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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.

Journal of Biomedical Optics
|July 10, 2010
PubMed
Summary
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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.

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  • 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.