Exploiting multi-photon excited fluorescence spectroscopy to solve problems in analytical chemistry
Bukowski, Eric John
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This dissertation puts forth a plan along with supporting experimental results using multi-photon excited fluorescence as a tool for chemical analysis in optically challenging samples. The document will compare data collected under one-photon (1PE), two-photon excitation (2PE), and multi-photon excitation (MPE) conditions as it applies to the development of novel detection strategies in optically dense samples. Most modern assays rely on spectroscopic measurements because they are simple and they can provide outstanding detection limits (down to the single molecule level). Unfortunately, optically dense samples make it almost impossible for one to obtain accurate analytical information directly through traditional spectroscopic techniques. If one thinks about this problem, it boils down to an inability of the spectroscopic probe beam from entering the sample due to strongly absorbing or scattering species which are intrinsic to the sample. Today, the solution to this problem is to remove the interfering species, but this approach increases time of analysis and it involves one or more pre-treatment procedures that can lead to sample adulteration and/or biasing. A more satisfying solution would involve direct analysis of the sample without any pre-treatment step. The characteristics of today's mode-locked titanium:sapphire (Ti:S) lasers make them an attractive light source for new types of spectroscopic measurements, including two-photon excitation, wherein the species of interest simultaneously absorbs n-longer wavelength photons to produce an excited electronic state. These advantages, when coupled with other techniques such as phase-resolved fluorescence spectroscopy, significantly reduce the background associated these types of measurements. This document also reports upon the addition of a time-correlated single photon counting detection set-up to our instrument. This new capability proved invaluable during our investigation of visible emission from various indole molecules. More specifically, our research presents the first ever visible emission from intrinsic tryptophan residues within proteins.