The development of multi-dimensional fluorescence microscopy and its application to the study of membrane lipid microdomains

This thesis describes the development and application of multi-dimensional fluorescence imaging to the study of membrane lipid microdomains. The work makes extensive use of spectrally- and temporally- resolved fluorescence microscopy and the novel, phase-sensitive, membrane-partitioning dye di-4-ANEPPDHQ. The methods are applied to live cell imaging including imaging of the immunological synapse between effector cells and their targets. It is demonstrated that the 60 nm spectral blue-shift in the fluorescence emission of di-4-ANEPPDHQ between the disordered and ordered phases that has been presented in the literature is accompanied by a 1900 ps lengthening in fluorescence lifetime. After optimising conditions, it is further demonstrated that the two contrast parameters are applicable to live cell microscopy and that they provide useful biological information about the order in the membrane. As the dye is excited in the blue spectral region and is relatively photostable, it can be used with single-photon excitation from commonly available sources and is compatible with widefield and TIRF microscopy. A rapid, optically-sectioning, line-scanning hyperspectral fluorescence lifetime imaging microscope is presented, characterised and demonstrated. As well as being able to extract intrinsic contrast from unstained biological tissue, this system is capable of imaging the spectral and lifetime changes of di-4ANEPPDHQ simultaneously in both artificial membrane constructs and live cells. From this, it is shown that it is likely that the dye is sensitive to the differential penetration of water into the lipid bilayer. The use of the probe is then demonstrated in a range of biologically-interesting examples. These include monitoring the change in lipid order during cholesterol depletion from the membrane using a high-speed, sectioning fluorescence lifetime imaging microscope, imaging diseased and healthy primary human T cells and imaging membrane nanotubes. Finally, membrane lipid order at the immune synapse between effector cells (T and NK cells) and their targets is studied. It is shown that order is patterned at the synapse in a distribution compatible with the so-called supramolecular activation clusters that have been observed in the protein distributions in these systems. These results show membrane lipid microdomains may play a key role in T cell activation and that multidimensional fluorescence microscopy and di-4-ANEPPDHQ are useful tools for imaging their spatial and temporal organisation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo