| Fluorescence spectroscopic methods, while lacking in the structural detail provided by the other techniques, are remarkably non-invasive and are characterized by high signal-to-noise, allowing for observation of complex and dynamic environments. In this dissertation, time resolved and single molecule fluorescence methods are developed which provide new tools for observing biomolecular dynamics and heterogeneity.; The first part of this dissertation focuses on the use of long lifetime fluorescence probes to ascertain the domain motions of proteins in different environments on the nanosecond time scale. Pyrene maleimide, which has a 100 ns lifetime component is shown to be useful for observing the dynamic motions of protein subunits on the 10 to 100 ns time scale.; Secondly, a technique is developed for the observation of internal dynamics in tyrosyl-containing peptides. Tyrosyl rotamer interchange rates are elucidated using a matrix formalism. For larger peptides, the interchange rates for two of the rotamers dominate the dynamics. Given this understanding of tyrosyl photophysics in peptides, tyrosine-to-tryptophan time resolved energy transfer is used to gain an understanding of the heterogeneity in a Leucine Enkephalin derivative in aqueous and membrane environments.; Next, we focus on the photophysical and dynamic properties of fluorescent dyes conjugated to oligonucleotides. We have assessed the utility of several commonly used dyes for rotational dynamics measurements of oligonucleotides important for analytical assays. We have shown that Texas Red, while not often used for such measurements, displays superior photophysical properties to other commonly used dyes. In addition, it demonstrates a fortuitous interaction with the double-stranded oligo in a non-specific groove-binding fashion allowing for observation of the oligo rotational dynamics.; Finally, a technique utilizing femtosecond fiber lasers for two photon imaging and fluorescence fluctuation spectroscopy, termed TP-FLEX (two photon fiber laser excitation) is developed. The two photon excitation signal from fiber lasers is shown to be of comparable stability to commonly used one photon excitation sources on timescales longer than 1 mus. Red-shifting the excitation wavelength of the laser with photonic crystal fibers allows for observation of spectral detail of stained cellular and tissue samples. |
