The work presented in this thesis follows two main branches. The first aims to develop instrumentation for 3D-STED microscopy and to apply it to the study of bulk diamond, nanoparticles and biological samples. The second aims to evaluate the application of fluorescence imaging and spectroscopy techniques to the study of luminescent defects in diamond.
Building on previous work in the Photonics Group at Imperial College London, spatial light modulator (SLM) technology was incorporated into a STED system in a novel configuration to provide a robust and convenient solution for 3D-STED microscopy. This system was applied to the first reported super-resolution imaging of the interaction between two cells in their natural state. The system was further applied to STED imaging of nitrogen vacancy centres in bulk diamond and to a proof of principle experiment for novel plasmon-assisted labels for STED microscopy.
The effects of wavefront aberration on STED microscopy were investigated and a predictive correction philosophy was developed based on spherical aberration induced by a refractive index mismatch. The flexibility offered by the SLM technology was taken advantage of to demonstrate recovery of STED imaging quality in glycerol and bulk diamond by active correction of spherical aberration experienced by the depletion point spread function.
Confocal intensity imaging, confocal fluorescence lifetime imaging (FLIM) and multispectral fluorescence lifetime measurement were applied to the imaging of fluorescent defects in bulk diamond. It was demonstrated that FLIM can provide information that is complimentary to intensity imaging in diamond and that it is possible to spectrally distinguish defects in diamond while simultaneously measuring their lifetime using multispectral lifetime measurement methods.
This thesis also presents the ongoing development of a system for STED of live samples that express green fluorescent protein (GFP).