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Updated: Sep 21, 2025
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A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales
Published on: May 10, 2022
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A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales.
Kenta Yamauchi1, Shinichiro Okamoto2, Megumu Takahashi3
1Department of Neuroanatomy, Juntendo University Graduate School of Medicine; Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine.
Journal of Visualized Experiments : Jove
|May 31, 2022
Summary
This study presents a protocol using ScaleSF hydrophilic tissue clearing to visualize neuronal structures in mouse brain slices. The method enables detailed imaging from neural circuits to subcellular components like dendritic spines.
Area of Science:
- Neuroscience
- Biotechnology
- Microscopy
Background:
- Visualizing neuronal structures across scales is crucial for understanding brain function.
- Existing tissue clearing methods may compromise fluorescence signal or structural integrity.
- A need exists for robust protocols to image both mesoscopic and microscopic neuronal details.
Purpose of the Study:
- To provide a detailed protocol for visualizing neuronal structures from mesoscopic to microscopic levels in optically cleared mouse brain slices.
- To adapt and optimize the ScaleS hydrophilic tissue clearing method (ScaleSF) for enhanced clearing and preservation.
- To develop a 3D-printed imaging chamber for reliable mounting and imaging of cleared brain tissues.
Main Methods:
- Mouse brains were injected with AAV-eGFP, fixed, and sectioned into 1-mm slices.
- Slices underwent hydrophilic clearing using ScaleSF protocol (ScaleS0, PBS(-), ScaleS4 solutions) for 10.5-14.5 hours.
- Cleared slices were mounted in a 3D-printed chamber with agarose and imaged using confocal microscopy with a multi-immersion objective.
Main Results:
- The ScaleSF method effectively cleared 1-mm thick mouse brain slices.
- High-level preservation of fluorescence signals and structural integrity was achieved.
- Mesoscopic visualization of neural circuits and microscopic imaging of subcellular structures (dendritic spines, axonal boutons) were successful.
Conclusions:
- The developed protocol enables comprehensive visualization of neuronal architecture from the circuit to the subcellular level.
- ScaleSF hydrophilic tissue clearing combined with a custom imaging chamber offers a powerful tool for neuroscience research.
- This method facilitates a deeper understanding of neuronal organization and connectivity in the brain.
