Cryo-EXLO Manipulation of FIB Specimens for Cryo-TEM

Abstract This work describes cryogenic ex situ lift out (cryo-EXLO) of focused ion beam (cryo-FIB) thinned specimens for analysis by transmission electron microscopy (cryo-TEM). The steps and apparatus necessary cryo-EXLO are described. Methods designed to limit ice contamination include use an anti-frost lid, a vacuum transfer assembly, cryostat. Cryo-EXLO is performed in cryostat with the cryo-shuttle holder positioned vapor phase above surface liquid N2 (LN2) using EXLO manipulation station installed inside glove box maintained at < 10% relative humidity inert (e.g., gas) conditions. Thermal modeling shows that specimen will remain vitreous within its FIB trench indefinitely while LN2 continuously supplied. Once cut off, over 4 min, allowing sufficient time cryo-transfer which take only seconds perform. was applied successfully cryo-FIB-milled preparation polymer sample plunge-frozen yeast cells. Cryo-TEM both minimal maintaining phase, illustrating potential cryo-FIB-TEM beam-sensitive, liquid, or biological materials.