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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 23 — Nov. 8, 2010
  • pp: 23818–23828

EMCCD-based spectrally resolved fluorescence correlation spectroscopy

Felix Bestvater, Zahir Seghiri, Moon Sik Kang, Nadine Gröner, Ji Young Lee, Kang-Bin Im, and Malte Wachsmuth  »View Author Affiliations

Optics Express, Vol. 18, Issue 23, pp. 23818-23828 (2010)

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We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.

© 2010 OSA

OCIS Codes
(040.1520) Detectors : CCD, charge-coupled device
(180.1790) Microscopy : Confocal microscopy
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence

ToC Category:

Original Manuscript: July 13, 2010
Revised Manuscript: September 29, 2010
Manuscript Accepted: October 1, 2010
Published: October 27, 2010

Virtual Issues
Vol. 6, Iss. 1 Virtual Journal for Biomedical Optics

Felix Bestvater, Zahir Seghiri, Moon Sik Kang, Nadine Gröner, Ji Young Lee, Kang-Bin Im, and Malte Wachsmuth, "EMCCD-based spectrally resolved fluorescence correlation spectroscopy," Opt. Express 18, 23818-23828 (2010)

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  1. M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. 4(3), 390–401 (1974). [CrossRef]
  2. E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974). [CrossRef]
  3. D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972). [CrossRef]
  4. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974). [CrossRef] [PubMed]
  5. K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29(1), 74–85 (2003). [CrossRef] [PubMed]
  6. M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. 57(1), 169–190 (2005). [CrossRef]
  7. J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008). [CrossRef]
  8. M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).
  9. J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995). [CrossRef]
  10. J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A 39(5), 2646–2652 (1989). [CrossRef] [PubMed]
  11. P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997). [CrossRef] [PubMed]
  12. M. Burkhardt, K. G. Heinze, and P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. 30(17), 2266–2268 (2005). [CrossRef] [PubMed]
  13. K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004). [CrossRef]
  14. L. C. Hwang, M. Leutenegger, M. Gösch, T. Lasser, P. Rigler, W. Meier, and T. Wohland, “Prism-based multicolor fluorescence correlation spectrometer,” Opt. Lett. 31(9), 1310–1312 (2006). [CrossRef] [PubMed]
  15. M. J. R. Previte, S. Pelet, K. H. Kim, C. Buehler, and P. T. C. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008). [CrossRef] [PubMed]
  16. M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006). [CrossRef] [PubMed]
  17. B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006). [CrossRef] [PubMed]
  18. D. Boening, T. W. Groemer, and J. Klingauf, “Applicability of an EM-CCD for spatially resolved TIR-ICS,” Opt. Express 18(13), 13516–13528 (2010). [CrossRef] [PubMed]
  19. G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009). [CrossRef] [PubMed]
  20. D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009). [CrossRef] [PubMed]
  21. D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006). [CrossRef] [PubMed]
  22. T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express 18(10), 10627–10641 (2010). [CrossRef] [PubMed]
  23. K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. 2(4), 287–305 (1990). [CrossRef]
  24. M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000). [CrossRef] [PubMed]
  25. T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002). [CrossRef]
  26. K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004). [CrossRef] [PubMed]
  27. J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008). [CrossRef] [PubMed]
  28. F. Christen, K. Kuijken, D. Baade, C. Cavadore, S. Deiries, and O. Iwert, “Fast Conversion Factor (Gain) Measurement of a CCD Using Images With Vertical Gradient,” in Scientific detectors for astronomy 2005 (Springer Netherlands, 2006).
  29. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005). [CrossRef] [PubMed]
  30. S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005). [CrossRef] [PubMed]
  31. P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008). [CrossRef]
  32. R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003). [CrossRef]
  33. J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005). [CrossRef] [PubMed]
  34. A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004). [CrossRef] [PubMed]
  35. S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006). [CrossRef] [PubMed]
  36. B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera,” Analytical Chemistry (2007).

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