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Published on: September 23, 2012
Multiphoton microscopy of cleared mouse organs
Sonia G Parra1, Thomas H Chia, Joseph P Zinter
1Yale University, Department of Biomedical Engineering, New Haven, Connecticut 06520, USA.
Abstract:
Typical imaging depths with multiphoton microscopy (MPM) are limited to less than 300 mum in many tissues due to light scattering. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having a similar index of refraction to that of proteins. We demonstrate MPM of intact, fixed, cleared mouse organs with penetration depths and fields of view in excess of 2 mm. MPM enables the creation of large 3-D data sets with flexibility in pixel format and ready access to intrinsic fluorescence and second-harmonic generation. We present high-resolution images and 3-D image stacks of the brain, small intestine, large intestine, kidney, lung, and testicle with image sizes as large as 4,096 x 4,096 pixels.
Insights
Optical clearing enables multiphoton microscopy (MPM) to image cleared mouse organs deeper than 2 mm. This technique overcomes light scattering, allowing for large, high-resolution 3D datasets of various organs.
Area of Science:
- Biomedical Imaging
- Optical Microscopy
- Tissue Optics
Background:
- Light scattering limits multiphoton microscopy (MPM) depth in tissues to <300 µm.
- Overcoming scattering is crucial for deep tissue imaging.
- Optical clearing methods aim to reduce scattering for enhanced penetration.
Purpose of the Study:
- To demonstrate deep-tissue multiphoton microscopy (MPM) in cleared, intact mouse organs.
- To achieve imaging penetration depths exceeding 2 mm.
- To showcase the capability for large-scale 3D imaging with high resolution.
Main Methods:
- Optical clearing of intact, fixed mouse organs by replacing water with a refractive index-matching fluid.
- Utilizing multiphoton microscopy (MPM) for imaging cleared tissues.
- Acquiring large 3D image datasets with high pixel dimensions (up to 4,096 x 4,096).
Main Results:
- Achieved MPM imaging penetration depths greater than 2 mm in cleared mouse organs.
- Demonstrated high-resolution 3D image stacks of brain, intestines, kidney, lung, and testicle.
- Captured large field-of-view images with intrinsic fluorescence and second-harmonic generation.
Conclusions:
- Optical clearing significantly enhances MPM penetration depth in biological tissues.
- This approach enables unprecedented large-scale, high-resolution 3D imaging of intact organs.
- The method provides valuable insights into organ morphology and structure at depths previously inaccessible.
