Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy

Abstract

Understanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30 nm spatial resolution.Aim: We developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30 nm lateral resolution.Approach: We used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM). Results:We demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30 nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites.Conclusions: Our optimized expansion microscopy approach improves the visualization and location of pre-and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.

Visualizing the trans-synaptic arrangement of synaptic proteins by expansion microscopy

“…Compared to conventional labeling for SR microscopy the post-labeling of decrowded synapses yields enhanced labeling efficiency by targeting so far inaccessible epitopes and visualization of individual clusters of BSN instead of bar-like shapes that are usually observed applying conventional immunolabeling and SR imaging without expansion of the sample (Siddig et al, 2020 ; Eilts et al, 2023 ). The visualization of distinct BSN clusters was also reported previously, but with a more complex protocol applying an iterative ExM process (Sarkar et al, 2022 ).…”

Special Section Guest Editorial: Frontiers in Neurophotonics

“…To that end, Caya-Bissonnette and Béïque discuss uncaging of glutamate and other photoactivation methods. 5 These methods are paralleled by the development and improvement of tools for high-resolution imaging ex vivo , such as the multicolor super-resolution expansion microscopy—the focus of perspective article by Eilts et al., 6 which is featured on the cover of Volume 10 Issue 4 .…”