Functional Biological Spectroscopy and Multiphoton Imaging Using Endogenous Optical Contrast

Endogenous optical signatures of tissue provide information relevant to biological and biomedical questions through measurements of intracellular and extracellular endogenous contrast. To develop sensitive diagnostic methods using this contrast, the important individual endogenous constituents were first evaluated. In particular, based on intracellular fluorescence consistent with NAD(P)H, FAD, and tryptophan in cell suspensions, fluorescence interrogation techniques were defined to determine cellular metabolism and response to metabolism altering treatments. Additionally, the main contributor to extracellular fluorescence, type I fibrillar collagen, was investigated with an in vitro model of collagen polymerization. By defining the fluorescence of collagen due to cross-linking and the structure of fibrillar collagen at a high resolution with second harmonic generation (SHG), sensitive techniques were developed to assess collagen integrity. Next, with tools developed for measurement of the individual endogenous optical constituents in tissue, two model systems were investigated with multiphoton microscopy in order to evaluate processes important in angiogenesis and ovarian carcinogenesis research. Starting with an in vitro model of angiogenesis, the individual intracellular and extracellular optical signals were combined for real-time imaging of angiogenesis related events. By localizing vessel associated cells with two-photon excited fluorescence (2PEF) and their interaction with collagen fibrils with SHG, the dynamics of matrix remodeling were assessed during sprouting events and neovessel growth. A dramatic reorganization of the collagen fibrils by early sprouts and differential collagen remodeling by neovessels was observed, revealing endothelial/matrix events that have previously been difficult to ascertain. Finally, in a model for investigating ovarian carcinogenesis, multiphoton microscopy of endogenous contrast in viable ovarian biopsies resulted in key features for differentiating normal and abnormal tissue. Features based on the morphology of surface epithelium imaged with 2PEF and the underlying collagen structure and integrity imaged SHG as well as cellular redox values determined by two-channel 2PEF were consistent with loss of organization and metabolic changes in the tissue that may aid in early detection and increased understanding of ovarian cancer. Results from the model applications reveal the utility of techniques sensitive to endogenous tissue signatures for non-invasive diagnostic interrogation of biological processes, specifically related to cellular function and cellular/extracellular matrix interactions