Practical Electron Microscopy and Database

Focused ion beam (FIB) systems have recently been applied in cryogenic environments in the temperature range of 83 to 123 K. For instance, it becomes a potential alternative to cryomicrotome for sectioning frozen biological samples to explore the engineering issues and is a possible tool to pattern organic materials in submicron scale. In this technique, ion milling of water ice should be taken into account. For a beam with 30 keV Ga ion at 80° incident angle in cryo-FIB, the sputtering yield on a water (H₂O) ice surface is about 18 molecules/ion.

Comparing with cryo-ultra microtome technique, the viewing area of lamella created with cryo-FIB technique is much smaller, but there are still some advantages with cryo-FIB technique as follows:
        i) Absence of artifacts caused by cryo-sectioning such as no crevasses and no compression. [1]
        ii) More uniform thickness. [2]
        iii) Localization of specific site. [2]

An example of cryo-FIB-SEM systems is that a Nova Nanolab 600 Dualbeam microscope (FEI Company, Eindhoven, The Netherlands) was equipped with a PP2000T cryo transfer system with a CHE2000 12 l Dewar as well as an Advanced Transfer Unit (ATU) (Quorum Technologies Ltd., Ringmer, UK) [3]. The cryo-temperature was achived with LN2 (liquid nitrogen). For ion milling, a flipper supporting the sample can be tilted to 38 degrees. To identify the crystalline phase, EBSD (Transmission-Electron BackScatter Diffraction) was used. In addition, to protect the sample from contamination, a cold trap at -175 °C was used to collect any water vapor that came within the vicinity of the sample.

Figure 2456. Sample stage in a cryo-FIB-SEM system. Adapted from [3]

[1] Al-Amoudi, A., Studer, D., Dubochet, J., 2005. Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy. J. Struct. Biol. 150 (1), 109–121.
[2] Jiménez, N., Van Donselaar, E.G., De Winter, D.A., Vocking, K., Verkleij, A.J., Post, J.A., 2010. Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers, by TEM and FIB–SEM. J. Microsc. 237 (2), 208–220.
[3] D. A. Matthijs de Winter, Rob J. Mesman, Michael F. Hayles, Chris T.W.M. Schneijdenberg, Cliff Mathisen, Jan A. Post, In-situ integrity control of frozen-hydrated, vitreous lamellas prepared by the cryo-focused ion beam-scanning electron microscope, Journal of Structural Biology 183 (2013) 11–18.