Control of ice thickness in cryo-EM via confinement

Achieving thin ice with optimal thickness is a prerequisite for successful cryo-EM analysis of embedded macromolecules. Despite decades of effort, precise control of ice thickness in cryo-EM remains a major challenge. Here we reveal that the intrinsic instability of sub-100-nm liquid film, a previously overlooked determinant before vitrification, plays a critical role in controlling ice thickness. We found that well-designed graphene reservoirs can confine liquid film to an optimal thickness and overcome the limitations posed by liquid film instability, achieving precise control of ice thickness with robust reproducibility and large-area uniformity. These graphene reservoirs demonstrate superior liquid retention capabilities than conventional techniques, enabling tunable ice thickness through reservoir depth modulation. Moreover, this approach yields uniform confined ice in graphene sandwiches with controllable thickness and high efficiency, facilitating cryo-EM imaging of a broad range of macromolecule particles with high contrast, reduced motion and improved orientations for more robust high-resolution reconstruction. This study explores the use of graphene reservoirs formed over perforated supporting films as a method to precisely control ice thickness during cryo-EM sample preparation.