Experimental techniques for measuring fluorescence lifetimes were evaluated for the purpose of studying the fluorescence quenching of the biologically important dye ethidium. A synchronously pumped cavity dumped dye laser was used to produce stable high repetition rate picosecond pulses for sample excitation. A system was established for recording nanosecond decay profiles by time correlated single photon counting. A careful investigation of the factors which contribute to the instrumental time jitter enabled an overall impulse response of 600 ps to be achieved. Software was developed for analysing fluorescence decay profiles by the method of convolution and curve fitting and its capability for accurately resolving multiple exponential components was evaluated using simulated data. Some improvement in accuracy was obtained by modifying the Poisson weights used in the curve fitting routine to allow for non-statistical errors, caused primarily by the wavelength dependence of the time response of the single photon counting photomultiplier. Fluorescence polarization effects are discussed. The rotational lifetime of Rhodamine 6G in ethylene glycol was determined by measuring the fluorescence polarization anisotropy decay and the Debye-Stokes-Einstein theory reaffirmed for small molecules at moderate viscosities. The torsional rigidity of DNA can be determined by measuring the depolarization of fluorescence of tightly bound (intercalated) ethidium, and this was not observed to change appreciably when the antitumour drug amsacrine also binds to the DNA. The fluorescence quenching of DNA intercalated ethidium by anticancer drugs was studied by accurately resolving the fluorescence decay profiles into exponential components. The lifetimes and proportions are interpreted in terms of current theories for drug binding and fluorescence quenching mechanisms. The fluorescence quenching behaviour by the antitumour drug amsacrine is consistent with a previously proposed electron transfer mechanism.
| Identifer | oai:union.ndltd.org:ADTP/275982 |
| Date | January 1984 |
| Creators | Davis, Lloyd Mervyn |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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