Figure 1. Schematic of the automated sequential cryoFIB milling workflow.

(A) FIB currents are aligned and calibrated, and the sample is loaded into the FIB-SEM instrument. (B) A target cell is identified on the grid with the SEM and FIB. (C) To correct for errors in mechanical stage movements, backlash correction of the stage is performed. The resulting stage location is saved in the stage navigator. (D) The stage is randomly moved out of position by the user. Using the saved coordinates in the stage navigator, the stage is autonomously moved back to the target. (E) The accuracy of this autonomous stage movement is determined by the user. If the target is not centered in the FIB image, backlash correction is repeated until accurate targeting is achieved (C–E). (F/F’) Rough milling, polishing and drift correction patterns are placed onto the image. Rough milling and polishing patterns are saved separately to the queue. The procedure (B–F’) is repeated to select additional targets. (G/H) Rough milling and lamellae polishing are executed automatically. (I) The grids with milled lamellae are removed and stored.

Figure 1—figure supplement 1. Schematic of the manual cryoFIB milling workflow.

(A) The sample is loaded into the FIB-SEM instrument. (B) A target is centered in the FIB image. (C–E ) The first pair of rough milling patterns is placed on the target and milling is executed (C). Lamellae milling is observed via a live FIB view to determine when a milling step is completed. The same procedure is repeated for the second (D) and third (E) rough milling patterns. After rough milling of the target is completed, additional targets can be milled by repeating steps (B–E). (F) Rough-milled lamellae are polished with a fourth set of milling patterns. Polishing is repeated for all rough-milled lamellae. (G) The grids with milled lamellae are removed and stored.