High-precision thickness control of ice layer on CVD grown bilayer graphene for cryo-TEM

A novel grid for cryogenic transmission electron microscopy (cryo-TEM) was developed using bilayer graphene to facilitate the formation of a thin ice layer over grid holes. To prepare samples, the bilayer graphene adhered on a holey carbon grid was treated by UV/ozone from the top, making the upper graphene layer hydrophilic, while maintaining the high conductivity via the intact lower layer. Virus solution was loaded on the hydrophilized-graphene surface of the grid, and a thin layer was successfully formed and frozen using an automated blotting system. Inspection in a monochromated cryo-TEM at low-acceleration voltage of 60 kV, provided image of viruses embedded in a very thin vitreous ice layer on bilayer graphene surface, at a high signal-to-noise ratio without charging drifts. The vitreous ice layer formed on the hydrophilized-bilayer graphene surface was homogeneously thin and reached as thin as 25 nm, as confirmed using scanning TEM with electron energy loss spectroscopy. Overall, the high hydrophilicity and homogeneous surface free energy of the UV/ozone-treated bilayer graphene is demonstrated to be highly suitable for thin ice layer preparation, and imaging using cryo-TEM, and especially for modern high-performance low-acceleration voltage TEM, which has higher contrast, but demand thinner ice layers on the grid.