Superresolution Imaging of Chemical Synapses in the Brain

Abstract

Determination of the molecular architecture of synapses requires nanoscopic image resolution and specific molecular recognition, a task that has so far defied many conventional imaging approaches. Here we present a super-resolution fluorescence imaging method to visualize the molecular architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction microscopy, the distributions of synaptic proteins can be measured with nanometer precision. Furthermore, the wide-field, volumetric imaging method enables high-throughput, quantitative analysis of a large number of synapses from different brain regions. To demonstrate the capabilities of this approach, we have determined the organization of ten protein components of the presynaptic active zone and the postsynaptic density. Variations in synapse morphology, neurotransmitter receptor composition, and receptor distribution were observed both among synapses and across different brain regions. Combination with optogenetics further allowed molecular events associated with synaptic plasticity to be resolved at the single-synapse level.

X10 expansion microscopy enables 25‐nm resolution on conventional microscopes

“…This level of resolution is usually only achieved in highly specialized applications of STED and STORM 23, 24 or in iterative expansion microscopy (iExM) 25 and is bettered substantially only by a recently developed tool, MINFLUX microscopy 26. When investigating the same protein in state‐of‐the‐art commercial STED and STORM setups, the image quality these techniques achieved was, at best, comparable to that of X10 (Figs 3A and EV2).…”

Morphological plasticity of astroglia: Understanding synaptic microenvironment

“…Although this type of approach could provide excellent spatial resolution (Table 1), its use in organised live tissue is limited, mainly because either the imaging conditions or the probe chemistry are not compatible with physiology. Nonetheless, there have been some spectacular revelations regarding the nanoscopic distribution of pre‐ and postsynaptic receptor proteins (Dani et al, 2010) documented in fixed tissue (Fig. 5C).…”

“…Adapted from (Volterra et al, 2014). ( C ) STORM imaging of pre‐ (Bassoon; red) and postsynaptic (Homer1; green) scaffolding proteins in the mouse main olfactory bulb glomeruli imaged using conventional fluorescence imaging (C1) and STORM (C2); adapted from (Dani et al, 2010). ( D , E ) dSTORM images of cultured (14 DIV) mixed glial cells from rat hippocampus (ProLong Diamond in Zeiss Elyra PS.1 microscope; Fiji Plugin ThunderSTORM, 3,000 frames); unpublished data by J. Heller.…”

Recent applications of superresolution microscopy in neurobiology

“…An exhaustive work used three-color 3D STORM to map the organization of 10 presynaptic and postsynaptic proteins in cryosections from different brain regions [17 ]. By averaging hundreds of synapses, the distance of the presynaptic protein Bassoon and the postsynaptic protein Homer1 was determined to be 153.8 nm.…”