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Flow Cytometry Protocols pp 67–84Cite as

Quantum Dots for Quantitative Flow Cytometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 699))

Abstract

In flow cytometry, the quantitation of fluorophore-tagged ligands and receptors on cells or at particulate surfaces is achieved by the use of standard beads of known calibration. To the best of our knowledge, only those calibration beads based on fluorescein, EGFP, phycoerythyrin and allophycocyanine are readily available from commercial sources. Because fluorophore-based standards are specific to the selected fluorophore tag, their applicability is limited to the spectral region of resonance. Since quantum dots can be photo-excited over a continuous and broad spectral range governed by their size, it is possible to match the spectral range and width (absorbance and emission) of a wide range of fluorophores with appropriate quantum dots. Accordingly, quantitation of site coverage of the target fluorophores can be readily achieved using quantum dots whose emission spectra overlaps with the target fluorophore.

This chapter focuses on the relevant spectroscopic concepts and molecular assembly of quantum dot fluorescence calibration beads. We first examine the measurement and applicability of spectroscopic parameters, ε, φ, and %T to fluorescence calibration standards, where ε is the absorption coefficient of the fluorophore, φ is the quantum yield of the fluorophore, and %T is the percent fraction of emitted light that is transmitted by the bandpass filter at the detector PMT. The modular construction of beads decorated with discrete quantities of quantum dots with defined spectroscopic parameters is presented in the context of a generalizable approach to calibrated measurements of fluorescence in flow cytometry.

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References

  1. Sklar, L. A. (2005) Flow Cytometry in Biotechnology, Oxford University Press, New York, NY.

    Google Scholar 

  2. Nolan, J. P. and Sklar L. A. (1998) The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. Nature Biotechnology 16, 633–8.

    Article  CAS  Google Scholar 

  3. Wang, L., Gaigalas, A. K., Abbasi, F., Marti, G., Vogt, R., and Schwartz, A. (2002) Quantitating fluorescence intensity from fluorophores: Practical use of MESF values. Journal of research of the National Institute of Standards and Technology 107, 339–53.

    CAS  Google Scholar 

  4. Schwartz, A., Wang, A., Early, E. et al. (2002) Quantitating fluorescence intensity from fluorophore: The definition of MESF assignment. Journal of research of the National Institute of Standards and Technology 107, 83–91.

    CAS  Google Scholar 

  5. Wu, Y., Campos, S. K., Lopez, G. P., Ozbun, M. A., Sklar, L. A., and Buranda, T. (2007) The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy. Analytical Biochemistry 364, 180–92.

    Article  CAS  Google Scholar 

  6. Wu, Y., Lopez, G. P., Sklar, L. A., and Buranda, T. (2007) Spectroscopic characterization of streptavidin functionalized quantum dots. Analytical Biochemistry 364, 193–203.

    Article  CAS  Google Scholar 

  7. Parker, C. A. (1968) Photoluminescence of Solutions, Elsevier, Amsterdam.

    Google Scholar 

  8. Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, 2nd edn., Plenum Press, New York, NY.

    Google Scholar 

  9. Gaponenko, S. V. (1998) Optical Properties of Semiconductor Nanocrystals, Cambridge University Press, Cambridge.

    Book  Google Scholar 

  10. Tonti, D., van Mourik, F., and Chergui, M. (2004) On the excitation wavelength dependence of the luminescence yield of colloidal CdSe quantum dots. Nano Letters 4, 2483–7.

    Article  CAS  Google Scholar 

  11. Bawendi, M. G. (1994) Chemistry and Physics of Semiconductor Nanoclusters. Abstracts of Papers of The American Chemical Society 21(208), 4-INOR.

    Google Scholar 

  12. Brus, L. E., Bawendi, M., Wilson, W. L. et al. (1991) Primary Photophysics of Quantum Semiconductor Crystallites. Abstracts of Papers of The American Chemical Society 14(201), 409-INOR.

