Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy

Abstract

SignificanceUnderstanding 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.AimWe developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30 nm lateral resolution.ApproachWe used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM).ResultsWe 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.ConclusionsOur 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.

Cite this article

@article{eilts_enhanced_2023,
  title = {Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy},
  volume = {10},
  issn = {2329-423X, 2329-4248},
  url = {https://www.spiedigitallibrary.org/journals/neurophotonics/volume-10/issue-4/044412/Enhanced-synaptic-protein-visualization-by-multicolor-super-resolution-expansion-microscopy/10.1117/1.NPh.10.4.044412.full},
  doi = {10.1117/1.NPh.10.4.044412},
  abstract = {SignificanceUnderstanding 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.AimWe developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30 nm lateral resolution.ApproachWe used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM).ResultsWe 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.ConclusionsOur 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.},
  number = {4},
  urldate = {2024-04-06},
  journal = {Neurophotonics},
  author = {Eilts, Janna and Reinhard, Sebastian and Michetschläger, Nikolas and Werner, Christian and Sauer, Markus},
  year = {2023},
  note = {Publisher: SPIE},
  pages = {044412},
}