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Related Experiment Videos

Second harmonic generation imaging of endogenous structural proteins.

William Mohler1, Andrew C Millard, Paul J Campagnola

  • 1Department of Genetics, Center for Biomedical Imaging Technology, University of Connecticut Health Center, 06030, Farmington, CT, USA.

Methods (San Diego, Calif.)
|January 25, 2003
PubMed
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Second harmonic generation (SHG) microscopy images structural proteins like collagen and tubulin in 3D with high resolution. This label-free technique offers reduced photodamage and deep tissue imaging capabilities for biological research.

Area of Science:

  • Biophysics
  • Microscopy
  • Cell Biology

Background:

  • Structural proteins like collagen, myosin, and tubulin form complex arrays in biological tissues.
  • Label-free imaging techniques are crucial for minimizing perturbation in biological samples.
  • Nonlinear optical microscopy offers advantages in optical sectioning and reduced phototoxicity.

Purpose of the Study:

  • To demonstrate the capability of laser-scanning second harmonic generation (SHG) microscopy for imaging structural protein arrays.
  • To evaluate SHG microscopy's performance in terms of contrast, resolution, and depth penetration in thick tissues.
  • To compare SHG imaging with fluorescence microscopy for understanding molecular origins of contrast and structural organization.

Main Methods:

  • Utilized laser-scanning second harmonic generation (SHG) microscopy.

Related Experiment Videos

  • Imaged endogenous structural proteins (collagen, myosin, tubulin) in three dimensions.
  • Combined SHG with two-photon excited green fluorescent protein (GFP) imaging in model organisms (C. elegans).

    • Main Results:

    • Achieved high-contrast and high-resolution 3D imaging of protein arrays without exogenous labels.
    • Demonstrated deep-tissue imaging (hundreds of micrometers) in muscle and bone.
    • Inferred molecular origins of SHG contrast in sarcomeres and studied microtubule organization in embryos, revealing symmetries not visible with GFP.

    Conclusions:

    • SHG microscopy is a powerful label-free tool for high-resolution 3D imaging of structural proteins in thick tissues.
    • SHG microscopy provides molecular-level insights into tissue organization and symmetry.
    • The technique offers advantages like intrinsic sectioning, reduced photobleaching, and low phototoxicity, making it suitable for in vivo imaging.