OSA's Digital Library

Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 14, Iss. 18 — Sep. 4, 2006
  • pp: 8111–8120

Polarization effect on position accuracy of fluorophore localization

Jörg Enderlein, Erdal Toprak, and Paul R. Selvin  »View Author Affiliations

Optics Express, Vol. 14, Issue 18, pp. 8111-8120 (2006)

View Full Text Article

Enhanced HTML    Acrobat PDF (376 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



The technique of determining the position of individual fluorescent molecules with nanometer resolution, called FIONA, has become an important tool for several biophysical applications such as studying motility mechanisms of motor proteins. The position determination is usually done by fitting a 2-D Gaussian (x-y vs. photon number) to the emission intensity distribution of the fluorescent molecule. However, the intensity distribution of an emitting molecule depends not only on its position in space, but also on its three-dimensional orientation. Here, we present an extensive numerical study of the achievable accuracy of position determination as a function of molecule orientation. We compare objectives with different numerical apertures and show that an effective pixel size of 100 nm or less per CCD pixel is required to obtain good positional accuracy. Nonetheless, orientation effects can still cause position errors for large anisotropy, as high as 10 nm for high numerical aperture objectives. However, position accuracy is significantly better (< 2.5 nm) when using objectives with a numerical aperture of 1.2. Of course, probes with lower anisotropy decrease the positional uncertainty.

© 2006 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(110.2990) Imaging systems : Image formation theory
(260.2510) Physical optics : Fluorescence

ToC Category:
Imaging Systems

Original Manuscript: April 21, 2006
Revised Manuscript: August 3, 2006
Manuscript Accepted: August 3, 2006
Published: September 1, 2006

