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Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.ORCID iD: 0000-0001-5738-0591
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
2014 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 89, e51463Article in journal (Refereed) Published
Abstract [en]

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Place, publisher, year, edition, pages
2014. no 89, e51463
Keyword [en]
Bioengineering, Issue 89, Cryoelectron Microscopy, Life Sciences (General), Cryo-microscopy, Focused ion beam, Sample preparation, TEM, FIB
National Category
Other Engineering and Technologies
Identifiers
URN: urn:nbn:se:uu:diva-247562DOI: 10.3791/51463ISI: 000349296100029PubMedID: 25146386OAI: oai:DiVA.org:uu-247562DiVA: diva2:796870
Available from: 2015-03-20 Created: 2015-03-20 Last updated: 2017-12-04Bibliographically approved

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Rubino, StefanoLeifer, Klaus

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