Time resolved emission spectroscopy experiments have numerous applications within the physical sciences. For example, intrinsic and extrinsic fluorescent molecules are used in chemical and biological assays, monitoring of reactions, and imaging of structures. The need to study chemical and biological systems of greater complexity with fluorescent molecules has required the development of more sophisticated time resolved emission spectroscopic instruments, experimental techniques, and data analysis methods. Current research continues to provide new information on fundamental principles and properties of fluorescent molecules, information that is critical to proper interpretation of data generated by these more complex experiments.
Chapter 4 of this dissertation discusses the development of a new instrument capable of performing full-spectrum fluorescence correlation spectroscopy. In Chapter 1 the advantages of full-spectrum fluorescence correlation spectroscopy, as well as proper interpretation of tryptophan fluorescence performed in Chapter 5, are discussed with conclusions, limitations, and concepts for future experiments presented in Chapter 6. Chapters 2 and 3 provide a background on fluorescence principles and multivariate analysis techniques as they apply to full-spectrum fluorescence correlation spectroscopy, and experiments involving tryptophan fluorescence interpretation that are presented in Chapter 5.
For the first time, full spectra are collected during the course of fluorescence correlation spectroscopy measurements, allowing multivariate analysis to be applied to the data. Measurement of instrumental parameters for full-spectrum fluorescence correlation spectroscopy such as observation volume size, two-photon excitation capabilities, concentration limitations, and the range of observable diffusion coefficients are performed and compared to traditional fluorescence correlation spectroscopy instruments. An experimental application of the instrument to fluorescently labeled polystyrene particle diffusion in a polymer solution with a second fluorescent dye is shown. Separation of the two overlapped fluorescence spectra in this mixture by multivariate analysis is performed and diffusion coefficients are calculated. The results show that spectral resolution obtained by multivariate analysis of data collected with this novel instrument provides a method to reduce cross-talk in multicolor fluorescence correlation spectroscopy experiments.
Experiments involving photokinetic matrix decomposition of time resolved emission decay matrices collected for the amino acid tryptophan and peptide mastoparan-X are performed. Tryptophan is a microenvironment sensitive fluorescent molecule that may naturally occur in proteins. The microenvironment sensitivity of tryptophan has been exploited to imply structural changes of proteins in time resolved fluorescence experiments, however tryptophan fluorescence photokinetics are complex. A controversy exists on the proper interpretation and use of tryptophan fluorescence, centered on the origin of the fluorescence from ground-state heterogeneity or excited-state reactions of the molecule. Photokinetic matrix decomposition of time resolved emission decay data has previously been able to differentiate between ground-state heterogeneity and excited state reaction photokinetics within other microenvironment sensitive fluorescent molecules, and is applied in this dissertation to the study of tryptophan fluorescence. Data collected with tryptophan and mastoparan-X suggest that ground-state heterogeneity is the source of complex tryptophan fluorescence decay in proteins.
|Advisor:||Neal, Sharon L.|
|Commitee:||Brown, Steven D., Galileo, Deni, Ridge, Douglas|
|School:||University of Delaware|
|Department:||Department of Chemistry and Biochemistry|
|School Location:||United States -- Delaware|
|Source:||DAI-B 72/04, Dissertation Abstracts International|
|Keywords:||Fluorescence correlation, Full-spectrum, Multivariate, Polymers, Proteins, Time resolved emission spectroscopies|
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