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Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA packaging motor.

Zhang H, Shu D, Browne M, Guo P - Biomed Microdevices (2010)

Bottom Line: A customized laser combiner was designed and constructed for dual channel single molecule imaging.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.Finally, techniques for dual laser alignment and tuning of laser power for dual-color excitation are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, OH 45221, USA.

ABSTRACT
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.

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Histograms summarized from single molecule studies for FRET efficiencies (a–d) and calculated distances (e–h) for the dsDNA 10 bp, 12 bp, 15 bp, and 17 bp, respectively
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Fig5: Histograms summarized from single molecule studies for FRET efficiencies (a–d) and calculated distances (e–h) for the dsDNA 10 bp, 12 bp, 15 bp, and 17 bp, respectively

Mentions: For all four DNA oligos (10, 12, 15, 17 bp), the individual FRET events were analyzed using the aforementioned equations. The results were then summarized for each sample and displayed in histograms (Fig. 5). Each graph shows either the distribution in FRET efficiency or in the deduced distances calculated from FRET efficiency. Both distributions followed Gaussian distribution and were subjected to Gaussian fitting. The peaks of the fitted curves indicate the mean values of the measurements, while the widths of the peaks represent the variation for each individual molecule in one sample (Fig. 5). The data is summarized in Table 1.Fig. 5


Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA packaging motor.

Zhang H, Shu D, Browne M, Guo P - Biomed Microdevices (2010)

Histograms summarized from single molecule studies for FRET efficiencies (a–d) and calculated distances (e–h) for the dsDNA 10 bp, 12 bp, 15 bp, and 17 bp, respectively
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2812712&req=5

Fig5: Histograms summarized from single molecule studies for FRET efficiencies (a–d) and calculated distances (e–h) for the dsDNA 10 bp, 12 bp, 15 bp, and 17 bp, respectively
Mentions: For all four DNA oligos (10, 12, 15, 17 bp), the individual FRET events were analyzed using the aforementioned equations. The results were then summarized for each sample and displayed in histograms (Fig. 5). Each graph shows either the distribution in FRET efficiency or in the deduced distances calculated from FRET efficiency. Both distributions followed Gaussian distribution and were subjected to Gaussian fitting. The peaks of the fitted curves indicate the mean values of the measurements, while the widths of the peaks represent the variation for each individual molecule in one sample (Fig. 5). The data is summarized in Table 1.Fig. 5

Bottom Line: A customized laser combiner was designed and constructed for dual channel single molecule imaging.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.Finally, techniques for dual laser alignment and tuning of laser power for dual-color excitation are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, OH 45221, USA.

ABSTRACT
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.

Show MeSH