OSA's Digital Library

Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 14, Iss. 25 — Dec. 11, 2006
  • pp: 12206–12216

Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations

Jérôme Wenger, Davy Gérard, Pierre-François Lenne, Hervé Rigneault, José Dintinger, Thomas W. Ebbesen, Annie Boned, Fabien Conchonaud, and Didier Marguet  »View Author Affiliations

Optics Express, Vol. 14, Issue 25, pp. 12206-12216 (2006)

View Full Text Article

Enhanced HTML    Acrobat PDF (151 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Single nanometric apertures in a metallic film are used to develop a simple and robust setup for dual-color fluorescence crosscorrelation spectroscopy (FCCS) at high concentrations. If the nanoaperture concept has already proven to be useful for single-species analysis, its extension to the dual-color case brings new interesting specificities. The alignment and overlap of the two excitation beams are greatly simplified. No confocal pinhole is used, relaxing the requirement for accurate correction of chromatic aberrations. Compared to two-photon excitation, nanoapertures have the advantage to work with standard fluorophore constructions having high absorption cross-section and well-known absorption/emission spectra. Thanks to the ultra-low volume analysed within one single aperture, fluorescence correlation analysis can be performed with single molecule resolution at micromolar concentrations, resulting in 3 orders of magnitude gain compared to conventional setups. As applications of this technique, we follow the kinetics of an enzymatic cleavage reaction at 2 µM DNA oligonucleotide concentration.We also demonstrate that FCCS in nanoapertures can be applied to the fast screening of a sample for dual-labeled species within 1 s acquisition time. This offers new possibilities for rapid screening applications in biotechnology at high concentrations.

© 2006 Optical Society of America

OCIS Codes
(050.1220) Diffraction and gratings : Apertures
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: October 6, 2006
Revised Manuscript: December 1, 2006
Manuscript Accepted: December 2, 2006
Published: December 11, 2006

