Unraveling the Nanoscopic Organization and Function of Central Mammalian Presynapses With Super-Resolution Microscopy

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.

Super-resolution imaging to reveal the nanostructure of tripartite synapses

“…Several super resolution methods have been developed, each with its own advantages and disadvantages. However, the discussion of these is beyond the scope of this review and the authors again point to some excellent reviews of this topic [44][45][46][47][48][49][50][51][52][53]. Most widely used methods include stimulated-emission depletion (STED) microscopy [54], structured illumination microscopy (SIM) [55], single molecule localisation microscopy (SMLM) [56][57][58], and expansion microscopy (ExM) [59,60].…”

“…We will also provide a short introduction into different super-resolution methods and recent advances in revealing the nanostructure of neuronal synapses. However, for further information we point the reader to some excellent, recent reviews on super-resolution microscopy and also its use in neuroscience research [44][45][46][47][48][49][50][51][52][53].…”

Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

“…In the following sections, we will describe methods for SPT and single-molecule SR imaging and showcase some examples of their applications for imaging of neurons and synapses. For extensive reviews of applications of these techniques in neuroscience, see e.g., Kim et al (2010), Maglione and Sigrist (2013), Tønnesen and Nägerl (2013), Bannai (2018), Nosov et al (2020), Carvalhais et al (2021), Werner et al (2021), andZieger andChoquet (2021).…”

Fluorescent Chemosensors for Ion and Molecule Recognition: The Next Chapter

“…Super-resolution-based technologies have revolutionized fluorescence imaging by circumventing the diffraction limit of light and thereby enabling visualization of structures with nanoscale accuracy within a cellular environment (Carvalhais et al, 2021). Amongst the super-resolution techniques available we would like to specifically highlight Stochastic Optical Reconstruction Microscopy (STORM) (Rust et al, 2006;Samanta et al, 2019) and photoactivated localization microscopy (PALM) (Betzig et al, 2006) as these techniques use probes with switchable fluorescence emission profiles (photoblinking).…”