Thinning of large mammalian cells for cryo-TEM characterization by cryo-FIB milling.
Focused ion beam milling at cryogenic temperatures (cryo-FIB) is a valuable tool that can be used to thin vitreous biological specimens for subsequent imaging and analysis by cryo-transmission electron microscopy (cryo-TEM) in a frozen-hydrated state. This technique offers the potential benefit of eliminating the mechanical artefacts that are typically found with cryo-ultramicrotomy. However, due to the additional complexity in transferring samples in and out of the FIB, contamination and devitrification of the amorphous ice is commonly encountered. To address these problems, we have designed a sample cryo-shuttle that directly and specifically accepts Polara TEM cartridges to simplify the transfer process between FIB and TEM. We optimized several parameters in the cryo-FIB and cryo-TEM processes using the quality of the samples' ice as an indicator and demonstrated high-quality milling with large mammalian cells. By comparing the results from HeLa cells to those from Escherichia coli cells, we discuss some of the artefacts and challenges we have encountered using this technique.
Department of Mechanical Engineering and Materials Science, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
K M
KM
Strunk
K
K
Wang
D
D
Ke
J L
JL
Gray
P
P
Zhang
D000818:Animals; D016477:Artifacts; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods; D016616:Cryoultramicrotomy / Q000379:methods; D004926:Escherichia coli / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D008322:Mammals; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation / Q000379:methods*; D013048:Specimen Handling / Q000379:methods