Cryo-EXLO Manipulation of FIB Specimens for Cryo-TEM

This work describes cryogenic ex situ lift out (cryo-EXLO) of cryogenic focused ion beam (cryo-FIB) thinned specimens for analysis by cryogenic transmission electron microscopy (cryo-TEM). The steps and apparatus necessary for cryo-EXLO are described. Methods designed to limit ice contamination include use of an anti-frost lid, a vacuum transfer assembly, and a cryostat. Cryo-EXLO is performed in a cryostat with the cryo-shuttle holder positioned in the cryogenic vapor phase above the surface of liquid N-2 (LN2) using an EXLO manipulation station installed inside a glove box maintained at < 10% relative humidity and inert (e.g., N-2 gas) conditions. Thermal modeling shows that a cryo-EXLO specimen will remain vitreous within its FIB trench indefinitely while LN2 is continuously supplied. Once the LN2 is cut off, modeling shows that the EXLO specimen will remain vitreous for over 4 min, allowing sufficient time for the cryo-transfer steps which take only seconds to perform. Cryo-EXLO was applied successfully to cryo-FIB-milled specimen preparation of a polymer sample and plunge-frozen yeast cells. Cryo-TEM of both the polymer and the yeast shows minimal ice contamination with the yeast specimen maintaining its vitreous phase, illustrating the potential of cryo-EXLO for cryo-FIB-TEM of beam-sensitive, liquid, or biological materials.

Automated summary

This work presents cryo-EXLO technology for pre-treatment of samples for cryo-TEM analysis. The steps and apparatus for cryo-EXLO are described, along with methods to limit ice contamination. The successful application of cryo-EXLO to polymer and plunge-frozen yeast cell specimens demonstrates its potential for beam-sensitive, liquid, or biological materials.