Cryo-Electron Tomography of Neurons, Glia, and Human Brain Tissue

Surprisingly little is known about the native structure of protein molecules inside cells. Cryo-electron tomography explores the structures of protein complexes and their interactions with other components in cells. The difficulty is to gain access to the appropriate cellular regions – neurons are too thick for imaging and some form of sectioning is needed to produce layers of vitrified cell structure capturing the cellular milieu but thin enough for cryo-electron microscopy, that is, well under a micron in thickness. The Fitzpatrick Lab seeks to address this challenge by utilizing the latest developments in cryo-electron tomography (cryo-ET) to visualize the interior of the cell at high-resolution.

Cryogenic Focussed ion beam (cryo-FIB) milling (see figure below), offers a way to produce relatively undisturbed layers of suitable thickness for cryo-electron tomography. Thin lamellas of vitrified cells containing protein complexes will be prepared by cryo-focused ion beam (FEI Aquilos cryo-FIB) and subsequently imaged in three dimensions by cryo-ET. Cryo-ET will enable the Fitzpatrick Lab to determine protein structures in situ, within pristinely preserved cellular environments at molecular resolution, shedding new light on the cellular mechanisms underlying neurodegeneration and memory.