Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7-August 11, 201

de Winter, M.

Hayles, M.

Humbel, B.

Luecken, U.

Meeldijk, J.

Persoon, H.

Schneijdenberg, C.

Verkleij, A.

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Cryo-FIB Machining: An Alternative to TEM Cryo-Sections Cut with Diamonds?Michael F. Hayles1, D. A. Matthijs de Winter2, Chris T. W. M. Schneijdenberg2, Johannes D. Meeldijk3, Hans Persoon1, Uwe Luecken1, Arie J. Verkleij2, Bruno M. Humbel2,41FEI Company, Achtseweg Noord 5, 5600 KA Eindhoven, The Netherlands 2Biomolecular Imaging, Institute of Biomembranes, Faculty of Sciences, Utrecht University, NL-3584 CH Utrecht, The Netherlands 3Inorganic Chemistry and Catalysis Group, Debye Institute for Nanomaterials Science, Faculty of Science, Utrecht University, NL-3584 CA Utrecht, The Netherlands 4Present address: Centre for Electron Microscopy, Faculty of Biology and Medicine, Lausanne University, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland Since the beginning of electron microscopy, sample preparation has been a matter of debate. In an electron micrograph every single detail down to about a few nanometers is visible; therefore the electron microscopists are constantly concerned to preserve biological matter as close as possible to nature. Today’s consent is that a physical arrest by cryo-fixation is the only method to preserve the living in its actual physiological state. Furthermore also the observation should be done in ice, as water is the natural solvent of life. During dehydration macromolecules may collapse [1] and the heavy metal stains tend to bind better to certain molecular sites, depicting more their distribution than the actual structure of the macromolecules. In structural biology this concept has become a standard since a long time [2]. For cell or tissue biology the implementation is more difficult because the sample has to be thin in order for the electron beam to pass through. Further, small objects like proteins can easily be vitrified in a thin film of water but cells and pieces of tissue require more advanced techniques. Therefore high-pressure freezing has become the method of choice [3, 4]. Depending on the natural antifreeze within the tissue, slices of 100 µm up to a 500 µm can be preserved. Subsequently, sections have to be prepared that allow TEM imaging. The group of Jacques Dubochet [5] has improved cryo-sectioning of vitrified biological samples and coined the term CEMOVIS, cryo-electron microscopy of vitreous sections. Every advantage has a disadvantage (after the famous Dutch soccer player Johan Cruijff): CEMOVIS allows the observation of cells vitrified in their physiological surrounding without the influence of chemicals, but the sectioning process induces physical damage like knife marks, compression and crevasses [6] that become worse with increasing thickness of the sections. For high resolution in situ analysis of molecular complexes the method is very valuable [7]. For cellular electron tomography, however, thicker cryo sections, up to 500 nm, are desired. To overcome the limitations of sectioning, Mike Marko and his group introduced a thinning technique using a focused ion beam (FIB). Cells were frozen on an electron microscopy grid, then the ice was thinned in the ion beam to allow cryo-electron tomography [8, 9]. A new approach was described by Rigort et al. [10]. They have established a correlative chain from imaging fluorescent cells grown on EM-grids, followed by coarse thinning in a cryo-ultramicrotome and final polishing with the FIB. We set out to make connectivity from samples cryo-fixed by high-pressure freezing until imaging in a cryo-TEM [11]. The heart is a newly developed cryo-stage for the FIB-SEM (Nova Nanolab 600, FEI Company), a cryo-nanolathe called CryoNanoBench. Samples are cryo-fixed in a copper tube by a high-pressure freezer (EMPact, Leica Microsystems), then the copper is trimmed off in a cryo-ultramicrotome. Thereafter, the sample is mounted in a specially designed ferrule, cryo-transferred into the CryoNanoBench. In the FIB-SEM a thin lamella is cut at the tip of the sample. Back through the newly developed transfer chain, including the Quorum cryo-transfer system (Quorum Technologies) the sample is loaded into a modified cryo-holder (Gatan, Inc) and transferred into the cryo-TEM for observation. In this presentation we will explain the setup in detail. 644doi:10.1017/S1431927611004090Microsc. Microanal. 17 (Suppl 2), 2011© Microscopy Society of America 2011https://doi.org/10.1017/S1431927611004090Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 11 Jul 2017 at 16:22:01, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.Yeast cells were cryo-fixed in a copper tube by high-pressure freezing and prepared according to the description above. The SE image on the left hand side depicts are row of thinned lamella. On the right hand side a TEM image of the frozen-hydrated yeast cells is shown. References1. Kellenberger, E., et al., Artefacts and morphological changes during chemical fixation. J. Microsc., 1992. 168(2): p. 181-201. 2. Dubochet, J., et al., Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys., 1988. 21: p. 129-228. 3. Müller, M. and H. Moor, Cryofixation of thick specimens by high pressure freezing, in Science of Biological Specimen Preparation 1983, J.P. Revel, T. Barnard, and G.H. Haggis, Editors. 1984, SEM Inc.: AMF O'Hare. p. 131-138. 4. Riehle, U., Über die Vitrifizierung verdünnter wässriger Lösungen. ETH Diss Nr 4271. 1968, Zürich: Federal Institute of Technology (ETH). 5. Al-Amoudi, A., et al., Cryo-electron microscopy of vitreous sections. EMBO J., 2004. 23(18): p. 3583-3588. 6. Al-Amoudi, A., D. Studer, and J. Dubochet, Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy. J. Struct. Biol., 2005. 150(1): p. 109-121. 7. Al-Amoudi, A., et al., The molecular architecture of cadherins in native epidermal desmosomes. Nature, 2007. 450: p. 832-837. 8. Hsieh, C.-E., et al., Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections. J. Struct. Biol., 2006. 153(1): p. 1-13. 9. Hsieh, C.-E., et al., Electron tomographic analysis of frozen-hydrated tissue sections. J. Struct. Biol., 2002. 138: p. 63-73. 10. Rigort, A., et al., Micromachining tools and correlative approaches for cellular cryo-electron tomography. J. Struct. Biol., 2010. 172(2): p. 169-179. 11. Hayles, M.F., et al., The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy. J. Struct. Biol., 2010. 172(2): p. 180-190. 12. We would like to acknowledge NWO Groot, FEI Company and Utrecht University for the generous funding of this research and Mrs. E van Donselaar for helping with HPF freezing.Microsc. Microanal. 17 (Suppl 2), 2011 645https://doi.org/10.1017/S1431927611004090Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 11 Jul 2017 at 16:22:01, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.

Cryo-FIB Machining: An Alternative to TEM Cryo-Sections Cut with Diamonds?

Cryo-FIB Machining: An Alternative to TEM Cryo-Sections Cut with Diamonds?

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