A simplified High-Pressure Freezing Workflow in Autogrids

Abstract Cryo-electron tomography (cryo-ET) is a powerful method for studying biological structures in near-native states, yet vitrification of thick and complex specimens remains a major limitation. High-pressure freezing (HPF) allows vitrification of up to ∼200 µm thick samples, 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. Combined with interchangeable lids for controlling sample thickness, this system enables robust vitrification of diverse specimens, including single-celled eukaryotes and tissue biopsies, while minimizing post-freezing handling. Integration with plasma-focused ion beam milling supports both on-grid lamella preparation and lift-out approaches. We demonstrate reliable preservation of cellular ultrastructure and macromolecular integrity by cryo-ET and subtomogram averaging. This resource provides a practical and scalable solution for integrating HPF into modern cryo-ET pipelines, expanding access to structurally complex biological systems and supporting future clinical applications.