    Google Scholar 

  13. Caruge, J. M., Chan, Y. T., Sundar, V., Eisler, H. J., and Bawendi, M.G. (2004) Transient photoluminescence and simultaneous amplified spontaneous emission from multiexciton states in CdSe quantum dots. Physical Review B 70, 085316.

    Article  Google Scholar 

  14. Dabbousi, B. O., RodriguezViejo, J., Mikulec, F. V. et al. (1997) (CdSe)ZnS core-shell quantum dots: Synthesis and characterization of a size series of highly luminescent nanocrystallites. Journal of Physical Chemistry. B 13, 9463–75.

    Article  Google Scholar 

  15. Danek, M., Jensen, K. F., Murray, C. B., and Bawendi, M. G. (1996) Synthesis of luminescent thin-film CdSe/ZnSe quantum dot composites using CdSe quantum dots passivated with an overlayer of ZnSe. Chemistry of Materials 8, 173–80.

    Article  CAS  Google Scholar 

  16. Efros, A. L., Rosen, M., Kuno, M., Nirmal, M., Norris, D. J., and Bawendi M. G. (1996) Band-edge exciton in quantum dots of semiconductors with a degenerate valence band: Dark and bright exciton states. Physical Review B 15, 4843–56.

    Article  Google Scholar 

  17. Kuno, M., Lee, J. K., Dabbousi, B. O., Mikulec, F. V., and Bawendi, M. G. (1997) The band edge luminescence of surface modified CdSe nanocrystallites: Probing the luminescing state. Journal of Chemical Physics 15, 9869–82.

    Article  Google Scholar 

  18. Norris, D. J., Nirmal, M., Murray, C. B., Sacra, A., and Bawendi, M. G. (1993) Size-dependent optical spectroscopy of Ii–Vi semiconductor nanocrystallites (quantum dots). Zeitschrift Fur Physik D Atoms Molecules and Clusters 26, 1–4.

    Google Scholar 

  19. Norris, D. J., Sacra, A., Murray, C. B., and Bawendi, M. G. (1994) Measurement of the size-dependent hole spectrum in CdSe quantum dots. Physical Review Letters 18, 2612–5.

    Article  Google Scholar 

  20. Norris, D. J. and Bawendi, M. G. (1995) Structure in the lowest absorption feature of CdSe quantum dots. Journal of Chemical Physics 103, 5260–8.

    Article  Google Scholar 

  21. Norris, D. J. and Bawendi, M. G. (1996) Measurement and assignment of the size-dependent optical spectrum in CdSe quantum dots. Physical Review B 15, 16338–46.

    Article  Google Scholar 

  22. Rumbles, G., Selmarten, D. C., Ellingson, R. J. et al. (2001) Anomalies in the linear absorption, transient absorption, photoluminescence and photoluminescence excitation spectroscopies of colloidal InP quantum dots. Journal of Photochemistry and Photobiology A: Chemistry 14, 2–3.

    Google Scholar 

  23. Ellingson, R. J., Blackburn, J. L., Yu, P. R., Rumbles, G., Micic, O. I., and Nozik, A. J. (2002) Excitation energy dependent efficiency of charge carrier relaxation and photoluminescence in colloidal InP quantum dots. Journal of Physical Chemistry B 15, 7758–65.

    Article  Google Scholar 

  24. Babbitt, S. E., Kiss, A., Deffenbaugh, A. E. et al. (2005) ATP hydrolysis-dependent disassembly of the 26 S proteasome is part of the catalytic cycle. Cell 20, 553–65.

    Article  Google Scholar 

  25. Buranda, T., Lopez, G. P., Simons, P., Pastuszyn, A., and Sklar, L. A. (2001) Detection of epitope-tagged proteins in flow cytometry: Fluorescence resonance energy transfer-based assays on beads with femtomole resolution. Analytical Biochemistry 298, 151–62.

