title: Investigation of photobleaching and saturation of single molecules by fluorophore recrossing events.
abstract: A method for investigation of photobleaching and saturation of single molecules by fluorophore recrossing events in a laser beam is described. The diffraction-limited probe volumes encountered in single-molecule detection (SMD) produce high excitation irradiance, which can decrease available signal. The single molecules of several dyes were detected and the data was used to extract interpeak times above a defined threshold value. The interpeak times revealed the number of fluorophore recrossing events. The number of molecules detected that were within 2 ms of each other represented a molecular recrossing for this work. Calcein, fluorescein and R-phycoerythrin were analyzed and the saturation irradiance and photobleaching effects were determined as a function of irradiance. This approach is simple and it serves as a method of optimizing experimental conditions for single-molecule detection.
authors: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, United States.
authors: Sean M
authors: SM
authors: Burrows
authors: Randall D
authors: RD
authors: Reif
authors: Dimitri
authors: D
authors: Pappas