OSA's Digital Library

Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 13, Iss. 19 — Sep. 19, 2005
  • pp: 7415–7423

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule

Kai Hassler, Marcel Leutenegger, Per Rigler, Ramachandra Rao, Rudolf Rigler, Michael Gösch, and Theo Lasser  »View Author Affiliations

Optics Express, Vol. 13, Issue 19, pp. 7415-7423 (2005)

View Full Text Article

Enhanced HTML    Acrobat PDF (475 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.

© 2005 Optical Society of America

OCIS Codes
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(240.6490) Optics at surfaces : Spectroscopy, surface
(300.2530) Spectroscopy : Fluorescence, laser-induced

ToC Category:
Research Papers

Original Manuscript: July 22, 2005
Revised Manuscript: September 2, 2005
Published: September 19, 2005

Kai Hassler, Marcel Leutenegger, Per Rigler, Ramachandra Rao, Rudolf Rigler, Michael Gösch, and Theo Lasser, "Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule," Opt. Express 13, 7415-7423 (2005)

Sort:  Journal  |  Reset  


  1. D. Magde, W. W. Webb, and E. Elson, �??Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,�?? Phys. Rev. Lett. 29, 705�??& (1972). [CrossRef]
  2. R. Rigler and E. L. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications (Springer, Berlin, 2001). [CrossRef]
  3. O. Krichevsky and G. Bonnet, �??Fluorescence correlation spectroscopy: the technique and its applications,�?? Rep. Progr. Phys. 65, 251�??297 (2002). [CrossRef]
  4. R. Rigler, U. Mets, J. Widengren, and P. Kask, �??Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,�?? Eur. Biophys. J. Biophys. Lett. 22, 169�??175 (1993). [CrossRef]
  5. R. Brock and T. M. Jovin, �??Fluorescence correlation microscopy (FCM) - Fluorescence correlation spectroscopy (FCS) taken into the cell,�?? Cell. Mol. Biol. 44, 847�??856 (1998). [PubMed]
  6. M. Auer, K. J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, �??Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS,�?? Drug Discov. Today 3, 457�??465 (1998).
  7. A. M. Lieto, R. C. Cush, and N. L. Thompson, �??Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,�?? Biophys. J. 85, 3294�??3302 (2003). [CrossRef] [PubMed]
  8. U. Kettling, A. Koltermann, P. Schwille, and 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]
  9. L. Edman, Z. Foldes-Papp, S.Wennmalm, and R. Rigler, �??The fluctuating enzyme: a single molecule approach,�?? Chem. Phys. 247, 11�??22 (1999). [CrossRef]
  10. N. L. Thompson, T. P. Burghardt, and D. Axelrod, �??Measuring Surface Dynamics of Biomolecules by Total Internal- Reflection Fluorescence with Photobleaching Recovery or Correlation Spectroscopy,�?? Biophys. J. 33, 435�??454 (1981). [CrossRef] [PubMed]
  11. K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, �??High count rates with total internal reflection fluorescence correlation spectroscopy,�?? Biophys. J. 88, L1�??L3 (2005). [CrossRef]
  12. D. Axelrod, E. H. Hellen, and R. M. Fulbright, �??Total Internal Reflection Fluorescence,�?? in Topics in Fluorescence Spectroscopy: Biochemical Applications, J. R. Lakowicz, ed., vol. 3 (Plenum Press, 1992).
  13. D. Axelrod, �??Total internal reflection fluorescence microscopy in cell biology,�?? Traffic 2, 764�??774 (2001). [CrossRef] [PubMed]
  14. D. Toomre and D. J. Manstein, �??Lighting up the cell surface with evanescent wave microscopy,�?? Tr. Cell Biol. 11, 298�??303 (2001). [CrossRef]
  15. R. L. Hansen and J. M. Harris, �??Total Internal Reflection Fluorescence Correlation Spectroscopy for Counting Molecules at Solid/Liquid Interfaces,�?? Anal. Chem. 70, 2565�??2575 (1998). [CrossRef] [PubMed]
  16. W. Lukosz, �??Light-Emission by Magnetic and Electric Dipoles Close to a Plane Dielectric Interface .3. Radiation-Patterns of Dipoles with Arbitrary Orientation,�?? J. Opt. Soc. Am. 69, 1495�??1503 (1979). [CrossRef]
  17. J. Enderlein, �??Fluorescence detection of single molecules near a solution/glass interface - an electrodynamic analysis,�?? Chem. Phys. Lett. 308, 263�??266 (1999). [CrossRef]
  18. N. L. Thompson, �??Fluorescence Correlation Spectroscopy,�?? in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed., vol. 1 (Plenum Press, New York, 1991).
  19. B. Richards and E. Wolf, �??Electromagnetic Diffraction in Optical Systems .2. Structure of the Image Field in an Aplanatic System,�?? Proc. Roy. Soc. A 253, 358�??379 (1959). [CrossRef]
  20. J. Mertz, �??Radiative absorption, fluorescence, and scattering of a classical dipole near a lossless interface: a unified description,�?? J. Opt. Soc. Am. B 17, 1906�??1913 (2000). [CrossRef]
  21. S. Weiss, �??Fluorescence Spectroscopy of Single Biomolecules,�?? Science 283, 1676�??1683 (1999). [CrossRef] [PubMed]
  22. K. Hassler, M. Leutenegger, M. Gösch, T. Lasser, Laboratoire d�??Optique Biomédicale, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland are preparing a manuscript to be called �??Mathematical Models for Total Internal Reflection Fluorescence Correlation Spectroscopy�??.
  23. C. T. Culbertson, S. C. Jacobson, and J. M. Ramsey, �??Diffusion coefficient measurements in microfluidic devices,�?? Talanta 56, 365�??373 (2002). [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.


Fig. 1. Fig. 2. Fig. 3.
Fig. 4.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited