TY - JOUR
T1 - 3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography
AU - Wang, Ke
AU - Strunk, Korrinn
AU - Zhao, Gongpu
AU - Gray, Jennifer L.
AU - Zhang, Peijun
N1 - Funding Information:
The authors thank Trevor Clark at the Materials Research Institute, Pennsylvania State University for help with cryo-FIB operation, Travis Wheeler and the machine shop at the Department of Cell Biology and Physiology for construction of the cryo-FIB shuttle, Drs. Sangmi Jun and XinMeng for help with 3D tomographic reconstruction, and Dr. Teresa Brosenitsch for critical reading of the manuscript. This work was supported by the National Institutes of Health ( RR024424 and GM085043 ).
PY - 2012/11
Y1 - 2012/11
N2 - Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB " lift-out"
AB - Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB " lift-out"
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U2 - 10.1016/j.jsb.2012.07.003
DO - 10.1016/j.jsb.2012.07.003
M3 - Article
C2 - 22796867
AN - SCOPUS:84867910247
SN - 1047-8477
VL - 180
SP - 318
EP - 326
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 2
ER -