Challenges in making ideal cryo-EM samples

Recognizing that interaction with the air-water interface (AWI) is a major challenge for cryo-EM, we first review current approaches designed to avoid it. Of these, immobilizing particles on affinity grids is arguably the most promising. In addition, we review efforts to gain more reliable control of the sample thicknesses, not the least important reason being to prevent immobilized particles from coming in contact with the AWI of the remaining buffer. It is emphasized that avoiding such a contact is as important for cryo-ET as for single-particle cryo-EM. Finally, looking to the future, it is proposed that immobilized samples might be used to perform time-resolved biochemical experiments directly on EM grids rather than just in test tubes or cuvettes.