Fig. 1: Overview of xenon plasma FIB milling workflow for high-pressure frozen samples.

a Orientation of the grid relative to the PFIB and scanning electron microscope (SEM) during ice contamination removal using xenon beam on an Arctis PFIB/SEM from the front (left) and back (right) of the grid. Bottom: schematic orientation of a grid square and the angle and currents used to remove the ice contamination. b SEM grid overview before ice contamination removal. Scalebar: 500 µm. n = 3 whole grids. c PFIB images before (left) and after (right) ice contamination removal by PFIB imaging. Scalebar: 100 µm. d Schematic overview of milling pattern placement (yellow striped rectangles) during the four trench milling steps. e SEM overview of the grid shown in b, after ice contamination removal, GIS deposition and trench milling steps (example milled sites marked with white arrows). Using a low magnification ion beam, all trenches are milled at the same time. Typically, more trenches are prepared than required, and sites with high front surface roughness are excluded from automated milling. Scalebar: 500 µm. f PFIB image of pattern placement for the 4th trench milling step. Milling patterns (yellow striped rectangles) are concurrently placed and milled in in horizontal rows, as tilting the grid varies the distance to the PFIB, resulting in a vertical focus gradient. Scalebar: 200 µm. g (top) Schematic overview of the grid orientation during automated milling relative to the PFIB and SEM beam. (bottom) Schematic overview of milling pattern placement relative to the forming lamella (black dashed line) during automated lamella preparation. h–s PFIB (top row) and SEM (bottom row) images of a lamella after the milling step indicated at the top. After the 4th trench milling step (h, n), automated lamella preparation for rough, medium and fine milling (i–k and o–q) at 4 nA, 1 nA and 0.3 nA respectively. After manually preparing notches at 0.1 nA (white arrow and bottom left of l) for stress-relief (I, r), two automated milling steps are performed at 30 pA (not shown) and 10 pA (m, s). Scalebars: 10 µm. n = 52 lamellae.