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Prism-Based Spectral Imaging of Single-Molecule Fluorescence from Gold-Nanoparticle/Fluorophore Complex

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Abstract

A wavelength-calibration method for prism-based spectral imaging of single-molecule (SM) fluorescence was developed. With this method, a wavelength reference is provided by photoluminescence from 50-nm-diameter gold nanoparticles (AuNPs) binding with fluorophores. The AuNPs each bound with a SM fluorophore, either Alexa488 or Cy3, to form AuNP/fluorophore complexes in tris-HCl buffer. Each complex was immobilized on a silica slide and then excited by total-internal-reflection illumination to make it emit SM fluorescence and AuNP photoluminescence. The portion of the AuNP photoluminescence transmitted by a band-pass filter gives the wavelength reference. A spectral-imaging system composed of a prism-based spectroscope (with a reciprocal dispersion of about 4 nm/μm) and a charge-coupled device with 6.45-μm-square pixels was used to obtain an SM-fluorescence spectrum and a wavelength-reference spectrum. Through smoothed differentiation of these two spectra, the peak location of the former in relation to the latter was determined with subpixel precision. After that, the SM fluorophore was classified as either Alexa488 or Cy3 according to the peak location. The error rate of the classification was estimated to be only 0.3%.

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Acknowledgments

We thank Dr. Miwako Nakahara and Dr. Osamu Kogi for their helpful advice on sample preparation.

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Authors and Affiliations

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

    Tsuyoshi Sonehara, Tomoyuki Sakai, Takanobu Haga & Takeshi Fujita

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

    Satoshi Takahashi

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

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Sonehara, T., Sakai, T., Haga, T. et al. Prism-Based Spectral Imaging of Single-Molecule Fluorescence from Gold-Nanoparticle/Fluorophore Complex. J Fluoresc 21, 1805–1811 (2011). https://doi.org/10.1007/s10895-011-0875-6

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  • DOI: https://doi.org/10.1007/s10895-011-0875-6

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