Graphene in cryo-EM specimen optimization

Specimen preparation is a critical but challenging step in high-resolution cryogenic electron microscopy (cryo-EM) structural analysis of macromolecules. In the past decade, graphene has gained much recognition as the supporting substrate to optimize cryo-EM specimen preparation. It improves macromolecule embedding in ice, reduces beam-induced motion, while imposing negligible background noise. Various types of graphene-coated cryo-EM grids were implemented to improve the robustness and efficiency of specimen preparation. Graphene functionalization by different means has been proved specifically useful in addressing challenges related to the air-water interface (AWI), such as preferential orientation and sample denaturation. Graphene sandwich specimen preparation sets a new direction to explore in cryo-EM analysis of biological specimens. In this review, we discuss the current challenges and future prospects of graphene application in cryo-EM analysis of macromolecules. •This work reviews the use of graphene as the supporting substrate for preparing cryo-EM specimens.•Graphene enables improved ice-embedding of particles, minimal background noise, and reduced motion and charging effect.•Graphene can be functionalized by various strategies to modulate the manner of interaction with target macromolecules.•Graphene functionalized with affinity ligands exhibits high affinity and specificity in binding to target macromolecules.