Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Abstract

Structure determination by cryo-EM has rapidly grown in the last decade, however sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. Many samples are refractory to this, however, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films, including amorphous carbon, graphene, and graphene oxide, can be applied to the grid to provide a surface which sample can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. We have recently reported the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimises the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. In this manuscript we provide step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.