title: Design and application of single fluorophore dual-view imaging system containing both the objective- and prism-type TIRF.
abstract: Simultaneous detection of two fluorescent markers is important in determination of distance, relative motion and conformational change of nanoparticles or nanodevices. We constructed an imaging system which combines deep-cooled sensitive EMCCD camera with both the objective- and prism-type TIRF. A laser combiner was introduced to facilitate laser controls for simultaneous dual-channel imaging by deliver lasers with different wavelength synchronically via an optic fiber to the sample. The system produces stable signal with extremely low background fluorescence for single-fluorophore detection. It has been applied to study the structure, stoichiometry, and function of the phi29 DNA packaging motor. Single-molecule photobleaching combined with binomial distribution analysis clarified the stoichiometry of pRNA on the motor and elucidated the mechanism of pRNA hexamer assembly. The feasibility of single-molecule FRET with this system was demonstrated. Distance rulers of dual-labeled molecule standards were used to evaluate the system. We have also re-engineered the energy conversion protein, gp16, of phi29 motor for single fluorophore labeling to facilitate the single-molecule studies of motor mechanism. The potential applications of single-molecule high-resolution imaging with photobleaching (SHRImP) and single molecule high resolution with co-localization (SHREC) approaches to the study of the phi29 nanomotor are under investigation.
authors.affiliation: Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, OH 45221, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Dan
authors.initials: D
authors.lastname: Shu
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Wang
authors.forename: Peixuan
authors.initials: P
authors.lastname: Guo