The SerialFIB software (Klumpe et al., 2021 (link)) was run on the microscope via the AutoScript 4 (Thermo Fisher Scientific) interface. To extend SerialFIB to plasma ion sources, the template pattern files that follow the Thermo Fisher standard were adjusted to a pFIB equivalent to allow reading and writing of patterns. Parameters that changed in those files between a system utilising gallium, that is, an Aquilos 2 dual-beam FIB/SEM (Thermo Fisher Scientific), to the system used in the current study utilising plasma-based milling were: total diameter, volume per dose, total beam area, sputter rates, and depth per pass. One crucial parameter that had to be adjusted when switch ion sources was the milling pattern pitch, as the pitch in plasma-based ion sources needs to be significantly higher than when milling with gallium-based liquid metal ion sources, likely due to the decrease in focussing capability of the pFIB. The pitch values were 9.5 nm for gallium and 116 nm for nitrogen and argon-based milling. Furthermore, the beam shift limits for the ion and electron column had to be adjusted to the limits on the Helios Hydra system.
The imaging script that allows for definition of a milling and another imaging position to allow for stage movements between the two steps in the volume imaging protocol was developed inside SerialFIB’s scripting interface. In brief, the function allowing for serial FIB/SEM volume imaging runs was modified to take one-stage position for slicing and one-stage position for imaging into account. While stage movements normally lead to adjustment to the SEM focus according to linked stage height, the focus was enforced to a user-defined or via autofocussed determined value to remove the necessity of autofocussing after every stage movement. In addition, the horizontal field width was locked to avoid slight changes when readjusting focus after stage movements to avoid changes in the pixel size.
The script and all code for operating the microscope utilised here is available on the SerialFIB GitHubhttps://github.com/sklumpe/SerialFIB/ (copy archived at Dumoux, 2023 ).
The imaging script that allows for definition of a milling and another imaging position to allow for stage movements between the two steps in the volume imaging protocol was developed inside SerialFIB’s scripting interface. In brief, the function allowing for serial FIB/SEM volume imaging runs was modified to take one-stage position for slicing and one-stage position for imaging into account. While stage movements normally lead to adjustment to the SEM focus according to linked stage height, the focus was enforced to a user-defined or via autofocussed determined value to remove the necessity of autofocussing after every stage movement. In addition, the horizontal field width was locked to avoid slight changes when readjusting focus after stage movements to avoid changes in the pixel size.
The script and all code for operating the microscope utilised here is available on the SerialFIB GitHub
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