A novel cryo-FIB lift-out procedure for cryo-TEM sample preparation S. Rubino 1 , S Akhtar 1 , P. Melin 2 , A. Searle 3 , P. Spellward 3 and K. Leifer 1 1. Department of Engineering Sciences, Uppsala University, Box 534, Uppsala, Sweden 2. Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, Uppsala, Sweden 3. Gatan Inc. 25 Nuffield Way, Abingdon Oxon, OX14 1RL, United Kingdom email.stefano.rubino@angstrom.uu.se Keywords: cryogenics, FIB The focused-ion-beam (FIB) is the method of choice for site-specific sample preparation for Transmission Electron Microscopy (TEM) in material sciences. A lamella can be physically lifted out from a specific region of a bulk specimen with submicrometer precision and thinned to electron transparency for high-resolution imaging in the TEM. The possibility to use this tool in life sciences applications has been limited by the lack of lift-out capabilities at the cryogenic temperatures often needed for biological samples. Conventional cryo-TEM sample preparation is mostly based on ultramicrotomy, a procedure that is not site-specific and known to produce artifacts. Here we demonstrate how a cooled nanomanipulator and a custom-built transfer station can be used to achieve cryo-preparation of TEM samples with the FIB, enabling high-resolution investigation of frozen-hydrated specimens in the TEM. FIB milling of frozen biological specimens has been reported, but only in two instances in connection with a transfer to a TEM for further higher resolution studies [1,2]. In both cases the entire sample (rather than just the thin area of interest) was transferred and traditional lift-out had been deemed impossible because of the difficulty in cooling down the nanomanipulator and in obtaining Pt deposition at cryogenic temperatures. Cryogenic Pt deposition has been recently demonstrated as a method of improving the quality of FIB cross-sections of frozen-hydrated samples [3]. We were able to obtain a cryogenic lift-out by modifying the nanomanipulator so that it could be cooled. We demonstrate the technique on Aspergillus niger spores stained with osmium tetroxide and potassium permanganate. A cryopreparation system (Gatan Alto 2500) was modified for this purpose. In this system, the tip of the manipulator can be kept at about 100 K. With this modification, common FIB/TEM sample preparation procedures can be adapted in a straightforward manner to frozen hydrated samples, with the entire preparation carried out at cryogenic temperatures (fig. 1). A cryo-transfer station has been developed to transfer the thinned lamella from the FIB/SEM to a cryo-TEM holder. Once in the TEM, high resolution images can be obtained at cryogenic temperatures with atomic resolution. It is also possible to perform elemental analysis (fig. 2) [4]. References [1] M Marko et al, Nat Methods 4 (2007), p. 215. [2] MF Hayles et al, J Struct Biol 172 (2010), p. 180. [3] MF Hayles et al, J Microsc 226 (2007), p. 263. [4] We thank Anna Otterström, Håkan Vikstrom, Marilyn Carey, Geoffrey Daniels, Fredric Ericson and Jan-Åke Gustafsson for technical support. We also thank the Knut and Alice Wallenberg foundation and the Higher Education Commission (HEC) of Pakistan for financial support.