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Prism-based Spectral Imaging of Four Species of Single-molecule Fluorophores by Using One Excitation Laser

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Abstract

A prism-based imaging system for simultaneously detecting four species of single-molecule (SM) fluorophores was developed. As for the detection method, four spectrally distinct species of BigDye fluorophores were bound to 50-nm-diameter gold nanoparticles (AuNPs) to form AuNP/BigDye complexes. Four species of complexes were randomly immobilized on different fused-silica slides. BigDyes were excited by an argon-ion-laser (excitation wavelengths: 488 and 514.5 nm) beam through total internal reflection on the slide surface. SM fluorescence emitted from a complex was spectrally dispersed through a prism to form an SM spot elongated in the spectral direction on a charge-coupled device. A scattered light spot generated by the AuNP of the same complex under 594-nm laser illumination was used as a wavelength reference, and the SM fluorescence spectrum was obtained from the pixel-intensity pattern of the elongated SM spot. Peak locations of fluorescence spectra of all the observed SM spots were obtained, and their histograms were distinctly separated according to species. SM spots can thus be classified as one of four species according to their peak locations. By statistically analyzing the histograms, the classification accuracy was estimated to be above 93.8 %. The number of pixels in the spectral direction required for classifying four species of SM fluorophores was estimated to be 10. As for the conventional system (which uses two excitation lasers), 15 pixels are required. Using BigDyes as the four fluorophores (which consist of donors linked to acceptors and can be excited by just an argon-ion laser) is the reason that such a small number of pixels was achieved. The developed system can thus detect 1.5 times more SM fluorophores per field of view; that is, its throughput is 1.5 times higher. The approach taken in this study, namely, using BigDye with a prism-type system, is effective for increasing the throughput of DNA microarray-chip analysis and SM real-time DNA sequencing.

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Notes

  1. The relation in Fig. 5 was calculated from the wedge angle of the dispersion prism (7°41′), the refractive index of the dispersion prism (BK7), the optical magnification (×28.3), and the pixel size of the CCD (6.45 × 6.45 μm2/pixel). The refractive index (n) of BK7 at wavelength λ (unit: μm) is calculated by using the Sellmeier equation: \( n={{\left\{ {{{{{{{1+1.04{\lambda^2}}} \left/ {{\left( {{\lambda^2}-0.006} \right)+0.232{\lambda^2}}} \right.}}} \left/ {{{{{\left( {{\lambda^2}-0.02} \right)+1.01{\lambda^2}}} \left/ {{\left( {{\lambda^2}-104} \right)}} \right.}}} \right.}} \right\}}^{{{1 \left/ {2} \right.}}}} \) .

  2. In reference [8], the DNA-sequencing result shows that seven out of 158 bases (i.e., 4 %) were identified as mismatch errors, which were mainly caused by fluorophore misclassifications. The classification accuracy of the conventional prism-type system is thus thought to be about 96 %.

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Acknowledgments

We thank Dr. Osamu Kogi for his helpful advice on sample preparation.

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Authors and Affiliations

  1. Central Research Laboratory, Hitachi, Ltd., 1-280 Higashi-Koigakubo, Kokubunji-shi, Tokyo, 185-8601, Japan

    Takanobu Haga, Tsuyoshi Sonehara & Takeshi Fujita

  2. Research and Development Division, Hitachi High-Technologies Corporation, 882 Ichige, Hitachinaka-shi, Ibaraki-ken, 312-8504, Japan

    Satoshi Takahashi

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  1. Takanobu Haga
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  2. Tsuyoshi Sonehara
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  3. Takeshi Fujita
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  4. Satoshi Takahashi
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Correspondence to Takanobu Haga.

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Haga, T., Sonehara, T., Fujita, T. et al. Prism-based Spectral Imaging of Four Species of Single-molecule Fluorophores by Using One Excitation Laser. J Fluoresc 23, 591–597 (2013). https://doi.org/10.1007/s10895-013-1208-8

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  • DOI: https://doi.org/10.1007/s10895-013-1208-8

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