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Abstract
Recent advances in multiphoton microscopy have revolutionized the field of biological optical imaging because of its submicrometer resolution and molecular specificity. At the same time, however, new contrasts are desperately needed to move the technology forward to become powerful tools for cell and tissue diagnosis. This dissertation focuses on the development of novel nonlinear optical imaging techniques and their application in a few specific areas.
Traditional pump probe technique has been improved with acousto-optic pulse shaping methods. The sensitivity of two-color pump probe experiments based on nonlinear absorption can measure a 10-6 absorption change easily. Two-color pump probe spectroscopy has been further adopted to a laser scanning microscopy setup. Detailed description of building the laser scanning microscope is given. The sensitive nonlinear absorption detection provides very useful contrast in biological cell and tissue imaging. Specifically, two systems were extensively studied: (1) melanin (the pigment in skin) and melanoma detection, demonstrating that nonlinear absorption imaging can be applied to melanin imaging in skin and has potential in diagnosing melanoma; (2) hemoglobin and its oxygenation in blood vessels, demonstrating imaging of oxyhemoglobin and deoxyhemoglobin in a live mouse, which can be a useful technique to monitor angiogenesis and hypoxia with appropriate tumor model systems.
In another application developing nonlinear molecular contrasts, stimulated Raman and its potential in cell and tissue imaging were studied. Detailed theoretical derivations and comparison to coherent anti-Stokes Raman scattering (CARS) are given. Preliminary results on cuvette samples and polystyrene beads showed that stimulated Raman microscopy is a promising technique to monitor molecular species in a biological environment.
Finally, various challenges and possible solutions in transferring those techniques from the laboratory into a clinical environment in the future are summarized.
