A streamlined workflow for automated cryo focused ion beam milling

TLDR

Cryo-electron tomography requires thinning of thick biological specimens by cryogenic focused ion beam milling, a process that is prone to contamination and often yields suboptimal results. The study introduces new hardware designed to overcome current limitations in cryo‑FIB milling. The authors developed a glove box with high‑vacuum cryo‑transfer, a stage heater, cryo‑shield, cryo‑shutter, and an automated software application to streamline cryo‑FIB milling. These improvements reduce ice contamination, simplify sample handling, enable high‑quality high‑throughput cryo‑FIB milling, and open possibilities for previously infeasible experiments.

Abstract

Cryo-electron tomography (cryo-ET) 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 (cryo-FIB). This procedure is prone to contaminations, which makes it a tedious process, often leading to suboptimal results. 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 reduces the ice contamination during the transfer and milling process 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. This paves the way for new types of experiments, which have been previously considered infeasible.