| Summary: | This thesis discusses the development of novel multi-dimensional fluorescence microscopy, particularly fluorescence lifetime imaging (FLIM) technology, and its application to imaging Förster Resonance Energy Transfer (FRET) events in live cells. Particular emphasis is placed on imaging activation of Ras family GTP-ases and binding to their effectors, including Phospholipase C Epsilon (PLCε). The early part of the thesis discusses FLIM-FRET experiments performed using a standard confocal microscope with time correlated single photon counting (TCSPC) to image interactions between PLCε and Ras. These early experiments suggested a weak interaction but this mode of imaging was too slow to capture dynamics of Ras activation in live cells. The long acquisition times required by the TCSPC microscope prompted the development of a high speed FLIM microscope using wide-field time-gated imaging, which was combined with a Nipkow disc confocal scan head to achieve optical sectioning. This system was characterised and its performance compared with commercially available TCSPC FLIM microscopes, demonstrating the enhancement in imaging speed for comparable accuracy of lifetime determination. This new microscope was subsequently applied to study the activation of the H-Ras oncogene in live cells following EGF stimulation. The latter part of the thesis discusses the development of a second novel microscope system for multiplexed FRET studies – using both FLIM and spectral ratiometric imaging to monitor two different FRET pairs expressed within a single live cell. A CFP-YFP cameleon FRET biosensor was used to probe calcium signals in cells expressing different PLC isoforms and this was complemented by several novel Ras activation sensors that were designed using fluorescent proteins in the red end of the visible spectrum. Calibration experiments were carried out to determine the optimal fluorophores and filter sets for imaging multiplexed biosensors and the potential for imaging dynamics of calcium flux and Ras activation within the same cell were investigated.
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