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Title: Quantitative fluorescence microscopy
Author: Elder, A. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2008
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The work presented here improves the level of quantification achievable with fluorescence microscopy by integrating novel technologies and developing new experimental and theoretical methodologies. Initial work focused on the use of fluorescence microscopy for the quantification of molecular interactions in living cells. This resulted in the development of an analysis routine for the quantification of Förster resonance energy transfer (FRET) by intensity-based sensitised acceptor emission measurements. The developed technique enabled quantification of the strength of interaction as well as the relative stoichiometry of free and bound fluorophores. The work culminated in the dynamic measurement of the cyclin – cyclin dependent kinase interaction through the course of the cell cycle. To improve the flexibility of microscopy techniques, a confocal microscopy system was designed and built which used novel fibre-based supercontinuum illumination technique and a prism-based spectrometer to provide wavelength resolved measurements. The multiparametric imaging approach which this system enabled was shown to aid in the quantification of complex systems. The remainder of this thesis considers the development of new frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) techniques. The advantages of lifetime imaging techniques were illustrated through their application to quantitative chemical analysis in microfluidic devices. Novel illumination technology was integrated into FD-FLIM systems; both in the form of inexpensive light emitting diodes and fibre-based supercontinuum technology. An in-depth theoretical analysis permitted the development of systems with much improved photon economy. Using extensions of the AB analysis technique, multicomponent lifetime data could be accurately quantified. finally, a new experimental technique was implemented, termed ø2FLIM, which enabled the rapid acquisition of alias-free fluorescence lifetime data.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available