A simplified high-pressure freezing workflow in Autogrids

Cryo-electron tomography (cryo-ET) enables structural analysis of biological samples in near-native states, yet vitrification of thick and complex specimens remains challenging. High-pressure freezing (HPF) can vitrify up to ∼200 μm thick specimens, but its seamless integration into cryo-electron microscopy (cryo-EM) workflows is hindered by planchettes that are not optimized for EM grids, resulting in extensive handling, reliance on user experience, and frequent grid damage. Here, we present a streamlined HPF workflow based on newly designed planchettes that directly accommodate clipped EM Autogrids. Interchangeable lids control sample thickness and support vitrification of diverse specimens, including single-celled eukaryotes and tissue biopsies, while minimizing post-freezing handling. We demonstrate that these planchettes enable reliable preservation of cellular ultrastructure and macromolecular integrity, as assessed by cryo-ET and subtomogram averaging. This resource provides a practical solution for integrating HPF into modern cryo-ET pipelines, expanding access to structurally complex specimens and supporting future clinical applications.