Applications and optimization of a two-photon excited fluorescence fiber-optic probe
Abstract
Fiber-optic remote sensing using two-photon induced molecular fluorescence was presented as a potential in situ monitoring technique for major fluorescent components in a simulated fermentation broth. It was shown that the two-photon induced signal is relatively unperturbed by the presence of a cellular matrix, allowing quantitation at very high cell densities. A small decline in two-photon induced signal with increasing cell density was hypothesized to be the result of mie-scattering. A fiber-based sample chamber for cryogenic two-photon measurements was developed and evaluated. The fiber-based system allowed the sample to be completely immersed in the cryogenic medium resulting in more efficient temperature control. The excellent signal stability afforded by this configuration will permit cryogenic measurements which were hitherto impossible using high average-power mode-locked lasers. Attempts were made to implement the sample cell for collection of two-photon excitation spectra. Optimization of the remote two-photon method was attempted by exploiting the greater sensitivity afforded at higher laser repetition rates. Although maximum two-photon sensitivity is achieved at a 4-MHz repetition rate, the pulse rate employed in prior remote two-photon studies was restricted to 160 kHz, as dictated by the maximum start rate of the time-to-amplitude converter (TAC) in the conventional-noninteractive arrangement. Operation of the TAC in the reverse-interactive mode permitted use of higher repetition rates, and yielded greater signal magnitude; however, the presence of a long-lived fiber background caused a large increase in the method blank with repetition rate. The nonlinearly-induced interferant resulted in severe signal-to-blank ratio degradation, prohibiting the implementation of higher experimental duty-cycles. A preliminary investigation of the background's origin was undertaken. The detection capabilities of the time-resolved, two-photon method were investigated in the absence of a fiber optic. Picomolar detection limits were attained using right-angle geometry and microscope objectives for focusing and collection optics. Time-filter processing was applied to the quantitative two-photon data to enhance method signal-to-noise. It was demonstrated that time-filtered detection may be successfully employed to reject a short-lived two-photon induced blank signal.
Degree
Ph.D.
Advisors
Lytle, Purdue University.
Subject Area
Analytical chemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.