Harmonic Optical Tomography of Nonlinear Structures

At a Glance

Researchers at Colorado State University in collaboration with the University of Illinois have developed Harmonic Optical Tomography (HOT) as a novel modality for imaging three-dimensional, microscopic, nonlinear, and inhomogeneous objects. The HOT principle of operation relies on interferometrically measuring the complex harmonic field and using a scattering inverse model to reconstruct the 3D distribution of harmonophores. The resulting HOT tomograms directly report a sample’s nonlinear susceptibility with no coherent artifacts, which has not been achieved before.

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Second-harmonic generation microscopy (SHGM) has emerged as a powerful technique for intrinsic contrast optical imaging of cells and tissues having the ability to report on molecules organized in filaments with high specificity. SHGM signals in biological tissues are dominated by specific types of collagen fibers, myosin in muscle fibers, and polarized microtubules. The ability to selectively image collagen in the tissue extracellular matrix has been exploited for studying tumor microenvironments as a marker for characterizing cancer aggressiveness; the list of diseases that can be characterized using SHGM continues to grow at a rapid pace.

Conventional laser scanning SHGM relies on the intrinsic optical sectioning capability that use tightly focused, short, laser pulses. This restricts the SHG scattering to a small volume at the focus of the incident light pulse. The resulting SHGM image is dependent on the spatial variation in the intensity and phase of the fundamental beam, as well as on the spatial distribution of harmonophores and phase matching. And thus, phase matching of the coherent SHG scattering process often obscures the quantitative interpretation of the images.

Early publications have demonstrated that a direct measurement of a complex SHG field eliminates image artefacts due to phase mismatch. Using this approach, holographic SHGM has been demonstrated on biological structures; however, a solution to the SHG scattering inverse problem has not been reported so far. In other words, the 3D images presented in the literature represent distributions of the SHG field, which is dependent on local phase matching, not on the nonlinear susceptibility itself.


  • transparent 3D nonlinear structures are visualized through computed solutions of the inverse problem
  • method has been demonstrated using microscopic defects that are embedded in a beta-barium borate (BBO) crystal and various tissues
  • technique demonstrates the substantial sectioning improvement over conventional SHGM


  • 3D imaging of biological tissue
  • Study tumor microenvironments, cancer, and other diseases
  • Living cell imaging
  • Diagnosis of disease (e.g., cancer)
Last Updated: March 2024

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​Randy Bartels
Jeffrey Field
Gabriel Popescu
Chenfei Hu
Varun Keiker

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