An experimental platform for exploring dual-jet vitrification mechanisms in cryo-EM sample preparation

Abstract Jet vitrification harnesses the exceptionally high cooling potential of rapidly moving liquid cryogens to achieve ultrafast sample freezing with superior cooling efficiency. Building on earlier work, this study provides a comprehensive investigation into the physical and technical principles underlying dual-jet vitrification. Developing ultra-rapid freezing methods that reliably produce vitreous cells, supports thicker samples, and integrate seamlessly into cryo-electron tomography (cryo-ET) workflows do represent important steps forward in the field. Here, we introduce an experimental platform designed to study the dynamics and efficacy of liquid-ethane jet vitrification at −180 °C. The system integrates multiple modules enabling controlled examination of the vitrification process at microsecond timescales, with a focus on quantifying temperature transitions, identifying optimal freezing parameters, and delineating the limitations of dual-jet configurations. The compact design allows operation within a standard biosafety cabinet and supports various sample holders compatible with biological specimens. Furthermore, we demonstrate how this setup can be directly incorporated into existing cryo-ET workflows using EM grids pre-mounted in AutoGrids. Overall, this work establishes a versatile platform for dissecting the mechanisms of ethane jet vitrification and provides methodological insights that support the preparation of thicker, well-preserved biological samples for in situ structural analysis.