  This thesis centres on the development of multidimensional fluorescence
imaging tools, with a particular emphasis on fluorescence lifetime imaging
(FLIM) microscopy for application to biological research. The key aspects of
this thesis are the development and application of tunable supercontinuum
excitation sources based on supercontinuum generation in microstructured
optical fibres and the development of stimulated emission depletion (STED)
microscope capable of fluorescence lifetime imaging beyond the diffraction
limit. The utility of FLIM for biological research is illustrated by examples
of experimental studies of the molecular structure of sarcomeres in muscle
fibres and of signalling at the immune synapse. The application of
microstructured optical fibre to provide tunable supercontinuum excitation
source for a range of FLIM microscopes is presented, including wide-field,
Nipkow disk confocal and hyper-spectral line scanning FLIM microscopes. The
application of supercontinuum generation to the first super-resolved FLIM
microscope is then described. This novel microscope exploited the concept of
STED with a femtosecond mode-locked Ti:Sapphire laser providing a tunable
excitation beam by pumping microstructured optical fibre for supercontinuum
generation and directly providing the (longer wavelength) STED beam. This STED
microscope was implemented in a commercial scanning confocal microscope to
provide compatibility with standard biological imaging, and exploited digital
holography using a spatial light modulator (SLM) to provide the appropriate
phase manipulation for shaping the STED beam profile and to compensate for
aberrations. The STED microscope was shown to be capable of recording
superresolution images in both the lateral and axial planes, according to the
settings of the SLM.

Instrumentation and Detectors;Biological Physics;Optics