OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 16, Iss. 19 — Sep. 15, 2008
  • pp: 14353–14368

The optics and performance of dual-focus fluorescence correlation spectroscopy

Thomas Dertinger, Anastasia Loman, Benjamin Ewers, Claus B. Müller, Benedikt Krämer, and Jörg Enderlein  »View Author Affiliations

Optics Express, Vol. 16, Issue 19, pp. 14353-14368 (2008)

View Full Text Article

Enhanced HTML    Acrobat PDF (774 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Fluorescence correlation spectroscopy (FCS) is an important spectroscopic technique which can be used for measuring the diffusion and thus size of fluorescing molecules at pico- to nanomolar concentrations. Recently, we introduced an extension of conventional FCS, which is called dual-focus FCS (2fFCS) and allows absolute diffusion measurements with high precision and repeatability. It was shown experimentally that the method is robust against most optical and sample artefacts which are troubling conventional FCS measurements, and is furthermore able to yield absolute values of diffusion coefficients without referencing against known standards. However, a thorough theoretical treatment of the performance of 2fFCS is still missing. The present paper aims at filling this gap. Here, we have systematically studied the performance of 2fFCS with respect to the most important optical and photophysical factors such as cover slide thickness, refractive index of the sample, laser beam geometry, and optical saturation. We show that 2fFCS has indeed a superior performance when compared with conventional FCS, being mostly insensitive to most potential aberrations when working under optimized conditions.

© 2008 Optical Society of America

OCIS Codes
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(180.1790) Microscopy : Confocal microscopy
(300.2530) Spectroscopy : Fluorescence, laser-induced

ToC Category:

Original Manuscript: July 3, 2008
Revised Manuscript: August 12, 2008
Manuscript Accepted: August 13, 2008
Published: August 29, 2008

