3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography.
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".
Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Ave, Pittsburgh, PA 15260, USA.
Ke
K
Wang
Korrinn
K
Strunk
Gongpu
G
Zhao
Jennifer L
JL
Gray
Peijun
P
Zhang
D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D005629:Frozen Sections; D006367:HeLa Cells; D006801:Humans