| The formation and development of many cancers and other diseases often involve associated structural changes to tissue and the extracellular matrix on a microscopic level. This work comprises the integrated use of light scattering measurements, Monte Carlo light transport simulations, 3D Second Harmonic Generation (SHG) imaging microscopy as well as physical models to relate experimental optical parameters to the underlying tissue structure. The overarching goal is the development of optical techniques to quantify tissue structure that could be used for disease diagnostics in the future. A novel approach is developed to measure the true single scattering anisotropy coefficient by experimental light scattering goniometry coupled with Monte Carlo simulations to decouple the effects of scattering and it is shown that multiple scattering affects the measurements even in the case of thin slices. Next, 3D SHG imaging microscopy is coupled with Monte Carlo simulations to decouple the effects of SHG creation and subsequent scattering. It is demonstrated that we can extract the ratio of the initial forward-to-backward (F/B) creation ratio and the reduced scattering coefficient of tissue by fitting experimental data with simulations. It is further shown that the wavelength dependency of both these parameters is related to the underlying tissue architecture. While, the experimental data in this study is for mouse tail tendon, it is expected that the approaches can be applied to more relevant disease models in future studies, particularly those involving collagen and the extracellular matrix. Furthermore, a theoretical model is presented for the wavelength dependence of the SHG emission directionality and the intensity of the SHG signal. It is shown that the only physical model that fits experimental data is where the collagen fibrils have a random orientation (either turning up or down), and the nonlinearity is limited to the outer shell of the fibrils. The model also allows making quantitative arguments about fibril sizing and shell thickness. Lastly, additional work is presented on wavefront correction and adaptive optics to correct for phase aberrations that occur when light travels through tissue, and work on a method for characterizing the shape of miniature tunable lenses is presented. |
