Development of instrumentation for autofluorescence spectroscopy and its application to tissue autofluorescence studies and biomedical research
Creators: name: De Jesus Reis Lagarto, Joao
Publisher: name: Imperial College London
Year: 2014

description: Autofluorescence spectroscopy is a promising non-invasive label-free approach to characterise biological samples and has shown potential to report structural and biochemical changes occurring in tissue owing to pathological transformations. This thesis discusses the development of compact and portable single point fibre-optic probe-based instrumentation for time-resolved spectrofluorometry, utilising spectrally resolved time-correlated single photon counting (TCSPC) detection and white light reflectometry. Following characterisation and validation, two of these instruments were deployed in clinical settings and their potential to report structural and metabolic alterations in tissue associated with osteoarthritis and heart disease was investigated. Osteoarthritis is a chronic and progressive disease of the joint characterised by irreversible destruction of articular cartilage for which there is no effective treatment. Working with the Kennedy Institute of Rheumatology, we investigated the potential of time-resolved autofluorescence spectroscopy as a diagnostic tool for early detection and monitoring of the progression of osteoarthritis. Our studies in enzymatically degenerated porcine and murine cartilage, which serve as models for osteoarthritis, suggest that autofluorescence lifetime is sensitive to disruption of the two major extracellular matrix components, aggrecan and collagen. Preliminary autofluorescence lifetime data were also obtained from ex vivo human tissue presenting naturally occurring osteoarthritis. Overall, our studies indicate that autofluorescence lifetime may offer a non-invasive readout to monitor cartilage matrix integrity that could contribute to future diagnosis of early cartilage defects as well as monitoring the efficacy of therapeutic agents. This thesis also explored the potential of time-resolved autofluorescence spectroscopy and steady-state white-light reflectometry of tissue to report structural and metabolic changes associated with cardiac disease, both ex vivo and in vivo, in collaboration with clinical colleagues from the National Heart and Lung Institute. Using a Langendorff rat model, the autofluorescence signature of cardiac tissue was investigated following different insults to the heart. We were able to correlate and translate results obtained from ex vivo Langendorff data to an in vivo myocardial infarction model in rats, where we report structural and functional alterations in the infarcted and remote myocardium at different stages following infarction. This investigation stimulated the development of a clinically viable instrument to be used in open-chest surgical procedures in humans, of which progress to date is described. 4 The impact of time-resolved autofluorescence spectroscopy for label-free diagnosis of diseased would be significantly enhanced if the cost of the instrumentation could be reduced below what is achievable with commercial TCSPC-based technology. The last part of this thesis concerns the development of compact and portable instrumentation utilising low-cost FPGA-based circuitry that can be used with laser diodes and photon-counting photomultipliers. A comprehensive description of this instrument is presented together with data from its application to both fluorescence lifetime standards and biological tissue. The lower potential cost of this instrument could enhance the potential of autofluorescence lifetime metrology for commercial development and clinical deployment.