BPS2026 – Cryo-vitrification of thick samples by turbo-plunging

Cryo-electron microscopy requires vitrifying samples in the frozen hydrated state. However, conventional cryo-plunging is limited to samples up to ∼10 μm in thickness, restricting access to many fully differentiated cell types. To address this limitation, we are developing enhanced cryo-plunging instruments designed to improve sample cooling rates and thereby increase vitrification depth. At the core of our approach is the computational observation that sample cooling is limited by an insulating layer of warmed up cryogen that remains attached to the sample during plunging. We hypothesize that disrupting this laminar layer by introducing turbulence during the plunging process would promote faster cooling and allow for deeper vitrification than what is currently possible. To this end, we are combining fluid dynamics simulations with advanced instrumentation in an iterative, synergistic workflow, where computational modeling informs design and experimental testing not only validates but also refines turbulence-enhanced cryo-plunger models—turbo-plungers. This work may pave the way toward cryo-plunging instruments that would enable deep and rapid cryo-vitrification at atmospheric pressure. Such instruments would facilitate cryo-imaging of differentiated cell types and complex multicellular systems inaccessible by current state-of-the-art methods.