A novel cryo-FIB lift-out procedure for cryo-TEM sample preparation

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].