No abstract

No abstract

No abstract

No abstract

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). 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 N2 (LN2) using an EXLO manipulation station installed inside a glove box maintained at < 10% relative humidity and inert (e.g., N2 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-EM of beam-sensitive, liquid, or biological materials.

No abstract

No abstract

Cryogenic focused ion beam (cryo-FIB) specimen preparation has been successfully used for cryogenic transmission electron microscopy (cryo-TEM) in both the life sciences and physical sciences [1][2][3][4][5][6][7][8][9][10][11]. The use of cryo-FIB in situ lift out (cryo-INLO) has become the norm despite its complex procedures and often poor reproducibility [11]. Conversely, it is well known that ambient ex situ lift out (EXLO) is fast, easy, reproducible, and capable of successful manipulations of > 20 specimens per hour [12]. Thus, we extended EXLO methods to cryo-FIB specimens and developed cryo-EXLO techniques and instrumentation for cryo-TEM.Cryo-EXLO was applied to cryo-FIB milled plunge-frozen yeast specimen. Conventional EXLO FIB milling procedures were used and a cryo-holder containing the FIB specimen was transferred to an EXLO station under controlled humidity and environmental conditions. After cryo-EXLO manipulation, the specimen was transferred to a cryo-TEM for analysis. Cryo-TEM results show minimal ice contamination while retaining the vitreous phase during the manipulation and transfer steps. In addition, thermal heat transfer modeling of the cryo-FIB process is consistent with, and supports, our cryo-EXLO experimental methods and results [13].