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Updated: May 18, 2026
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.
Abstract:
Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.(1,2) However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light(3) do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.(4) Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.
Insights
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.
