Development of novel analytical applications for single molecule fluorescence spectroscopy

The molecular recrossing of single molecules in a defined probe volume was used to investigate photobleaching and saturation of single molecules. The normalized recrossing ratio, Nr/Nt, was defined as the number of molecules that reenter the probe volume (Nr) to the total number of molecules detected (Nt). Saturation irradiance and photobleaching effects were determined as a function of irradiance for Calcein, Fluorescein, R-Phycoerythrin and Streptavidin R-Phycoerythrin-AlexaFluor-647. The light tolerance and the energy transfer process in phycobiliproteins were studied as a function of excitation irradiation and irradiation time. Normalized molecular recrossings showed that energy transfer to a tandem conjugate could reduce the formation of triplet states in R-Phycoerythrin and extend the light tolerance of certain phycobiliproteins. Measuring normalized recrossing ratios serves as a method of optimizing experimental conditions for single molecule detection and examining the light tolerance and energy transfer in single molecular systems. Single molecule fluorescence anisotropy (SMFA) is described to quantify free and bound probe molecules from a complexation reaction. Initially the error on SMFA measurements attributed to photon shot noise and molecular counting error was investigated. The ability to quantify binding was investigated by formulating a ratio of bound to total probe molecules sampled (Nb/Nt ratio). We report on a comparison of three methods to extract fluorescent bursts from single molecules from a ten-minute time trace. The impact on the Nb/Nt ratio using either anisotropy values alone or anisotropy combined with the difference in detector counts (∆n) were investigated. The data analysis methods reduced the systematic error due to scatter. Biotin-Rhodamine 110 (BR110) was used as the labeled probe for these studies. Increasing amounts of the target protein, Neutravidin, were added to a constant amount of BR110. A competitive reaction between labeled BR110 probe and unlabeled Biotin was also investigated. The use of SMFA as a tool to probe molecular complexation will be useful in performing sensitive immunoassays, in drug discovery to investigate and enhance the binding of drugs to their substrates, and to study other molecular interactions.