3-Dimensional Electron Microscopy of Biological Specimens

Three-dimensional (3D) imaging is an important tool in electron microscopy, especially in biological specimens where the main focus is the structure of the cells. Many times important information is lost because the exact orientation of a specimen is unknown. We tested two different 3D imaging techniques, focused ion beam (FIB) slice and view, and cryo-FIB thinning of samples for use in cryo transmission electron tomography (cryo-TEM) and cryo-electron tomography (cryo-ET).\ud \ud We began our research with room temperature FIB slice and view, with an intention to move onto slice and view at cryogenic temperatures. We found this technique to be difficult to control and the time required to produce results was simply too high. We moved on to investigating cryo-FIB milling as a tool for thinning cryo-ET specimens. Advances in cryo-ET have enabled high-resolution 3D imaging of complex assemblies and determination of cellular architectures in their close-to-native states. However, one major limitation, the accessible specimen thickness, has hindered its broader application in cellular biology. Recent efforts have been made to create thin, frozen-hydrated sections using cryo-ultramicrotomy, but with many mechanical artifacts and low yields. Here, we report a method that applies a focused ion beam (FIB) at cryogenic temperature (cryo-FIB) to reduce the thickness of frozen-hydrated cells, including mammalian cells, to a degree suitable for cryo-ET