruction.
SN  - 1548-7091
SN  - 1548-7105
ER  -