Cryo-EXLO for Cryo-TEM of FIB Specimens

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].