Design and application of single fluorophore dual-view imaging system containing both the objective- and prism-type TIRF.

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.