Optimization of Biophotonics and Optomechanics for Frequency Domain Tissue Fluorescence Measurements

Abstract

Collagens and extra cellular matrix (ECM) proteins play important roles in the structural composition of tissues, and their modifications are implicated in diseases including cancer metastasis, tissue fibrosis, diabetic ulcers. Current tissue collagen analyses are based on destructive histological methods, whereas the ECM proteins’ intrinsic autofluorescences can provide a label-free detection and imaging to differentiate tissue compositions, modifications, and disease states. Fluorescence lifetime spectroscopy has been demonstrated as a key technique to distinguish ECM proteins in tissue. However, the large, expensive, and often operationally challenging instrumentation required to measure fluorescent lifetimes from tissues prevents the technique from gaining traction in biomedical applications.To this end, this thesis represents three major efforts to address this roadblock in tissue lifetime instrumentation by 1) demonstrate a facile fluorescence lifetime technique via frequency domain analysis, 2) realizing a hand-held multispectral lifetime probe for point detection on the skin, 3) realizing a low-cost, low complexity LED based imaging system to enable FLIM (fluorescence lifetime imaging microscopy) for histology without daunting instrumentation.In the first effort, the frequency domain (FD) fluorescence lifetime technique was demonstrated for the differentiation of collagens I/III and elastin, three major biophotonic components in wound tissues that undergo change during healing. In the second effort, a compact multispectral xsystem demonstrated the FD lifetime’s utility as a point-of-care device, and added additional wavelength-specific lifetime spectroscopy to further improve differentiation. The third effort expands theFD technique to an imaging modality, by leveraging LED scanning optomechanics to create histological images of tissues without labeling. Additionally, the scanning of a diabetic ulcer showed evidence that the technique can provide quantitative information on disease modification of the tissue that current immunohistochemical methods are ill-equipped to provide. By providing a quantitative, more nuance model of tissue biophotonics while using low-cost, low complexity FD instrumentation, the works in this thesis hopes to increase accessibility and popularity of label-free ECM lifetime spectroscopy for a number of biomedical and clinical applications.