In-Chamber Sublimation: A Practical Approach for Mitigating Ice and Curtaining in Cryo-Electron Tomography Lamellae Preparation

Abstract Surface ice contamination is a persistent challenge in cryo-electron tomography (cryo-ET) workflows, where it can obscure regions of interest and contribute to curtaining artefacts during focused ion beam (FIB) milling. We demonstrate using high-pressure frozen yeast cells that a sublimation step within the scanning electron microscope (SEM) chamber before lamella milling visually removes surface ice and reduces sample roughness without detectable devitrification. While sublimation has been widely applied in cryo-SEM and volume imaging, it is not common on cryo-ET samples due to concerns about devitrification. Using tomographic reconstructions, we show that controlled sublimation improves lamella quality by reducing surface roughness and minimizing curtaining without compromising sample vitrification. Furthermore, subtomogram averaging of the 80S ribosome confirmed lamellae quality are preserved after sublimation. This approach offers a practical refinement to existing cryo-ET preparation protocols, requiring no additional instrumentation or workflow modifications.