Abstract
The complex, nanoscopic scale of neuronal function, taking place at dendritic spines, axon terminals, and other minuscule structures, cannot be adequately resolved using standard, diffraction-limited imaging techniques. The last couple of decades saw a rapid evolution of imaging methods that overcome the diffraction limit imposed by Abbe’s principle. These techniques, including structured illumination microscopy (SIM), stimulated emission depletion (STED), photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), among others, have revolutionized our understanding of synapse biology. By exploiting the stochastic nature of fluorophore light/dark states or non-linearities in the interaction of fluorophores with light, by using modified illumination strategies that limit the excitation area, these methods can achieve spatial resolutions down to just a few tens of nm or less. Here, we review how these advanced imaging techniques have contributed to unprecedented insight into the nanoscopic organization and function of mammalian neuronal presynapses, revealing new organizational principles or lending support to existing views, while raising many important new questions. We further discuss recent technical refinements and newly developed tools that will continue to expand our ability to delve deeper into how synaptic function is orchestrated at the nanoscopic level.
Highlights
Imaging in neurosciences has come a long way since 19th-century neuroanatomist Ramon y Cajal made detailed drawings of silver-stained neurons and postulated that they were not continuous, but instead connected through gaps
We find structured illumination microscopy (SIM) and its variations, stimulated emission depletion (STED) and reversible saturable optical fluorescence transition (RESOLFT) microscopy
Though the number and dynamics of these modules changes rapidly with synaptic plasticity, they remain aligned (Hruska et al, 2018). These results suggest that structural plasticity linked to synaptic potentiation could be mediated by addition of building blocks made of unitary synaptic nanomodules
Summary
Imaging in neurosciences has come a long way since 19th-century neuroanatomist Ramon y Cajal made detailed drawings of silver-stained neurons and postulated that they were not continuous, but instead connected through gaps. Unraveling their detailed molecular composition and functional dynamics (for example, the trafficking of receptors or channels at the membrane or the changes in protein cluster number or size with synaptic activity) requires imaging at the nanoscale, using so-called super-resolution microscopy (SRM) techniques, that were developed to surpass the diffraction-limited resolution of conventional light microscopy while attempting to retain its versatility.
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