A streamlined workflow for automated cryo focused ion beam milling

Cryo-electron tomography is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ. Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions. This procedure is prone to frost and amorphous ice depositions which makes it a tedious process, leading to suboptimal results especially when larger batches are milled. Here, we present new hardware that overcomes the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This tremendously reduces the ice depositions during transfer and milling, and simplifies the handling of the sample. In addition, we tested a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling.