SA-FIB: A Semi-automatic Program for Rapid Positioning in Cryo-Focused Ion Beam Milling

Methods

SA-FIB was developed on the Thermo Fisher Scientific Aquilos 2 cryo dual-beam SEM platform using the Autoscript scripting package, and consists of three functional modules: coincidence point adjustment with milling-site recording, a milling assistant that supports both automated and manual operation, and a map module for on-site annotation. Coincidence point adjustment is completed semi-automatically, with no requirement for stage tilting.

When the stage tilt and rotation angles remain unchanged, the working distances of the electron beam (EB) and ion beam (IB), as well as the stage tilt angle, can be saved as a reusable reference. Next, the target is centered and focused in the IB field of view for coarse correction via the "add point" function, then moved to the center of the EB field of view for fine correction through the "E correct" function, which requires at most two rounds of correction.

We used Saccharomyces cerevisiae and mouse sperm as test specimens. After sputter coating and gas-injection coating, lamellae were milled via a gradient beam-current process (30 kV; 0.5 nA to 50 pA), thinning from an initial size of approximately 13 μm × 2 μm to a final thickness of less than 200 nm. We also measured the positioning errors and grid-loading errors of the two microscopes. Results Adjusting and recording a single coincidence site takes only 15 to 20 s, and all sites are automatically labeled on the low-magnification overview map of the grid.

In semi-automatic mode, one site is automatically thinned to approximately 500 nm within 10 to 15 minutes, and then manually thinned to below 200 nm; after the lamella is transferred to the TEM, the map allows the target lamella to be located within a few minutes. In fully automatic mode, since the coincidence point has already been adjusted, the three preparation steps of AutoTEM Cryo (Eucentric tilt, Milling angle and Image acquisition) can be skipped, saving approximately three  minutes per site.

The positioning errors of Aquilos 2 and Titan Krios G3i are about one micrometer and one hundred nanometers respectively, while the repeated grid-loading errors are 12 to 26 micrometers for the SEM and 60 to 141 micrometers for the TEM. Since the commonly used 200-mesh grids have a mesh size of approximately 100 micrometers, the largest TEM loading error exceeds this 100-micrometer criterion and makes coordinate-based targeting unfeasible; accordingly, SA-FIB only provides a relative-position map.

Conclusion SA-FIB enables rapid coincidence point adjustment and milling-site annotation without stage tilting, simplifies the cryo-FIB milling workflow, facilitates lamella localization after sample transfer, and can be used either as a stand-alone aid for manual milling or in combination with fully automatic packages such as AutoTEM Cryo. It provides a convenient solution for cryo in situ structural analysis.