Nonlinear imaging in scattering media with pump-probe and fluorescence-based techniques

Thesis (Ph. D.)--University of Rochester. Institute of Optics, 2011.Nonlinear optical biomedical imaging techniques have attracted a great amount of interest starting with the first demonstration of two-photon fluorescence (TPF) imaging by Webb’s group in the 1990s. The various imaging formats and applications reported have revolutionized the field of biomedical imaging, offering great advantages including high resolution, internal three-dimensional sectioning ability, and functional imaging capability. This thesis focuses on exploring the development and novel application of nonlinear optical techniques and extending previous work in transmission mode nonlinear absorption imaging. The topical pharmacological introduction of molecules and drugs to the ocular surface is a common means of assessing and treating a variety of pathological ocular conditions. However, no reliable method exists to date for the quantification of actual delivered dosage with high enough resolution. We developed a two-photon fluorescence (TPF) system capable of quantifying, with micron-level axial resolution, the distribution and concentration of fluorescent or fluorescently-tagged chemicals and drugs in live feline corneas. With this high-resolution method, we were able to measure, for the first time, both the penetration depth and concentrations of molecules applied topically to the ocular surface, either with an intact or removed epithelial layer. As a proof of concept, we tested two classes of fluorescent molecules – Fluorescein and Riboflavin – which are commonly used in ophthalmologic practice. Finally, we used our TPF instrument to test the barrier function of the corneal epithelium and to measure the concentration of non-fluorescent molecules (in this case, dextrans) conjugated to fluorescent dyes as they diffused across the cornea. A pump-probe based technique has been applied in biomedical imaging by Prof. Warren Warren’s group recently. They reported images with endogenous contrast agents (hemoglobin and melanin) in biological tissue in a transmission mode with two pulsed laser systems to generate two wavelengths for the pump and probe beam. We built two simplified systems with only one Ti:Sapphire laser. In both systems, the pump and probe beam were selected from a broadband source, which was generated by either broadening the spectrum with a holey fiber or a 27 fs KM laser, which has a broad spectrum itself. We explored the capability of imaging in tissue-like turbid media in the backscattering mode, and studied the achievable imaging depth for the first time. By simulating using Monte Carlo based methods, we further optimized the detection geometry and improved the photon collection efficiency. Also, we compared this nonlinear absorption technique with the more commonly used TPF method. We finally obtained pump probe signals and images using quantum dots as a nonlinear medium. This could be important in future studies of toxicity in skin-care products