Harmonic optical tomography (HOT) is a new technique for imaging microscopic, nonlinear, and inhomogeneous objects. It uses holographic information to generate 3D images of the sample. It is the result of a collaboration between researchers at the University of Illinois at Urbana-Champaign’s Beckman Institute for Advanced Science and Technology and Colorado State University. “Our lab specializes in using holographic data to investigate live cells and tissues,” professor Gabriel Popescu said. “We wanted to extend this technique to nonlinear samples by combining the holographic data and new physics models.” The HOT technique interferometrically measures the complex harmonic field and uses a scattering inverse model to reconstruct the 3D distribution of harmonophores. The researchers began by developing theoretical models to describe how to image the tissue. They discovered a capability for 3D imaging that arose, counterintuitively, when the sample was illuminated with blurry, out-of-focus laser light. To collect data, researchers at Colorado State University designed and built a a custom, high-power laser. The data was reconstructed using computational imaging algorithms. The researchers used two types of samples to test and demonstrate the HOT principle — a manufactured crystal that is typically used for generating nonlinear signals, and a muscle tissue sample. Although the experiments were based on second-order nonlinear materials, the experimental results showed that the approach could apply to any coherent nonlinear process. According to the researchers, the experiments verified a new form of optical tomography that validated the experimental predictions. Imaging the muscles in mice with standard techniques (left), and with harmonic optical tomography (right). The new technique is better at revealing the structure of the tissue. Courtesy of Gabriel Popescu/University of Illinois at Urbana-Champaign. “This new type of tomographic imaging could prove to be very valuable for a wide range of studies that currently rely on two-dimensional images to understand collagen fiber orientation, which has been used as a reporter for a number of types of cancer,” CSU researcher Jeff Field said. “Unlike typical laser-scanning microscopes, an additional benefit of HOT is that its speed makes it much less vulnerable to vibrations and unwanted microscope drift, which leads to sharper images and increased repeatability,” Kimani Toussaint, a former professor in the College of Engineering at Illinois and now professor in the School of Engineering at Brown University, said. The research was published in Nature Photonics (www.doi.org/10.1038/s41566-020-0638-5).