Automated LN₂ refill device for uninterrupted cryoFIB-SEM operations.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN₂ to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN₂ refill system that is compatible with gas-cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling and might be particularly beneficial in BSL-3 or BSL-4 laboratories where minimizing physical presence is essential for biosafety reasons.