Figure 4∣. Cryo-stage setup.

a, Schematic of the cryo stage setup inside of cryo-FIB dual beam microscope. The cryo-stage has 5 degrees of freedom which are highlighted in its close-up view. b, The heat exchanger with the liquid N₂ dewar. The N₂ flow tubes are enclosed within hoses which are kept under vacuum to provide an insulating environment. c, 5 degrees of freedom of the cryo-stage. d, The eucentric height is the stage height at which the sample remains centered even when the stage is tilted. For a well-aligned microscope, the electron beam and FIB should be coincident at eucentric height. Therefore, milling should be performed at this height to enable the visualization of the evolving lamella using SEM imaging. Since the grids are held in the shuttle at 45° angles, when moving stage to other areas on EM grids, stage Z height must be changed to bring the areas of interest to be Eucentric height.