The combination of fluorescence spectroscopy and imaging provides a powerful approach to study the morphology and biochemistry of living biological systems. For example, fluorescence spectroscopic imaging technique offers the capability to reveal the important biochemical and morphological changes during pathological processes based on unique properties of fluorescent molecules such as spectral lineshape, lifetime and anisotropy. This dissertation aims to advance the current fluorescence spectroscopy and microscopy techniques for employment in basic bioscience realm, and to translate these technologies to clinical diagnostic applications. Two specific biological questions are addressed, namely the application of autofluorescence to diagnose epithelial precancer, and the use of a...[ Read more ]

The combination of fluorescence spectroscopy and imaging provides a powerful approach to study the morphology and biochemistry of living biological systems. For example, fluorescence spectroscopic imaging technique offers the capability to reveal the important biochemical and morphological changes during pathological processes based on unique properties of fluorescent molecules such as spectral lineshape, lifetime and anisotropy. This dissertation aims to advance the current fluorescence spectroscopy and microscopy techniques for employment in basic bioscience realm, and to translate these technologies to clinical diagnostic applications. Two specific biological questions are addressed, namely the application of autofluorescence to diagnose epithelial precancer, and the use of autofluorescnece to extract microvasculature in tissue in vivo.

In the study of fluorescence detection of precancer, we first investigated the feasibility of non-invasive monitoring cellular metabolism based on the time-resolved fluorescence spectroscopy. Then, we conducted a systematic study of the single- and two-photon excited autofluorescence from epithelial tissues. Finally, using a DMBA treated hamster cheek pouch model of oral cancer, we studied the noninvasive diagnosis of epithelial precancer based on multimodal two-photon excitation fluorescence imaging technique. Our study demonstrated that the morphological analysis of microscopic images provided both qualitative and quantitative assessments of the cancer development. The NADH fluorescence lifetime reduced with the carcinoma progression. Moreover, the nonlinear optical signals from collagen matrix provide diagnostic information for precancer differentiation. We recently discovered for the first time that hemoglobin, an essential protein molecule in blood, emits strong high-energy Soret fluorescence and can be potentially used as an endogenous contrast agent for imaging of microvasculature. We first characterized the spectral and lifetime properties of hemoglobin fluorescence by using a time-resolved spectroscopic imaging system. Then, the in vivo label-free three-dimensional imaging of microvasculature was performed based on the unique spectral and temporal characteristics of hemoglobin fluorescence. Finally, we explored the potential of in vivo label-free blood count based on the TPEF signals from hemoglobin in erythrocytes and tryptophan in leukocytes. In conclusion, we demonstrated that the multiphoton excitation fluorescence spectroscopy and microscopy of endogenous agents has the tremendous potential to become a routine technique for life science research and clinical diagnosis of diseases.