Cryo-electron microscopy (cryo-EM) visualizes the atomic structure of macromolecules that are embedded in vitrified thin ice at their close-to-native state. However, the homogeneity of ice thickness, a key factor to ensure high image quality, is poorly controlled during specimen preparation and has become one of the main challenges for high-resolution cryo-EM. Here we found that the uniformity of thin ice relies on the surface flatness of the supporting film, and developed a method to use ultraflat graphene (UFG) as the support for cryo-EM specimen preparation to achieve better control of vitreous ice thickness. We show that the uniform thin ice on UFG improves the image quality of vitrified specimens. Using such a method we successfully determined the three-dimensional structures of hemoglobin (64 kDa), α-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) at a resolution of 3.5 Å, 2.6 Å and 2.2 Å, respectively. Furthermore, our results demonstrate the potential of UFG for the fields of cryo-electron tomography and structure-based drug discovery.

Technological breakthrough in electron microscopy (EM) has started a resolution revolution in EM imaging. Nowadays, the promotion of resolution demands a robust background‐noise‐free EM support for specimen preparation, which is a primary bottleneck of high‐resolution EM imaging. Owing to the atomic thickness and excellent physical properties, graphene has attracted widespread attention in the EM field to achieve high‐resolution multi‐dimensional imaging. However, it is still challenging to prepare high‐quality suspended graphene membranes. Problems like breakage, contamination and wrinkling reduce the quality of suspended graphene membranes, which inhibit their wide and killer applications in EM imaging. In this review, suspended graphene membranes are looked deeply into for multi‐dimensional EM imaging. This study begins with a brief introduction to EM development, followed by a discussion of the synthesis of high‐quality graphene. Then it summarizes various approaches to produce suspended graphene membranes and their killer applications in multi‐dimensional EM characterization, including high‐resolution 2D imaging, cryo‐EM 3D reconstruction, and 4D in situ liquid EM. Based on current achievements, the prospects of graphene membranes for more cutting‐edge applications are finally proposed.