    Article  CAS  Google Scholar 

  26. Buranda, T., Huang, J. M., Perez-Luna, V. H., Schreyer, B., Sklar, L. A, and Lopez, G. P. (2002) Biomolecular recognition on well-characterized beads packed in microfluidic channels. Analytical Chemistry 74, 1149–56.

    Article  Google Scholar 

  27. Piyasena, M. E., Buranda, T., Wu, Y., Huang, J. M., Sklar, L. A., and Lopez, G. P. (2004) Near-simultaneous and real-time detection of multiple analytes in affinity microcolumns. Analytical Chemistry 76, 6266–73.

    Article  Google Scholar 

  28. Simons, P. C., Shi, M., Foutz, T. et al. (2003) Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry. Molecular Pharmacology 64, 1227–38.

    Article  CAS  Google Scholar 

  29. Simons, P., Vines, C. M., Key, T. A. et al. (2005) Analysis of GTP-binding protein-coupled receptor assemblies by flow cytometry, in Flow Cytometry in Biotechnology (Sklar, L. A., ed.), Oxford University Press, New York, NY, pp. 323–46.

    Google Scholar 

  30. Buranda, T., Jones, G., Nolan, J., Keij, J., Lopez, G. P., and Sklar, L. A. (1999) Ligand receptor dynamics at streptavidin coated particle surfaces: A flow cytometric and spectrofluorimetric study. The Journal of Physical Chemistry. B 103, 3399–410.

    Article  CAS  Google Scholar 

  31. Kasha, M. (1950) Characterization of electronic transitions in complex molecules. Discussions of the Faraday Society 9, 14–9.

    Article  Google Scholar 

  32. Magde, D., Rojas, G. E., and Seybold, P. G. (1999) Solvent dependence of the fluorescence lifetimes of xanthene dyes. Photochemistry and Photobiology 70, 737–44.

    Article  CAS  Google Scholar 

  33. Buranda, T., Waller, A., Wu, Y. et al. (2007) Some mechanistic insights into GPCR activation from detergent-solubilized ternary complexes on beads. Advances in Protein Chemistry 74, 95–135.

    Article  CAS  Google Scholar 

  34. Babbitt, S. E., Kiss, A., Deffenbaugh, A. E. et al. (2005) ATP hydrolysis-dependent disassembly of the 26 S proteasome is part of the catalytic cycle. Cell 121, 553–65.

    Article  CAS  Google Scholar 

  35. Christopoulos, A. and Kenakin, T. (2002) G protein-coupled receptor allosterism and complexing. Pharmacological Reviews 54, 323–74.

    Article  CAS  Google Scholar 

  36. Walboomers, J. M. M., Jacobs, M. V., Manos, M. M. et al. (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. Journal of Pathology 189, 12–9.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by NIH K25AI60036, U54MH074425, U54MH084690, HL081062, NSF CTS0332315, Dedicated Health Research Funds of the University of New Mexico School of Medicine (C-2294-T). Images in this paper were generated in the University of New Mexico Cancer Center Fluorescence Microscopy Facility, supported as detailed on the Web page: http://hsc.unm.edu/crtc/microscopy/Facility.html.

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Authors and Affiliations

  1. Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM, USA

    Tione Buranda, Yang Wu & Larry A. Sklar

Authors
  1. Tione Buranda
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  2. Yang Wu
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  3. Larry A. Sklar
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Correspondence to Tione Buranda .

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Editors and Affiliations

  1. Medical Center, Flow Cytometry Core Facility, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Teresa S. Hawley

  2. Medical Center, Dept. Anatomy & Regenerative Biology, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Robert G. Hawley

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Buranda, T., Wu, Y., Sklar, L.A. (2011). Quantum Dots for Quantitative Flow Cytometry. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 699. Humana Press. https://doi.org/10.1007/978-1-61737-950-5_4

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  • DOI: https://doi.org/10.1007/978-1-61737-950-5_4

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-949-9

  • Online ISBN: 978-1-61737-950-5

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