Abstract: Herein are innovations that enable facile cryo-EM analysis of diverse samples. Methods of functionalizing sample grids for cryo-EM are described, including methods of creating high quality graphene oxide films on cryo-EM substrates. The cryo-EM sample substrates are functionalized with affinity molecules that efficiently concentrate sample molecules and other specimen types on the grid, away from the air-water interface. Affinity groups include amines and proteins such as tagging system proteins and peptides that can be used to capture diverse sample types with high affinity. Optionally, spacers such as PEG chains are used to place sample particles away from the substrate surface, reducing substrate-induced artifacts.