Fib quanta 3d feg

Two dual-beam FIB microscopes were used for this work: 1) a FIB Quanta 3D FEG (FEI) equipped with a Quorum PP3000T cryo-system (Quorum Technologies, Laughton, United Kingdom) and a homemade 360° rotatable cryo-stage cooled by an open nitrogen circuit (Rigort et al., 2010a) ; 2) a FIB Scios (FEI) equipped with a complete cryo-system prototyped by FEI, including a rotatable cryo-stage cooled by an open nitrogen circuit. Both systems utilize FEI Autogrid specimen cartridges modified for FIB preparation under shallow milling angles (Rigort et al., 2012) . Two plunge-frozen grids were clipped into Autogrids and mounted into a shuttle under LN 2 (Rigort et al., 2010b) . The shuttle was mounted into the FIB using the cryo-transfer system. Samples were typically first sputter-coated with Pt in the Quorum prep chamber (10 mA, 30 -60 s) to improve the overall conductivity of the sample. An organometallic Pt layer was then deposited inside the microscope chamber using the in situ gas injection system (GIS, FEI). Coating procedures are described in detail in sections 2.2 (pre-milling coating) and 2.4 (post-milling coating). During FIB operation, samples were kept at constant LN 2 temperature. Lamellas were produced using the gallium ion beam at 30 kV with stage tilt angles of 15°-20°, employing procedures described in detail in 2.3 Cryo-FIB milling.