Three-Dimensional Visualization of Whole Synapses by Stem Tomography

Abstract

Scanning transmission electron microscope (STEM) tomography enables determination of 3D ultrastructure from 1 or 2-micrometer thick sections of cells and tissues. These sections are considerably thicker than can be analyzed by conventional electron tomography, where resolution is limited by chromatic aberration due to multiple inelastic scattering. In STEM tomography a probe of small angular convergence gives a large depth of field throughout the thickness of the specimen while maintaining a probe diameter of approximately 2 nm; and the use of an on-axis bright-field detector reduces the effects of beam broadening and thus improves the spatial resolution compared to that attainable by STEM dark-field tomography. We have found that STEM tomography is ideal for visualizing entire synapses in the nervous system, and for making quantitative measurements on the numbers, sizes and shapes of synaptic components. We applied the technique to study the architecture of ribbon synapses in retina, and the structure of postsynaptic densities in brain cortex. For the first time, it was possible to determine a full 3D architecture of ribbon synapses in mammalian (rat) rod bipolar cells, in which regular docked and tethered vesicles, as well as larger (possibly pre-fused) vesicles were visualized. Quantitative analysis revealed a readily releasable pool of vesicles, which correlates structurally with previous physiological data. We have also applied STEM tomography to reconstruct entire spine postsynaptic densities in mouse hippocampus, both in control preparations as well as in preparations, where RNAi knockdown eliminates specific PSD scaffolding proteins to illustrate their key role in organizing the PSD. STEM tomography of thick sections thus provides a novel approach for correlating the nanoscale structure of synapses with function.This work was supported by the Intramural Research Programs of NIBIB and NINDS, NIH.