Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

Area of Science:

• Neuroscience
• Molecular Biology
• Microscopy

Background:

• Understanding synaptic plasticity requires detailed molecular mapping of brain regions.
• Current 3D super-resolution microscopy techniques face limitations in imaging deep into tissues.
• Presynaptic active zones (AZ) are crucial for synaptic function and plasticity.

Purpose of the Study:

• To develop and validate a method for deep volumetric imaging of presynaptic AZs in large neuronal structures.
• To quantify molecular organization differences in AZs across distinct hippocampal regions.
• To enable high-resolution analysis of entire, large mossy fiber boutons.

Main Methods:

• Developed targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM).
• Optimized fast axial scanning and sequential fluorescent labeling protocols.
• Applied method to 25 µm thick mouse brain sections for imaging Bassoon and GFP.

Main Results:

• Successfully imaged and quantified presynaptic AZs in large (up to 10 µm) hippocampal mossy fiber boutons.
• Revealed significant regional variations in Bassoon cluster size and density within the hippocampus.
• Identified the largest Bassoon clusters in mossy fiber boutons.

Conclusions:

• The developed 3D-dSTORM method enables deep tissue imaging of synaptic structures.
• Significant molecular heterogeneity exists in presynaptic AZs across hippocampal subregions.
• This technique provides a powerful tool for studying synaptic organization and plasticity.