Second-harmonic generation (SHG) imaging can help reveal interactions between collagen fibers and cancer cells. Quantitative analysis of SHG images of collagen fibers is challenged by the heterogeneity of collagen structures and low signal-to-noise ratio often found while imaging collagen in tissue. The role of collagen in breast cancer progression can be assessed post acquisition via enhanced computation. To facilitate this, we have implemented and evaluated four algorithms for extracting fiber information, such as number, length, and curvature, from a variety of SHG images of collagen in breast tissue. The image-processing algorithms included a Gaussian filter, SPIRAL-TV filter, Tubeness filter, and curvelet-denoising filter. Fibers are then extracted using an automated tracking algorithm called fiber extraction (FIRE). We evaluated the algorithm performance by comparing length, angle and position of the automatically extracted fibers with those of manually extracted fibers in twenty-five SHG images of breast cancer. We found that the curvelet-denoising filter followed by FIRE, a process we call CT-FIRE, outperforms the other algorithms under investigation. CT-FIRE was then successfully applied to track collagen fiber shape changes over time in an in vivo mouse model for breast cancer.
We explore combining Coherent anti-Stokes Raman Scattering
with Optical Coherence Tomography ranging when low numerical aperture scanning is used, and demonstrate ranging of layers in a Raman-active medium.
Diagnostic imaging trends are progressing toward the molecular level with the advent of molecular imaging techniques. Optical molecular imaging techniques that utilize targeted exogenous contrast agents or detect endogenous molecular signatures will significantly extend our ability to detect early pathological changes in biological tissue, and treat diseases early when they are most amenable to be cured. We have developed a technique called Nonlinear Interferometric Vibrational Imaging (NIVI) that takes advantage of the coherent nature of coherent anti-Stokes Raman scattering (CARS) processes and the coherent optical ranging and imaging capabilities of optical coherence tomography (OCT). OCT uses interferometry and heterodyne detection in the time or spectral domain to localize reflections of near-infrared light deep from within highly-scattering tissues. OCT has found wide biological and medical applications, and recently, molecular imaging methods have been developed. By utilizing the molecular-sensitivity of CARS, NIVI performs optical ranging and multi-dimensional molecular imaging with OCT-like optical systems, enabling the retrieval of not only χ(3) [chi(3)] amplitude but also phase information, the rejection of problematic non-resonant background four-wave-mixing signals, enhanced sensitivity from heterodyne detection, and a relaxation of the high-numerical aperture focusing requirements present in CARS microscopy. We present recent progress and advances in NIVI, including depth-ranging capabilities that extend significantly deeper than current CARS microscopy methods and are potentially more suitable for cross-sectional deep-tissue in vivo imaging.
Vibrationally-sensitive spectroscopic techniques are becoming important clinical tools for real-time, in vivo diagnostics. The molecular information made available with these techniques can provide early diagnostic signs of disease, often before morphological changes occur. We model and experimentally demonstrate a new technique for measuring optical spectroscopy signals using interferometric ranging. This new technique, nonlinear interferometric vibrational imaging (NIVI), uses principles from coherent anti-Stokes Raman scattering (CARS) spectroscopy and optical coherence tomography (OCT) to achieve cross-sectional imaging of the distribution of specific molecular species within a sample. Two CARS signals are generated, one from a known reference molecular species and a second from the unknown molecules in a sample. These coherent signals are interfered with each other using an interferometer setup. The intensity envelope of the interference signal provides a measure of the concentration of selected bonds present in the sample focal volume. The interference fringes themselves can provide phase information that will allow for the exact reconstruction of the vibrational characteristics of the molecules in the sample focal volume. Theoretical background to CARS interferometry is presented, the experimental laser systems are described, and a depth-resolved scan line of a benzene filled cuvette is demonstrated. The experimental results show close resemblance to the theoretical models. The advantages of NIVI over existing vibrational imaging systems and its clinical implications are discussed.
We present our progress in developing a novel technique and instrument that images specific molecular species in biological tissues using Optical Coherence Tomography (OCT). Standard OCT instruments measure only the scattering from structural features, such as refractive index changes. We utilize Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopy, a nonlinear optics technique that can selectively stimulate molecular groups, to gather compositional information from the sample. Being a coherent process, our instrument will produce interference between the nonlinear anti-Stokes signal produced in the sample and a reference molecular sample to both exclude background and nonresonant signals and range features in the tissue. Because of this, we will also gain the benefits of sensitivity that interferometry can provide. By utilizing the tunability of an optical parametric oscillator, we can address a range of molecular resonances from 1500 cm-1 to 3500 cm-1. This frequency range offers the possibility of measuring the distributions and densities of proteins, lipids, and nuclear material that we believe will be useful for determining the early presence of epithelial carcinomas. We demonstrate the principle of this imaging method by producing interference
between two separately produced CARS signals from the same probe and