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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)
http://dx.doi.org/10.1364/OE.14.008111


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Abstract

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

History
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

Citation
Joerg Enderlein, Erdal Toprak, and Paul R. Selvin, "Polarization effect on position accuracy of fluorophore localization," Opt. Express 14, 8111-8120 (2006)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-18-8111


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