Fluorescence lifetime imaging is an established imaging technique that has been extensively explored over the past few decades but has yet to break through as a diagnostic tool in clinical applications. This work will characterize and analyze the quasi-fluorescence lifetime technique with improvements in contrast by a novel approach to manipulate LEDs photonic characteristics to push forward a real time video rate relative lifetime imaging technique. This thesis establishes the framework of using quasi-fluorescence lifetime imaging in the specific long gate time acquisition mode and instating the parameters for any future research of multi-spectral lifetime imaging. Results and analysis of changes in spectral lifetime signatures of collagen and elastin in dry, hydrated, and in acetone chemical environments are presented for the first time using this technique.

Failed wounds in the extremities are characterized with abnormal collagen production and inappropriate bone growth in soft tissue. This condition can occur after trauma to a limb and can cause a disruption to the healing process, and is known as Heterotopic Ossification (HO). In HO, regions in the tissue start to mineralize and form microscopic bone-like structures. These structures continue to calcify and develop into large, non-functional bony masses that cause pain, limit limb movement, and expose the tissue to reoccurring infections; in the case of open wounds this can lead to amputation as a result of a failed wound. Both Magnetic Resonance Imaging (MRI) and X-ray imaging have poor sensitivity and specificity for the detection of HO, thus delaying therapy and leading to poor patient outcomes. This work presents a bi-modality approach using real-time fluorescence lifetime imaging and Raman imaging for tissue differentiation. The fluorescence system is based on relative lifetime contrast generation to characterize surface tissue morphology and detect margins of abnormal tissue, specifically connective tissue biomarkers. The Raman system utilizes a biocompatible, fast, and a large field of view (1 cm2) acquisition to differentiate bone tissue from soft tissue without using a spectrometer, this is in contrast to conventional Raman microscopy systems. This capability may allow for the development of instrumentation which permits bedside complementary diagnosis of HO.