Virtual Issues
Vol. 1, Iss. 10 Virtual Journal for Biomedical Optics

Joerg Enderlein, Erdal Toprak, and Paul R. Selvin, "Polarization effect on position accuracy of fluorophore localization," Opt. Express 14, 8111-8120 (2006)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. A. Yildiz, and P. R. Selvin, "Fluorescence imaging with one nanometer accuracy, Application to molecular motors," Acc. Chem. Res. 38, 574-582 (2005). [CrossRef] [PubMed]
  2. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand, single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003). [CrossRef] [PubMed]
  3. H. Park, G. T. Hanson, S. R. Duff, and P. R. Selvin, "Nanometre localization of single ReAsH molecules," J. Microsc. 216, 199-205 (2004). [CrossRef] [PubMed]
  4. G. E. Snyder, T. Sakamoto, J. A. Hammer, J. R. Sellers, and P. R. Selvin, "Nanometer localization of single green fluorescent proteins, Evidence that Myosin V walks and-over-hand via Telemark Configuration," Biophys. J. 87, 1776-1783 (2004).
  5. M. P. Gordon, T. Ha, and P. R. Selvin, "Single-molecule high-resolution imaging with photobleaching," Proc. Nat. Acad. Sci. USA 101, 6462-6465 (2004). [CrossRef] [PubMed]
  6. H. Balci, T. Ha, H. L. Sweeney, and P. R. Selvin, "Interhead distance measurements in Myosin VI via SHRImP support a simplified hand-over-hand model," Biophys. J. 89, 413-417 (2005). [CrossRef] [PubMed]
  7. B. Muls, H. Uji-i, S. Melnikov, A. Moussa, W. Verheijen, J. P. Soumillion, J. Josemon, K. Müllen, and J. Hofkens, "Direct measurement of the end-to-end distance of individual polyfluorene polymer chains," ChemPhysChem. 6, 2286-2294 (2005). [CrossRef]
  8. M. J. Saxton, and K. Jacobson, "Single-particle tracking: Applications to membrane dynamics," Ann Rev Biophys Biomol Struct 26, 373-399 (1997). [CrossRef]
  9. M. Speidel, A. Jonas, and E. L. Florin, "Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging," Opt. Lett. 28, 69-71 (2003). [CrossRef] [PubMed]
  10. R. E. Thompson, D. R. Larson, and W. W. Webb, "Precise nanometer localization analysis for individual fluorescent probes," Biophys. J. 82, 2775-2783 (2002). [CrossRef] [PubMed]
  11. M. K. Cheezum, W. F. Guilford, and W. H. Walker, "Quantitative comparison of Algorithms for tracking single fluorescent particles," Biophys. J. 81, 2378-2388 (2001). [CrossRef] [PubMed]
  12. R. J. Ober, S. Ram, and E. S. Ward, "Localization accuracy in single-molecule microscopy," Biophys. J. 86, 1185-1200 (2004). [CrossRef] [PubMed]
  13. A. P. Bartko, and R. M. Dickson, "Imaging three-dimensional singe molecule orientations," J. Phys. Chem. B 103, 11237-11241 (1999). [CrossRef]
  14. M. Böhmer, and J. Enderlein, "Orientation imaging of single molecules by wide-field epi-fluorescence microscopy," J. Opt. Soc. B 20, 554-559 (2003). In Eq. (11) of this publication, the factor i is erroneous and has to be replaced by -1. J.E. thanks Rolfe Petschek for finding this typo. [CrossRef]
  15. 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]
  16. J. Enderlein, "Theoretical study of detecting a dipole emitter through an objective with high numerical aperture," Opt. Lett. 25, 634-636 (2000). [CrossRef]
  17. J. Enderlein, and M. Böhmer, "Influence of interface-dipole interactions on the efficiency of fluorescence light collection near surfaces," Opt. Lett. 28, 941-943 (2003). [CrossRef] [PubMed]
  18. W. Lukosz, and R. E. Kunz, "Light emission by magnetic and electric dipoles close to a plane interface II. Radiation patterns of perpendicular oriented dipoles," J. Opt. Soc. Am. 67, 1615-1619 (1977). [CrossRef]
  19. W. Lukosz, "Light emission by magnetic and electric dipoles close to a plane interface III. Radiation patterns of dipoles with arbitrary orientation," J. Opt. Soc. Am. 69, 1495-1503 (1997). [CrossRef]
  20. W. Lukosz, "Light emission by multipole sources in thin layers. I. Radiation patterns of electric and magnetic dipoles," J. Opt. Soc. Am. 71, 744-54 (1981). [CrossRef]
  21. E. H. Hellen, and D. Axelrod, "Fluorescence emission at dielectric and metal-film interfaces," J. Opt. Soc. Am. B 4, 337-350 (1987). [CrossRef]
  22. J. D. JacksonClassical Electrodynamics (John Wiley, New York, 1975).
  23. M. Abramowitz, and I. A. Stegun, eds., Handbook of mathematical functions (Harry Deutsch, Thun and Frankfurt/Main, 1984).
  24. 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]
  25. J. Enderlein, T. Ruckstuhl, and S. Seeger, "Highly efficient optical detection of surface-generated fluorescence," Appl. Opt. 38, 724-732 (1999). [CrossRef]
  26. A. M. van Oijen, J. Köhler, J. Schmidt, M. Müller, and G. J. Brakenhoff, "3-Dimensional super-resolution by spectrally selective imaging," Chem. Phys. Lett. 292, 183-187 (1998). [CrossRef]
  27. S. W. Hell, and E. H. K. Stelzer, "Properties of a 4Pi-confocal fluorescence microscope," J. Opt. Soc. Am. A 9, 2159-2166 (1992). [CrossRef]
  28. S. W. Hell, and J. Wichmann, "Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy," Opt. Lett. 19, 780-782 (1994). [CrossRef] [PubMed]
  29. M. G. Gustafsson, D. A. Agard, and J. W. Sedat, "I/sup 5/M: 3D widefield light microscopy with better than 100 nm axial resolution," J Microsc. 195, 10-16 (1999). [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