High-data throughput using laser-based, time-resolved fluorometry
Abstract
High-data throughput can achieve two goals in time-resolved fluorescence. It can produce very high signal-to-noise ratio data in collection times normally associated with slower variants of the experiment. And second, it can obtain decays very quickly at signal-to-noise ratios normally associated with those slower approaches. Historically, fluorescence-decay data could only be obtained over a period of time encompassing several minutes. This has precluded its application to problems involving reacting systems, turbulent flows, and chromatographic and flow-cytometric effluent. One method which possesses near-ideal qualities as a trace-level, time-resolved detection scheme is time-correlated single-photon counting with time-filtered detection. Unfortunately, this system has received little attention as a trace-level, on-the-fly detection scheme as a result of low data-processing rates. Low-processing rate limitations are surmounted in time-correlated single-photon counting by configuring the timing electronics in a reverse-interactive configuration. This configuration yields a six-fold enhancement in sensitivity over the conventional-noninteractive mode. With such improvements, the detection scheme is capable of obtaining temporal and quantitative information in 10 ms. This latter result prompted our interest into designing a new detection scheme capable of processing temporal information in shorter collection periods. This work demonstrates that photomultiplier anode-current sampling with a digital oscilloscope is capable of collecting decay data in 8.058 $\mu$s. The excited-state lifetime is often employed in conjunction with other spectroscopic data to probe molecular conformations and photophysical dynamics of emitting substances. Typically, the decay data observed from complex emitting systems will be comprised of distributions of lifetime parameters. A high-precision decay is required for the recovery of these distributions. This thesis also demonstrates that the method of maximum entropy is successful at recovering distributions of decay parameters from decay data emitted by a large protein molecule.
Degree
Ph.D.
Advisors
Lytle, Purdue University.
Subject Area
Chemistry|Analytical chemistry
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