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Forskningsartikel2014Vetenskapligt granskadÖppen tillgång

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Rubino, Stefano; Melin, Petter; Spellward, Paul; Leifer, Klaus

Sammanfattning

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.

Nyckelord

Bioengineering; Issue 89; Cryoelectron Microscopy; Life Sciences (General); Cryo-microscopy; Focused ion beam; Sample preparation; TEM; FIB

Publicerad i

Journal of Visualized Experiments
2014, nummer: 89
Utgivare: JOURNAL OF VISUALIZED EXPERIMENTS