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-par ticle cryoEM. 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.

Automated summary

This article primarily discusses the challenge of interacting with the air-water interface (AWI) in cryo-EM and reviews current approaches to avoid it. Immobilizing particles on affinity grids is considered the most promising method. Efforts to gain better control over sample thicknesses are also reviewed, with the important goal of preventing immobilized particles from contacting the AWI of the remaining buffer. It is emphasized that avoiding such contact is crucial for both cryo-ET and single-particle cryoEM. Looking ahead, the use of immobilized samples for time-resolved biochemical experiments directly on EM grids is proposed.