This thesis describes the development and application of multidimensional fluorescence imaging to signalling events at the Natural Killer cell immunological synapse. The primary techniques used in this work are intensity imaging, ratiometric spectral fluorescence imaging and fluorescence lifetime imaging, which have been applied to live and fixed cells.
It is shown that although protein accumulation at the immunological synapse can simply be determined by intensity imaging, the presence of protein does not indicate that signalling events are occuring. Signalling at the inhibitory synapse as determined by KIR2DL1 receptor phosphorylation is imaged by means of confocal FLIM. The resolution achievable using this technique is then improved upon by the use of optical tweezers for cell reorientation.
A comparison of the sectioning abilities of single point confocal and multiphoton microscopy with multipoint spinning disk based systems is made and a means of achieving an increased rate of imaging for the gold standard of FLIM methods, TCSPC FLIM, is proposed.
The proposed multifocal multiphoton TCSPC FLIM system is first simulated and then implemented, with a comparison to widefield time-gated FLIM being carried out. The system is then used to image test samples, and to acquire cell-level metabolic information with the highest time resolution achieved to date via autofluorescence imaging of NADH.
Membrane order at activating and inhibitory NK cell immunological synapses is examined by means of ratiometric imaging of a lipid phase-sensitive dye, and software is developed for the analysis of NK cell spreading patterns, and this software was used to demonstrate that the spreading behaviour of NK cells is affected by the type ofligands encountered in terms of the symmetry and dynamics of spreading
