Enhanced Detection Using Femtosecond Laser Driven Excitation of Single- and Two- photon Activated Fluorescent Dyes

This work examines fluorescence processes activated by high intensity femtosecond laser pulses. Fluorescence processes have a wide range of scientific uses. One notable use is PCR and CRISPR based methods for detection of COVID-19. The work reported in this thesis utilizes both single-photon and two-photon activated dyes. Issues inherent to high intensity femtosecond laser excitation are investigated. Some issues include photobleaching of fluorescent dye and solvent breakdown causing a shift in peak wavelength of fluorescence emissions. The solvent breakdown is quantitatively examined using a gas chromatograph/mass spectrometer. A two-photon dye that can only be activated using the high intensities available with a pulsed laser source is used to bypass these issues. Other areas examined are the optical detection of fluorescence emissions, and ways to improve detection. Improvements to optical detection that were investigated include use of an integrating sphere, different photodetectors, different optical filters, and lock-in amplification schemes. Implementing these improvements resulted in a lowest detectable dye concentration of 1.44 x 1012 copies/µL using a two-photon activated dye. Advisor: Craig A. Zuhlk