Related Experiment Video
Updated: May 18, 2026
10:03
Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP
Published on: September 23, 2012
Multiphoton microscopy of cleared mouse brain expressing YFP.
Sonia G Parra1, Sam S Vesuna, Teresa A Murray
1Department of Biomedical Engineering, Yale University, CT, USA.
Journal of Visualized Experiments : Jove
|October 2, 2012
Summary
This study presents a novel optical clearing protocol for whole mouse brains. The method preserves yellow fluorescent protein (YFP) signals in neurons for deep tissue multiphoton microscopy.
Area of Science:
- Neuroscience
- Biomedical Imaging
- Optical Engineering
Background:
- Multiphoton microscopy enables deep tissue imaging of intrinsic fluorescence and second harmonic generation (SHG).
- Standard optical clearing methods using methanol and benzyl alcohol:benzyl benzoate (BABB) can damage fluorescent proteins like YFP.
- Preserving fluorescent signals is crucial for imaging genetically encoded reporters in cleared tissues.
Purpose of the Study:
- To develop a novel optical clearing protocol for whole mouse brains that preserves YFP fluorescence.
- To enable high-resolution multiphoton imaging of neuronal YFP expression deep within the mouse brain.
Main Methods:
- Modified optical clearing protocol using an ethanol dehydration series.
- Clearing agent: benzyl alcohol:benzyl benzoate (BABB).
- Protection from light during the clearing process.
Main Results:
- Ethanol-based dehydration significantly reduces damage to fluorescent proteins compared to methanol.
- The optimized protocol successfully preserves YFP fluorescence in mouse brain tissue.
- High-resolution multiphoton images were acquired more than 2 mm deep in the brain.
Conclusions:
- An optimized, light-shielded optical clearing protocol using ethanol dehydration preserves YFP fluorescence in mouse brains.
- This method facilitates deep-tissue multiphoton imaging of neuronal structures.
- The protocol advances the capability for studying neuronal circuits in vivo.