Virtual Issues
Vol. 2, Iss. 1 Virtual Journal for Biomedical Optics

Jérôme Wenger, Davy Gérard, Pierre-François Lenne, Hervé Rigneault, José Dintinger, Thomas W. Ebbesen, Annie Boned, Fabien Conchonaud, and Didier Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12206-12216 (2006)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. M. Eigen, R. Rigler, "Sorting single molecules: Application to diagnostics and evolutionary biotechnology," Proc. Natl. Acad. Sci. USA 91, 5740-5747 (1994). [CrossRef] [PubMed]
  2. P. Schwille, F.-J. Meyer-Almes, R. Rigler, "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution," Biophys. J. 72, 1878-1886 (1997). [CrossRef] [PubMed]
  3. T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, J. Langowski, "Analysis of Ligand Binding by Two-Colour Fluorescence Cross-Correlation Spectroscopy," Single Mol. 3, 49-61 (2002). [CrossRef]
  4. K. Bacia, S. A. Kim, P. Schwille, "Fluorescence cross-correlation spectroscopy in living cells," Nat. Methods 3, 83-89 (2006). [CrossRef] [PubMed]
  5. U. Kettling, A. Koltermann, P. Schwille, M. Eigen, "Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy," Proc. Natl. Acad. Sci. USA 95, 1416-1420 (1998). [CrossRef] [PubMed]
  6. A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, M. Eigen, "Rapid assay processing by integration of dualcolor fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity," Proc. Natl. Acad. Sci. USA 95, 1421-1426 (1998). [CrossRef] [PubMed]
  7. T. Winkler, U. Kettling, A. Koltermann, M. Eigen, "Confocal fluorescence coincidence analysis: an approach to ultra high throughput screening," Proc. Natl. Acad. Sci. USA 96, 1375-1378 (1999). [CrossRef] [PubMed]
  8. N. Baudendistel, G. M¨uller, W. Waldeck, P. Angel, J. Langowski, "Two-Hybrid Fluorescence Cross-Correlation Spectroscopy Detects ProteinProtein Interactions In Vivo," Chem. Phys. Chem. 6, 984-990 (2005). [CrossRef] [PubMed]
  9. K.G. Heinze, A. Koltermann, P. Schwille, "Simultaneous two-photon excitation of distinct labels for dual-color fluorescence cross-correlation analysis," Proc. Natl. Acad. Sci. USA 97, 10377-10382 (2000). [CrossRef] [PubMed]
  10. S. A. Kim, K. G. Heinze, M. N. Waxham, P. Schwille, "Intracellular calmodulin availability accessed with twophoton crosscorrelation," Proc. Natl. Acad. Sci. USA. 101, 105-110 (2004). [CrossRef]
  11. D. R. Larson, J. A. Gosse, D. A. Holowka, B. A. Baird,W.W. Webb, "Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells," J. Cell Biol. 171, 527-536 (2005). [CrossRef] [PubMed]
  12. T. Kogure, S. Karasawa, T. Araki, K. Saito, M. Kinjo, A. Miyawaki, "A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy," Nat. Biotechnol. 24, 577-581 (2006). [CrossRef] [PubMed]
  13. M. Burkhardt, K. G. Heinze, P. Schwille, "Four-color fluorescence correlation spectroscopy realized in a gratingbased detection platform," Opt. Lett. 30, 2266-2268 (2005). [CrossRef] [PubMed]
  14. L. C. Hwang, M. Leutenegger, M. G¨osch, T. Lasser, P. Rigler, W. Meier, T. Wohland, "Prism-based multicolor fluorescence correlation spectrometer," Opt. Lett. 31, 1310-1312 (2006). [CrossRef] [PubMed]
  15. M. Leutenegger, H. Blom, J. Widengren, C. Eggeling, M. G¨osch, R. A. Leitgeb, T. Lasser, "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," J. Biomed. Opt. 11, 040502 (2006). [CrossRef] [PubMed]
  16. M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, W.W. Webb, "Zero-mode waveguides for single-molecule analysis at high concentrations," Science 299, 682-686 (2003). [CrossRef] [PubMed]
  17. K. T. Samiee, M. Foquet, L. Guo, E. C. Cox, H. G. Craighead, "Lambda repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides," Biophys. J. 88, 2145-2153 (2005). [CrossRef]
  18. H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T. W. Ebbesen, P.-F. Lenne, "Enhancement of single-molecule fluorescence detection in subwavelength apertures," Phys. Rev. Lett. 95, 117401 (2005). [CrossRef] [PubMed]
  19. J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Dintinger, T.W. Ebbesen, "Single molecule fluorescence in rectangular nano-apertures," Opt. Express 13, 7035-7044 (2005). [CrossRef] [PubMed]
  20. M. Leutenegger,M. G¨osch, A. Perentes, P. Hoffmann, O. J. F.Martin, T. Lasser, "Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture," Opt. Express 14, 956-969 (2006). [CrossRef] [PubMed]
  21. J. B. Edel, M. Wu, B. Baird, H. G. Craighead, "High spatial resolution observation of single molecule dynamics in living cell membranes," Biophys. J. 88, L43-L45 (2005). [CrossRef] [PubMed]
  22. J. Wenger, H. Rigneault, J. Dintinger, D. Marguet, P. F. Lenne, "Single-fluorophore diffusion in a lipid membrane over a subwavelength aperture," J. Biol. Phys. 32, SN1-SN4 (2006). [CrossRef] [PubMed]
  23. K. T. Samiee, J. M. Moran-Mirabal, Y. K. Cheung, H. G. Craighead, "Zero Mode Waveguides for Single- Molecule Spectroscopy on Lipid Membranes," Biophys. J. 90, 3288-3299 (2006). [CrossRef] [PubMed]
  24. E. Popov, M. Nevi`ere, J. Wenger, P.-F. Lenne, H. Rigneault, P. Chaumet, N. Bonod, J. Dintinger, T.W. Ebbesen, "Field enhancement in single subwavelength apertures," J. Opt. Soc. Am. A 23, 2342-2348 (2006). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited