Optimization of critical parameters for cryo-focused ion beam milling

Although cryo-electron microscopy (cryo-EM) is widely used to determine the three-dimensional structures of isolated and purified biomacromolecules, high-resolution structural studies of cells and tissues in situ still require the preparation of high-quality ultrathin sections. Focused ion beam (FIB) milling has emerged as a key technique for producing ultrathin cryo-lamellae from in situ samples because of its minimal artifacts and precise targeting capability. This study systematically investigates the critical factors influencing the quality of cryo-lamellae prepared by FIB milling, including sample vitrification quality, accurate temperature control of the cryo-system, and ice deposition and contamination.

We studied the entire process from sample preparation to lamella assessment using an Aquilos 2 cryo-FIB/SEM microscope. Diverse biological samples—yeast cells, 293T cells, and isolated muscle fibers—were prepared. Cells were vitrified by plunge freezing, and muscle fibers were pretreated with glycerol before vitrification in an ethane/propane mixture for homogeneous vitrification. A systematic FIB milling protocol was established: initial coarse milling (1–3 nA beam current) to create trenches and stress relief cuts, followed by sequential thinning with a beam current reduced stepwise from 1 nA to 50 pA, and final polishing at beam currents as low as 10 pA to produce 100–200 nm lamellae. With this protocol, key operating parameters were meticulously optimized based on experimental results: (1) Sample vitrification quality was first assessed during FIB–SEM milling and then correlated with cryo-ET outcomes to identify failure signatures. (2) A temperature sensor was directly instrumented on the shuttle to measure the true thermal conditions at the sample, guiding the optimal waiting period before loading according to the cooling kinetics profile. (3) The ice deposition inside the microscope chamber was evaluated by imaging the lamella at regular intervals after coarse milling. Rapid ice accumulation nearly doubled lamella thickness within 1.5 h and caused edge curling, both of which degraded sample quality. (4) On the basis of a study of ice contamination mechanisms, a custom integrated loading device with an anti-contamination lid was designed to reduce ice formation during the sample package and transfer procedure and fully tested against standard methods.

The primary challenge in cryo-sample preparation stems from the inherent limitations of plunge freezing. Effective vitrification requires a coordinated strategy of sample pretreatment and freezing-medium optimization, whereas for larger biological specimens, high-pressure freezing is essential to achieve uniform vitrification. The system temperature was consistently underreported relative to the actual grid temperature, necessitating direct temperature calibration. A major finding was the detrimental impact of rapid ice deposition on lamella integrity. Enhancing the chamber vacuum via system upgrades effectively mitigated this issue, maintaining stable lamella thickness throughout extended chamber sessions. An integrated loading device was developed to reduce ice contamination during packaging and transfer. Additionally, key technical parameters—platinum coating uniformity, ion beam milling settings, and machining precision—were identified as critical for producing high-quality lamellae.

By integrating systematic analysis with experimental data, we demonstrate that complete vitrification is a prerequisite for successful milling, reveal the critical discrepancy between the displayed system temperature and the actual sample temperature and its practical implications, and confirm that enhancing chamber vacuum is vital for controlling ice deposition, thereby providing an effective solution to reduce ice contamination. This work presents a reliable workflow and optimized strategies, offering concrete guidance to improve the robustness and reproducibility of FIB-based thinning for in situ structural biology.