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

Hui Zhang; Dan Shu; Wenjuan Wang; Peixuan Guo

doi:10.1117/12.847457

17 February 2010 Design and application of single fluorophore dual-view imaging system containing both the objective- and prism-type TIRF

Hui Zhang, Dan Shu, Wenjuan Wang, Peixuan Guo

Author Affiliations +

Proceedings Volume 7571, Single Molecule Spectroscopy and Imaging III; 757107 (2010) https://doi.org/10.1117/12.847457
Event: SPIE BiOS, 2010, San Francisco, California, United States

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 singlefluorophore 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 singlemolecule high-resolution imaging with photobleaching (SHRImP) and single molecule high resolution with colocalization (SHREC) approaches to the study of the phi29 nanomotor are under investigation.

Citation Download Citation

Hui Zhang, Dan Shu, Wenjuan Wang, and Peixuan Guo "Design and application of single fluorophore dual-view imaging system containing both the objective- and prism-type TIRF", Proc. SPIE 7571, Single Molecule Spectroscopy and Imaging III, 757107 (17 February 2010); https://doi.org/10.1117/12.847457

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