Autofluorescent proteins have become an invaluable research tool throughout the biological sciences. Sensitive spectroscopic methods for detecting and analyzing these molecules can provide insight into their potential as well as their limitations. Single-molecule spectroscopy is an emerging example of these methods that unearths information normally discarded by traditional measurements on large ensembles. The work in this thesis is dedicated to a series of projects involving autofluorescent proteins which provide information regarding their chemical and physical properties, and demonstrate a previously unreported application of single-molecule spectroscopy in living bacteria.; The prior discovery of the DsRed protein from Discosoma genus of coral was an important step in the development of genetically encoded, red-emitting chromophores that can be efficiently detected within the autofluorescence present in biological systems. This work documents a series of early studies on the photophysics of the Ds Red chromophore, demonstrating that single tetrameric units are relatively bright, stable emitters for single-molecule detection. Also, the rapid energy transfer among the complex's four chromophores was analyzed.; In the following chapter, the fluorescent properties of a blue-emitting fluorescent protein are investigated with respect to elevated pressures and lowered temperatures. Applying either condition constrains the rotation of the protein's inherent chromophore, thus increasing its quantum yield of fluorescence and disfavoring quenching processes, as monitored by analysis of the fluorescence decay in both the picosecond and nanosecond regimes. Understanding the mechanisms behind the performance limitations at room temperature and atmospheric pressure provides insight towards the development of more effective biocompatible blue emitters.; Finally, a series of experiments are described which outline the tracking of single autofluorescent fusion proteins within the membrane of the bacterium Caulobacter crescentus. This is the first single-molecule study within live bacteria of which we are aware, and demonstrates the collection of information that would otherwise be obscured by ensemble averaging. Apparent diffusion coefficients as a function of developmental stage or subcellular position are extracted, with implications regarding the regulation of an important protein in the organism's life cycle.
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