Development of two-photon fluorescence microscopy for quantitative imaging in turbid tissues

Two-photon laser scanning fluorescence microscopy (TPM) is a high resolution, non-invasive biological imaging technique that can be used to image turbid tissues both in vitro and in vivo at depths of several hundred microns. Although TPM has been widely used to image tissue structures, no one has focused on using TPM to extract quantitative information from turbid tissues at depth. As a result, this thesis addresses the quantitative characterization of two-photon signals in turbid media. Initially, a two-photon microscope system is constructed, and two-photon images that validate system performance are obtained. Then TPM is established as an imaging technique that can be used to validate theoretical observations already listed in the literature. In particular, TPM is found to validate the exponential dependence of the fluorescence intensity decay with depth in turbid tissue model systems. Results from these studies next prompted experimental investigation into whether TPM could be used to determine tissue optical properties. Comparing the exponential dependence of the decay with a Monte Carlo model involving tissue optical properties, TPM is shown to be useful for determining the optical properties (total attenuation coefficient) of thick, turbid tissues on a small spatial scale. Next, a role for TPM for studying and optimizing wound healing is demonstrated. In particular, TPM is used to study the effects of perturbations (growth factors, PDT) on extracellular matrix remodeling in artificially engineered skin tissues. Results from these studies combined with tissue contraction studies are shown to demonstrate ways to modulate tissues to optimize the wound healing immune response and reduce scarring. In the end, TPM is shown to be an extremely important quantitative biological imaging technique that can be used to optimize wound repair.