Expansion microscopy: A powerful nanoscale imaging tool for neuroscientists

Abstract

One of the biggest unsolved questions in neuroscience is how molecules and neuronal circuitry create behaviors, and how their misregulation or dysfunction results in neurological disease. Light microscopy is a vital tool for the study of neural molecules and circuits. However, the fundamental optical diffraction limit precludes the use of conventional light microscopy for sufficient characterization of critical signaling compartments and nanoscopic organizations of synapse-associated molecules. We have witnessed rapid development of super-resolution microscopy methods that circumvent the resolution limit by controlling the number of emitting molecules in specific imaging volumes and allow highly resolved imaging in the 10–100 nm range. Most recently, Expansion Microscopy (ExM) emerged as an alternative solution to overcome the diffraction limit by physically magnifying biological specimens, including nervous systems. Here, we discuss how ExM works in general and currently available ExM methods. We then review ExM imaging in a wide range of nervous systems, including Caenorhabditis elegans , Drosophila, zebrafish, mouse, and human, and their applications to synaptic imaging, neuronal tracing, and the study of neurological disease. Finally, we provide our prospects for expansion microscopy as a powerful nanoscale imaging tool in the neurosciences.

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].…”

“…Several advances have been made in our understanding of neuronal synapses using super-resolution microscopy (for recent reviews see [ 45–48 , 52 ]).…”

“…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].…”

A connectome is not enough – what is still needed to understand the brain ofDrosophila?

“…Genetic and optical techniques such as t-GRASP (i.e. targeted GFP reconstitution across synaptic partners; Shearin et al, 2018) and expansion microscopy (Gallagher and Zhao, 2021) can look at a single connection across many animals, with enough throughput for multiple animals and time points. Repeated EM of small circuits (such as a column of the medulla) can show variation of that one circuit across several animals.…”

Nanoscale Imaging of Biomolecules using Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence Microscopy

“…During the rst step of the process, molecular handles are covalently attached to speci c biomolecules and/or labels that enables their anchoring to a swellable hydrogel network that is subsequently synthesized throughout the specimen. 2 These molecular handles are either conjugated antibodies or reactive chemicals prone to quick hydrolysis in water, 2,18,19 with one exception that contains a mixture of formaldehyde and acrylamide that is only effective for un xed specimens 5 . Most ExM protocols use Proteinase K digestion to enable expansion, which destroys endogenous epitopes.…”