Monophoton decay time fluorometry : theory, design, and application to biochemical systems Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/2514np641

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  • In the determination of binding heterogeneity and rotational diffusion relaxation times, fluorescence decay curves often take the form of sums of exponential decay times. An instrument is described which includes features permitting the collection of high precision time decay data, which may successfully be resolved into component decays. The instrument incorporates several novel features that significantly improve the monophoton technique. A decay curve is built up by successively adding to the memory.of a pulse height analyzer events, which represent the time between a brief excitation pulse and the detection of a single emitted photon from the sample. This decay curve represents the true probability of emission as a function of time only if the events measured are single photon events. By measuring the pulse height of the detected signals and rejecting all events not characteristic of single photons, we are able to collect single photon data at a rate significantly higher than that required for avoiding multi-photon events by using signal attenuation. The performance of the instrument is demonstrated by a number of tests. In all cases the experimental data are analyzed by a method of moments. We have successfully measured the decay time of NADH in water and obtained a value, 0.48 ± .05 nanoseconds, which is much shorter than the three nanosecond half width of our excitation pulse. Subnanosecond discrimination is demonstrated by the difference in the reproducible decay times of air equilibrated and deoxygenated solutions of anthracene in benzene, 3.65 and 4.00 nanoseconds respectively. We rigorously demonstrate the ability to resolve a double exponential decay curve by successively adding to the memory of the pulse height analyzer data from a solution of quinine and a solution of anthranilic acid. The data from each are analyzed and the results compared with a double exponential analysis of the summed decay curve. Finally we give the first example of the analysis of a single polarized component of emission yielding the zero point anisotropy, the excited state lifetime, and the rotational relaxation time of a dye protein complex. These values for an ANS-apomyoglobin complex are 0.33, 16.5 nanoseconds, and 28.4 nanoseconds respectively.
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