Virtual Issues
Vol. 3, Iss. 11 Virtual Journal for Biomedical Optics

Thomas Dertinger, Anastasia Loman, Benjamin Ewers, Claus B. Müller, Benedikt Krämer, and Jörg Enderlein, "The optics and performance of dual-focus fluorescence correlation spectroscopy," Opt. Express 16, 14353-14368 (2008)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. D. Magde, E. Elson, and W. W. Webb "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708, (1972). [CrossRef]
  2. E. L. Elson and D. Magde "Fluorescence Corelation Spectroscopy I. Conceptual Basis and Theory," Bioploymers 13, 1-27 (1974). [CrossRef]
  3. D. Magde, E. Elson, and W. W. Webb "Fluorescence Corelation Spectroscopy II. An Experimental Realization," Biopolymers 13, 29-61 (1974). [CrossRef] [PubMed]
  4. J. Widengren and ??. Mets, Single-Molecule Detection in Solution - Methods and Applications, Eds. C. Zander, J. Enderlein, and R. A. Keller (Wiley-VCH, 2002) pp. 69-95. [CrossRef]
  5. R. Rigler and E. Elson, Eds. Fluorescence Correlation Spectroscopy (Springer, 2001). [CrossRef]
  6. A. Benda, M. Benes, V. Marecek, A. Lhotsky, W.T. Hermens, and M. Hof, "How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy," Langmuir 19, 4120-4126 (2003). [CrossRef]
  7. J. Humpolicková, E. Gielen, A. Benda, V. Fagulova, J. Vercammen, M. VandeVen, M. Hof, M. Ameloot, and Y. Engelborghs, "Probing Diffusion Laws within Cellular Membranes by Z-Scan Fluorescence Correlation Spectroscopy," Biophys. J. Biophys. Lett. 91, L23-L25 (2006).
  8. J. Ries and P. Schwille, "Studying Slow Membrane Dynamics with Continuous Wave Scanning Fluorescence Correlation Spectroscopy," Biophys. J. 91, 1915-1924 (2006). [CrossRef] [PubMed]
  9. Z. Petrasek and P. Schwille, "Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy," Biophys. J. 94, 1437-1448 (2008). [CrossRef]
  10. T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," ChemPhysChem 8, 433-443 (2007). [CrossRef] [PubMed]
  11. A. Loman, T. Dertinger, F. Koberling, and J. Enderlein, "Comparison of optical saturation effects in conventional and dual-focus fluorescence correlation spectroscopy," Chem. Phys. Lett. 459, 18-21 (2008). [CrossRef]
  12. M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001). [CrossRef]
  13. B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005). [CrossRef] [PubMed]
  14. G. Nomarski, "Interference Microscopy - State of Art and Its Future," J. Opt. Soc. Am. 60, 1575-1575 (1970).
  15. E. Wolf, "Electromagnetic diffraction in optical systems I. An integral representation of the image field," Proc. Roy. Soc. London A 253, 349-357 (1959). [CrossRef]
  16. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. Roy. Soc. London A 253, 358-379 (1959). [CrossRef]
  17. P. R. T. Munro and P. Török, "Vectorial, high numerical aperture study of Nomarski's differential interference contrast microscope," Opt. Express 13, 6833-47 (2005). [CrossRef] [PubMed]
  18. P. Török, Z. Varga, G. R. Laczik, and J. Booker, "Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: an integral representation," J. Opt. Soc. Am. A 12, 325 (1995). [CrossRef]
  19. P. Török and P. Varga, "Electromagnetic diffraction of light focused through a stratified medium," Appl. Opt. 36, 2305 (1997). [CrossRef] [PubMed]
  20. A. Egner, M. Schrader, and S. W. Hell, "Refractive index mismatch induced intensity and phase variations in fluorescence confocal, multiphoton and 4Pi-microscopy," Opt. Commun. 153, 211 (1998). [CrossRef]
  21. O. Haeberlé, "Focusing of light through a stratified medium: a practical approach for computing microscope point spread functions. Part II: confocal and multiphoton microscopy," Opt. Commun. 235, 1 (2004). [CrossRef]
  22. O. Haeberlé, M. Ammar, H. Furukawa, K. Tenjimbayashi, and P. Török, "The point spread function of optical microscopes imaging through stratified media," Opt. Express 11, 2964 (2003). [CrossRef] [PubMed]
  23. I. Gregor and J. Enderlein "Focusing astigmatic Gaussian beams through optical systems with a high numerical aperture," Opt. Lett. 30, 2527-9 (2005). [CrossRef] [PubMed]
  24. J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of Fluorescence Correlation Spectroscopy for Measuring Diffusion and Concentration," ChemPhysChem 6, 2324-2336 (2005). [CrossRef] [PubMed]
  25. M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, "Fast focus field calculations," Opt. Express 14, 11277-11291 (2006). [CrossRef] [PubMed]
  26. I. Gregor, D. Patra, and J. Enderlein, "Optical Saturation in Fluorescence Correlation Spectroscopy under Continuous-Wave and Pulsed Excitation," ChemPhysChem 6, 164-70 (2005). [CrossRef] [PubMed]
  27. P. Török, P. D. Higdon, and T. Wilson, "Theory for confocal and conventional microscopes imaging small dielectric scatterers," J. Mod. Opt. 45, 1681-1698 (1998). [CrossRef]
  28. P. D. Higdon, P. Török, and T. Wilson, "Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes," J. Microsc. 193, 127-141 (1999). [CrossRef]
  29. P. Török, "Propagation of electromagnetic dipole waves through dielectric interfaces," Opt. Lett. 25, 1463-1465 (2000). [CrossRef]
  30. M. Leutenegger and T. Lasser, "Detection efficiency in total internal reflection fluorescence microscopy," Opt. Express 16, 8519-8531 (2008). [CrossRef] [PubMed]
  31. M. Wahl, I. Gregor, M. Patting, and J. Enderlein, "Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting," Opt. Express 11, 3583-91 (2003). [CrossRef] [PubMed]
  32. C. B. Müller, K. Wei??, W. Richtering, A. Loman, and J. Enderlein, "Calibrating Differential Interference Contrast Microscopy with dual-focus Fluorescence Correlation Spectroscopy," Opt. Express 16, 4322-9 (2008). [CrossRef] [PubMed]
  33. C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, "Precise Measurement of Diffusion by Multi-Color Dual-Focus Fluorescence Correlation Spectroscopy," Eur. Phys. Lett. 83, 46001 (2008). [CrossRef]
  34. G. Donnert, C. Eggeling, and S. W Hell, "Major signal increase in fluorescence microscopy through dark-state relaxation," Nature Meth. 4, 81-86 (2007). [CrossRef]
  35. G. Nishimura and M. Kinjo "Systematic error in fluorescence correlation measurements identified by a simple saturation model of fluorescence," Anal. Chem. 76, 1963-1970 (2004). [CrossRef] [PubMed]
  36. K. Berland and G. Shen, "Excitation Saturation in Two-Photon Fluorescence Correlation Spectroscopy," Appl. Opt. 42, 5566-5576 (2003). [CrossRef] [PubMed]
  37. I. Gregor, D. Patra, and J. Enderlein, "Optical Saturation in Fluorescence Correlation Spectroscopy under Continuous-Wave and Pulsed Excitation," ChemPhysChem 6, 164-170 (2005). [CrossRef] [PubMed]

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