Robust ultraclean atomically thin membranes for atomic-resolution electron microscopy

Liming Zheng,Hailin Peng,Peng Gao,Jincan Zhang,Wenhui Dang,Yanbin Li,Ning Li,Yi Cui,Nan Liu,Mingzhan Wang,Xiaoyin Gao,Shipu Xu,Bing Deng,Xiaoding Wei,Hao Yang,Hongwei Wang ,Luzhao Sun,Junjie Liu,Zi Yang,Yanan Chen,Congwei Tan

doi:10.1038/s41467-020-14359-0

Abstract

The fast development of high-resolution electron microscopy (EM) demands a background-noise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. Yet the preparation of robust and ultraclean graphene EM grids remains challenging. Here we present a polymer- and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room- and cryo-temperature. The same kind of hydrophilic graphene grid allows the formation of ultrathin vitrified ice layer embedded most protein particles at the graphene-water interface, which facilitates cryo-EM 3D reconstruction of archaea 20S proteasomes at a record high resolution of ~2.36 Å. Our results demonstrate the significant improvements in image quality using the graphene grids and expand the scope of EM imaging.

Highlights

The fast development of high-resolution electron microscopy (EM) demands a backgroundnoise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate
The graphene membrane suspended on the spaced holes was formed with an ultraclean surface, thanks to the transfer-free and polymer-free process
The robustness of suspended graphene membranes is essential for an EM specimen support

Summary

Introduction

The fast development of high-resolution electron microscopy (EM) demands a backgroundnoise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. We present a polymer- and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room- and cryo-temperature. During the cryo-EM specimen preparation, unsupported protein particles tend to adsorb at the air–water interfaces, which might cause preferential orientation and denaturation of proteins, limited the attainable resolution of the reconstructions[3,4,5] To address this issue, conventional amorphous carbon films were often used to support the proteins. It is critical to develop a robust and scalable method to produce ultraclean and hydrophilic graphene grids for more general application in high-resolution EM imaging

Methods

Results

Conclusion