Developing endoscopic instrumentation and techniques for in vivo fluorescence lifetime imaging and spectroscopy

• Main Author: Thompson, Alexander James
• Other Authors: Dunsby, Christopher ; French, Paul ; Neil, Mark ; Elson, Dan
• Published: Imperial College London 2013
• Subjects: 530
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616820

Confocal fluorescence endomicroscopes employ fibre optics along with miniaturised scanning and focussing mechanisms to allow microscopic investigation of remote tissue samples with sub-cellular resolution. For this reason they are widely used in biomedical research, both in clinical studies and in small animal imaging experiments. Fluorescence lifetime imaging microscopy (FLIM) has been shown to provide contrast between normal and unhealthy tissue in several diseases including gastro-intestinal (GI) cancer. As such, there is significant interest in developing instrumentation that will allow endoscopic confocal FLIM as this would permit the in vivo investigation of human GI tissue. This thesis describes the development and use of several instruments and techniques aimed at clinically viable in vivo fluorescence lifetime spectroscopy and confocal endomicroscopy. This research has consisted of two broad branches: the study of the fluorescence signature of healthy and diseased tissue both ex vivo and in vivo; and the development of a novel method for achieving beam scanning in confocal endomicroscopy. Firstly the tissue studies are discussed. This begins with the application of a compact steady-state diffuse reflectance/fluorescence spectrometer and a fibre-optic-coupled time-resolved spectrofluorometer to an in vivo investigation of the spectral signatures of skin cancer. This study – which involved the interrogation of 27 clinically diagnosed lesions – was carried out in collaboration with researchers at Lund University in Sweden and revealed significant differences between healthy and diseased tissue both in terms of fluorescence lifetime and steady state reflectance and fluorescence spectra. Further to this study, work is presented charting the development of a clinically viable spectrometer, which measures time-resolved fluorescence spectra with two excitation wavelengths (375 nm and 435 nm) as well as diffuse reflectance spectra. The entire system is contained within a compact trolley (120 x 70 x 55 cm) for easy transportation and safe use in a clinic. It utilises a fibre optic probe to deliver/collect light that can be inserted into the working channel of a medical endoscope meaning that the system can be used to measure diffuse reflectance and time-resolved fluorescence spectra in the GI tract in vivo. The development and testing of this system are discussed and data are presented from both ex vivo and in vivo studies of GI cancer. The second broad section of this thesis focuses more closely on confocal endomicroscopy. Firstly current methods used in this field are discussed and the sources of several drawbacks are explained. A novel approach to laser scanning endomicroscopy is then presented, which requires no moving parts and can be implemented without the need for any distal scanners or optics. This technique is similar in concept to the use of adaptive optics to focus through turbid media: it utilises a proximal spatial light modulator to correct for phase variations across a fibre imaging bundle and then to encode for arbitrary wavefronts at the distal end of that fibre bundle. Thus, it is possible to realise both focussing and beam scanning at the output of the fibre bundle with no distal components, permitting extremely compact endoscopic probes to be developed. Proof-of-principle results are presented illustrating the imaging capabilities of this novel system as well as simulations showing the achievable resolution and field of view in several feasible endoscopic configurations. Overall, this thesis contains work from two quite different projects both aimed at developing novel optical techniques for clinical diagnostic use in endoscopic procedures. The first is aimed at investigating the temporal and spectral properties of the fluorescence and reflectance signatures of cancer, while the goal of the second is to develop improved confocal endomicroscopes.