Bailey, EJ; (2015) Polarised Fluorescence and Stimulated Emission Depletion Studies of Excited State Dynamics. Doctoral thesis , UCL (University College London).

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Abstract

This thesis presents the research undertaken in time-resolved fluorescence inten¬sity and anisotropy techniques and their application. New information is revealed concerning the emission behaviour of a synthetic multipolar branched chromophore (AF257). Investigation of the relative radiative rates of fluorescing components in fluorescent proteins EGFP and mCherry was carried out using stimulated emission depletion (STED). Previous assumptions in the modelling of both continuous wave (CW) and pulsed STED are shown to be insufficient, and refinements are proposed and tested. The fluorescence techniques employed in this thesis utilise photoselection of an or¬dered excited state through polarised single and two-photon laser excitation, in addition to polarised, time-resolved measurement of sample fluorescence. Chapter one introduces the fluorescence process, the time-evolution of the alignment in the excited state, and the core measurement processes used. Chapter two examines in detail the absorption-emission mechanism of AF257 using both two-photon and single-photon excitation. By combining fluorescence intensity and anisotropy mea¬surements, it was possible to resolve the emission into that from two distinct excited state geometries. Such a thorough analysis has not been previously published for a molecule of this type; this synthesis of information enables the proposition of a new model for the absorption-emission process. The second half of the thesis concerns the use of STED, which incorporates the orientationally sensitive de-excitation of the excited state distribution. Chapter 3 employs CW STED to probe the fluorescence lifetimes and radiative rates of the commonly used fluorescent probes EGFP and mCherry. It is shown that each of these molecules consist of two fluorescing components with individual radiative rates and varying amplitudes (with STED power). A single ‘average’ lifetime and radiative rate is often used for these molecules in biological applications, and the differences observed here could have major implications for the accuracy of results obtained in their application in the biosciences. The chapter goes on to assess the assumption of an average STED rate in CW STED. Using fluorescence anisotropy measurements and numerical simulation, the importance of orientational dependence of fluorescence lifetimes in the analysis of STED measurements is shown. Chapter 4 investigates the effects of pulse stretching and solvent viscosity in pulsed STED. Analysis of this data shows the breakdown of the previously used model with the use of very long (of the order of 2-10 times the rotational correlation time) STED pulses. Revisions to the model are developed and tested. From the results of these revised models it is concluded that the rotation of the higher order spherical harmonic moments in the excited state distribution has a highly significant effect on the observed depolarisation of the excited state alignment, and that further improvements to the model are needed to incorporate this effect.

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