Research

(This work was done in Center for Biological Imaging, Institute of Biophysics, CAS)

Design of the cryo-FIB transfer station, the transfer shuttle and the “D”-shaped grid. (A) The drawing of the cryo-FIB transfer station. (B) The drawing of the cryo-FIB transfer shuttle with the specimen protection cover close (left) and open (right), respectively. The angle between the grid-loading slot and the horizontal plane is 30°. The crosshead fixing screw shown left is used during the storage of the shutter in the liquid nitrogen. (C) The design of a “D”-shaped molybdenum grid with one quarter trimmed and regions labeled. (D) The grids mounted onto the AutoGrid (left) and the standard side-entry cryo-holder (right). (E) A snapshot of the SEM (FEI Helios Nanolab 600i Dual Beam) chamber with the cryo-FIB transfer shuttle mounted onto the Quorum PP3000T cryo-stage.
Movie at up right: Slicing view of tomogram of cryo-FIB milled insect SF9 cell lamella.
Movie at bottom right: Slicing view of tomogram of cryo-FIB milled MEF cell lamella.
Cryo-electron tomography (cryo-ET) provides great insights into the ultrastructure of cells and tissues in their native state and provides a promising way to study the in situ 3D structures of macromolecular complexes. However, this technique has been limited on the very thin specimen, which is not applicable for most cells and tissues. Besides cryo-sectioning approach, cryo focused ion beam (cryo-FIB) appeared recently to achieve ‘artifact-free’ thin frozen hydrated lamella via fabrication. Considering that the current cryo-FIB methods need modified holders or cartridges, here, with a “D-shaped” molybdenum grid and a specific shutter system, we developed a simple cryo-FIB approach for thin frozen hydrated lamella fabrication, which fits both standard transmission cryo-electron microscopes with side-entry cryo-holders and state-of-the-art ones with AutoGrids. Our approach will expand the usage of cryo-FIB approach in many labs.
Reference:
Zhang J., Ji G., Huang X., Xu W.* and Sun F.* (2016), An improved cryo-FIB method for fabrication of frozen hydrated lamella. Journal of Structural Biology 194(2): 218-223. doi: 10.1016/j.jsb.2016.02.013.