A flexible wide-field fluorescence lifetime imaging (FLIM) endoscope is developed for clinical applications. For remote imaging, a coherent fibre optic bundle was used which required techniques and protocols to be developed to minimise artefacts associated with dispersion and recover accurate lifetime estimates. Potentially gain switched pulsed laser diodes are better suited for the clinic than light sources used conventionally for FLIM endoscopy because they are more compact and lower in cost. By imaging ex vivo human tissue samples, it was demonstrated that gain-switched pulsed laser diodes are a viable alternative excitation light source. The system was able to acquire ∼mm-scale spatial FLIM images from fresh ex vivo diseased human larynx biopsies. For wide-field FLIM with the fastest update rates, gated optical intensifiers (GOIs) are used as detectors. This thesis documents the characterisation of next generation GOI technology that can achieve larger than existing standard GOI standard gate-widths for improved light efficiency and that use a magnetic field to enhance spatial resolution beyond that of proximity focusing alone. For intra vital microscopy, access to tissue surfaces deep inside hollow or solid organs is challenging for microscopes. This thesis applies a commercially available confocal laser scanning endomicroscope (CLSE) adapted for time correlated single photon counting (TCSPC) FLIM to study protein interactions using based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins. Protocols to minimise artefacts associated with dispersion in the CLSE’s fibre probe to recover accurate lifetime estimates are presented. To validate the design, the CLSE was used for a time-lapse study of live cancer cells in vitro to monitor their response to an inhibitor of the ERK intracellular signalling pathway by FLIM FRET of an extracellular signal-regulated kinase (ERK) biosensor.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:749116 |
| Date | January 2015 |
| Creators | Sparks, Hugh |
| Contributors | French, Paul ; Dunsby, Chris |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/28962 |
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