<p>In this dissertation, we develop pump probe spectroscopy as a method to differentiate different chemical varieties of melanin, a common biopigment, and exploit these differences to improve the accuracy of melanoma diagnosis. This method gives insight into the chemical makeup and secondary structure of melanins. Pump probe spectroscopy is implemented in a scanning laser microscope as a form of multiphoton imaging, where it is used to image biopsies of human pigmented cutaneous lesions. Melanoma diagnosis is clinically challenging: the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. Using pump probe imaging, significant chemical and morphological changes were found between melanoma and melanocytic nevi, including increased eumelanin content, chemical heterogeneity and general pigmentation. Signal processing methods revealed further differences between melanoma and melanocytic nevi on the cellular scale. Pump probe imaging directly in H&E stained biopsy samples allows integration of this technique with existing histopathology protocols. High resolution imaging found chemical heterogeneity of melanin within pigmented cells. We show that oxyhemoglobin and deoxyhemoglobin may also be differentiated by pump probe imaging. Epi mode imaging of eumelanin, pheomelanin and microvasculature is demonstrated in vivo in human xenograft mouse models of melanoma.</p> / Dissertation

Optical imaging methods have the potential to detect ovarian cancer at an early, curable stage. Optical imaging has the disadvantage that high resolution techniques require access to the tissue of interest, but miniature endoscopes that traverse the natural orifice of the reproductive tract, or access the ovaries and fallopian tubes through a small incision in the vagina wall, can provide a minimally-invasive solution. We have imaged both rodent and human ovaries and fallopian tubes with a variety of endoscope-compatible modalities. The recent development of fiber-coupled femtosecond lasers will enable endoscopic multiphoton microscopy (MPM). We demonstrated two-and three-photon excited fluorescence (2PEF, 3PEF), and second-and third-harmonic generation microscopy (SHG, THG) in human ovarian and fallopian tube tissue. A study was undertaken to understand the mechanisms of contrast in these images. Six patients (normal, cystadenoma, and ovarian adenocarcinoma) provided ovarian and fallopian tube biopsies. The tissue was imaged with three-dimensional optical coherence tomography, multiphoton microscopy, and frozen for histological sectioning. Tissue sections were stained with hematoxylin and eosin, Masson's trichrome, and Sudan black. Approximately 1 mu m resolution images were obtained with an excitation source at 1550 nm. 2PEF signal was absent. SHG signal was mainly from collagen. 3PEF and THG signal came from a variety of sources, including a strong signal from fatty connective tissue and red blood cells. Adenocarcinoma was characterized by loss of SHG signal, whereas cystic abnormalities showed strong SHG. There was limited overlap of two-and three-photon signals, suggesting that three-photon imaging can provide additional information for early diagnosis of ovarian cancer.

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