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Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons
Published on: October 31, 2020
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Visualizing Synaptic Multi-Protein Patterns of Neuronal Tissue With DNA-Assisted Single-Molecule Localization
Kaarjel K Narayanasamy1,2, Aleksandar Stojic1, Yunqing Li2
1Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany.
Frontiers in Synaptic Neuroscience
|July 5, 2021
Summary
Super-resolution microscopy (SRM) now visualizes multiple proteins in neuronal tissue using DNA-assisted single-molecule localization microscopy (SMLM). This method enhances structural cell biology by overcoming limitations of traditional multiplexing techniques.
Area of Science:
- Cellular and Molecular Biology
- Microscopy Techniques
- Neuroscience
Background:
- Super-resolution microscopy (SRM) advances biomolecular structure and function understanding.
- Multiplexing targets in SRM is crucial for elucidating spatial localization and functional co-dependencies.
- Current SRM multiplexing methods face limitations in fluorophore capacity and antigen loss during antibody restaining.
Purpose of the Study:
- To develop a novel DNA-assisted single-molecule localization microscopy (SMLM) method for multi-target protein visualization.
- To overcome the limitations of traditional organic dye multiplexing in SRM.
- To enable high-resolution imaging of protein interactions within neuronal tissue.
Main Methods:
- Utilized DNA-assisted SMLM for multi-target protein imaging in 350-400 nm neuronal tissue sections.
- Employed a single labeling step with DNA oligonucleotide-conjugated antibodies.
- Facilitated sequential exchange of fluorophore-labeled complementary oligonucleotides in the imaging buffer.
Main Results:
- Successfully visualized multiple protein targets within the pre- and postsynapse in a single labeling step.
- Achieved a lateral resolution better than 25 nm.
- Demonstrated robustness for multi-target imaging in semi-thin tissue sections, avoiding chromatic aberration.
Conclusions:
- The DNA-assisted SMLM method enables efficient and high-resolution multi-target protein visualization in neuronal tissue.
- This approach circumvents issues associated with multiple immunolabeling rounds and chromatic aberration.
- Paves the way for advanced structural cell biology studies using single-molecule SRM.
