Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA packaging motor.
A customized laser combiner was designed and constructed for dual channel single molecule imaging. The feasibility of a combiner-incorporated imaging system was demonstrated in studies of single molecule FRET. Distance rulers made of dual-labeled dsDNA were used to evaluate the system by determining the distance between one FRET pair. The results showed that the system is sensitive enough to distinguish between distances differing by two base pair and the distances calculated from FRET efficiencies are close to those documented in the literature. The single molecule FRET with the dual-color imaging system was also applied to reconstructed phi29 motor pRNA monomers. Finally, techniques for dual laser alignment and tuning of laser power for dual-color excitation are discussed.
Zhang
Hui
H
Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, OH 45221, USA.
Shu
Dan
D
Browne
Mark
M
Guo
Peixuan
P
D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005240:Feasibility Studies; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D007834:Lasers; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*
D004279:DNA, Viral; D012367:RNA, Viral