Fabrication of Frozen-Hydrated Sections by Focused Ion Beam(FIB) Method

Cryo-electron tomography is an important research method to study the cell ultrastructure in a near native state, especially in the structural biology and cell biological fields[1]. There are a few cellular specimens that bacterial and eukaryotic cells can be examined directly by cryo-TEM [2, 3], but eukaryotic and tissue are difficult to realize for the specimen thickness. The primary method to thin the frozen-hydrated cells and tissues is cryo-ultramicrotomy [4-6]. This method prepares the specimen by high pressure frozen, and then cutting the specimen by the cryo-ultramicrotomy, but there are some unavoidable problems and produced some artifacts.The specimen preparation is the limitation for the tomography technical development. Here, we continue the development of the FIB method milling cryo-hydrated single cell specimen.First, we design three-quarters of the molybdenum grid with lacey support film that can keep the grid direction of FIB milling and data collection. Second, this grid is hard enough for the transfer process and has the less limitation of data collection TEM.