Abstract
This thesis presents the development and application of multi-dimensional ˛uorescence imaging based on confocal microscopy and time-correlated single photon counting (TCSPC) instrumenta-tion, to reveal the relationship between microenvironment and order parameters of lipid bilayers, which are the fundamental entities of cell membranes. The objective is to use all the informa-tion available from the ˛uorescence signal - emission wavelength, lifetime and polarisation - to independently interrogate several biophysical properties of biomembranes, simultaneously.The photophysical characterisation of membrane dye laurdan was carried out using ˛uorescence spectroscopy, and this showed that its emission wavelength and ˛uorescence lifetime report on dif-ferent characteristics of its environment, namely, hydration and polarity. Time-resolved anisotropy measurements were used to report on two membrane order parameters related to the hindered ro-tational di˙usion of the ˛uorophore in the bilayer. An experimental and analytical framework was implemented in order to measure and quantify these four parameters, on the same ˝eld of view. This method was then applied to the extensive characterisation of the relationship between these membrane properties in arti˝cial bilayers, and showed distinct dependence of membrane microen-vironment and order to temperature and to membrane chemistry, i.e. the degree of saturation and cholesterol content. This approach also proved informative in resolving the dynamics of domain mixing in ternary lipid mixtures. Applied to the membranes of live cells, simultaneous imaging of membrane micro-environments and order parameters showed that while plasma and internal membranes display di˙erent membrane polarity and order parameter values, linked to their di˙er-ent chemical composition, the relationship between these parameters is preserved throughout the cell. The role of cholesterol in the preservation of these parameters was investigated, showing that cholesterol strongly determines membrane hydration and polarity, while the order parameters are less sensitive to modulation of cholesterol content. Interpretation of spectral and lifetime informa-tion allowed discrimination between the e˙ect of cholesterol on membrane hydration and polarity. Cell-derived plasma membrane vesicles were also characterised as a system to study lipid bilayers with compositions more faithful to that of the cell membrane, and results suggest that the lipid content of such vesicles is strongly determined by the modalities of their formation.
Lastly, strategies to improve the analysis of multi-dimensional time-resolved ˛uorescence image data were explored. A novel processing routine based on principal component analysis was created, allowing sensitive detection of lifetime contrast in photon-limited TCSPC images, and the potential to analyse time-resolved anisotropy data in a pixel-wise fashion was also demonstrated.
| Date of Award | 2018 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Klaus Suhling (Supervisor) & Dylan Owen (Supervisor) |