Labeling Postsynaptic Densities for Super-Resolution Microscopy With Minimal Signal-Loss and Offset

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Accurate labeling of postsynaptic sites is crucial for high-resolution imaging but challenging due to dense synaptic environments.
• Conventional antibodies face limitations in accessing and precisely labeling postsynaptic densities (PSDs).

Purpose of the Study:

• To present a novel protocol using synthetic peptide probes (Sylites) for precise and efficient labeling of excitatory and inhibitory postsynaptic densities.
• To overcome limitations of antibody-based labeling in high-resolution synaptic imaging.

Main Methods:

• Utilized Sylites, small synthetic peptide probes (3 kDa), with high affinity for postsynaptic markers PSD-95 (eSylites) and Gephyrin (iSylites).
• Developed eSylites targeting PDZ1/PDZ2 domains of PSD-95 for excitatory PSDs (ePSDs) and iSylites targeting Gephyrin's E-domain for inhibitory PSDs (iPSDs).
• Integrated Sylite labeling into standard immunocytochemistry workflows compatible with primary neurons and brain tissue.

Main Results:

• Sylites enabled precise and efficient labeling of ePSDs and iPSDs, overcoming steric hindrance.
• The small size of Sylites reduced linkage error, enhancing accessibility and spatial resolution in super-resolution microscopy.
• Demonstrated clear separation of PSD-95 nanodomains and compatibility with multiplexed imaging using standard antibodies.

Conclusions:

• The Sylite protocol provides robust and reproducible labeling of excitatory and inhibitory synapses with improved spatial resolution.
• This method facilitates detailed visualization of synaptic nanodomains and is adaptable for expansion microscopy and live-cell applications.
• Offers a significant advancement for studying synaptic organization and function in neuroscience research.