The Design and Construction of Novel Near -Infrared Time -Correlated Single Photon Counting Devices for the Identification of Analytes in Multiplexed Applications.
Date of Award
Doctor of Philosophy (PhD)
This manuscript details the design, construction, and application of novel near infrared time correlated single photon counting devices to the identification of analytes in analytical separations. The thrust of this research is to provide a simple, low cost technique for the high-speed identification of DNA sequencing bases that are labeled with a series of unique near infrared fluorophores. These fluorophores are unique because they possess the same emission and absorption maxima, but different fluorescence lifetimes. Consequently, they allow analytes to be discriminated by fluorescence lifetime as opposed to color. The first goal of this dissertation research was to implement a time correlated single photon counting system with the use of single mode fiber optics. Utilizing a passively mode locked Ti: Sapphire Laser, a single photon avalanche diode, single mode fiber optics and a mechanical switch a fiber optic based time correlated single photon counting device with subnanosecond resolution was constructed. The experimental results showed that group velocity dispersion was low and that it was possible to perform multiple time correlated single photon counting experiments with a limited number of excitation sources and detectors. It was determined that the average instrumental response of each channel was 181 picoseconds. The fluorescence lifetime of a near infrared dye, aluminum tetrasulfonated naphthalocyanine was determined to be 3.08 nanoseconds. The second phase of this doctoral research involved the construction and characterization of a near infrared time correlated single photon counting scanning device. This integrated device consisted of a pulsed diode laser, single photon avalanche diode, and a time correlated single photon counting board. The instrument response function of this system was determined to be less than 300 ps. The sensitivity and ability to discriminate between various fluorophores was determined. In addition to its application for scanning solid surfaces such as DNA microarrays, the device was utilized to detect analytes in a micro-capillary electrophoresis separation. The fluorescence lifetimes of these analytes were determined on-line.
Waddell, Emanuel Austin Jr, "The Design and Construction of Novel Near -Infrared Time -Correlated Single Photon Counting Devices for the Identification of Analytes in Multiplexed Applications." (2000). LSU Historical Dissertations and Theses. 7305.