Three-Dimensional Ultrastructure of Micrometer Thick Cellular Subvolumes by STEM Tomography

Abstract

Electron tomography (ET) is a powerful technique for determining 3D ultrastructure at the supramolecular scale in sections of rapidly frozen, freeze-substituted, stained, embedded and sectioned cells and tissues. The application of conventional ET is limited to sections of thickness less than about 400 nm due to image blurring of multiply scattered electrons that are affected by the chromatic aberration of the objective lens. Many important structures such as pre- and post-synaptic nerve terminals in brain have larger dimensions so cannot be imaged in their entirety by conventional ET methods. We have therefore developed and applied tomography based on the scanning transmission electron microscope (STEM) operating at a beam energy of 300 keV. In STEM, the absence of imaging lenses after the specimen enables ET to be performed on plastic sections up to 1.5 micrometers in thickness without the deleterious effect of chromatic aberration on electrons that have undergone multiple inelastic scattering. Furthermore, it was found that the spatial resolution of 3D reconstructions obtained with an axial bright-field STEM detector was greatly improved relative to that obtained with a standard annular dark-field STEM detector, for regions in the lower half of micrometer thick sections. This advantage is attributable to the exclusion of electrons that have undergone multiple elastic scattering from the collection angles subtended by the axial bright-field detector. We have applied STEM tomography to image entire postsynaptic densities (PSDs) in dissociated cultures of rat hippocampus, in which specific proteins had been knocked down by RNAi. The technique allows multiple PSDs to be reconstructed from contiguous regions of neurons. The 3D ultrastructure reveals the role of key scaffolding proteins in the organization of PSDs.This work was supported by the Intramural Research Programs of NIBIB and NINDS, NIH.