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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 3 — Feb. 29, 2012

Single-image axial localization precision analysis for individual fluorophores

Michael C. DeSantis, Shannon Kian Zareh, Xianglu Li, Robert E. Blankenship, and Y. M. Wang  »View Author Affiliations

Optics Express, Vol. 20, Issue 3, pp. 3057-3065 (2012)

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Bio-mechanism investigations demand single particle tracking with high spatial and temporal resolutions which require single fluorophore 3D localization measurements with matching precision and speed. Although the precision for lateral-localization measurements is well described by an analytical expression, for the axial direction, it is often obtained by repeating location measurements or by estimating a lower bound. Here, we report a precision expression for an axial-localization method that analyzes the standard deviations of single fluorophores’ intensity profiles. Like the lateral-localization precision, this expression includes all relevant experimental effects measurable from a Gaussian intensity profile of the fluorophore. This expression completes the precision analysis for single-image 3D localization of individual fluorophores and lifts the temporal resolution to the typical exposure timescales of milliseconds.

© 2012 OSA

OCIS Codes
(100.6640) Image processing : Superresolution
(100.6890) Image processing : Three-dimensional image processing
(110.2960) Imaging systems : Image analysis
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy

ToC Category:

Original Manuscript: September 6, 2011
Revised Manuscript: January 13, 2012
Manuscript Accepted: January 19, 2012
Published: January 25, 2012

Virtual Issues
Vol. 7, Iss. 3 Virtual Journal for Biomedical Optics

Michael C. DeSantis, Shannon Kian Zareh, Xianglu Li, Robert E. Blankenship, and Y. M. Wang, "Single-image axial localization precision analysis for individual fluorophores," Opt. Express 20, 3057-3065 (2012)

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  1. Y. M. Wang, R. H. Austin, and E. C. Cox, “Single molecule measurements of repressor protein 1D diffusion on DNA,” Phys. Rev. Lett.97, 048302 (2006). [CrossRef] [PubMed]
  2. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-overhand: Single fluorophore imaging with 1.5-nm localization,” Science300, 2061–2065 (2003). [CrossRef] [PubMed]
  3. C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai, and T. Ha, “Advances in single-molecule fluorescence methods for molecular biology,” Annu. Rev. Biochem.77, 51–76 (2008). [CrossRef] [PubMed]
  4. B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science319, 810–813 (2008). [CrossRef] [PubMed]
  5. M. C. DeSantis, S. H. DeCenzo, J.-L. Li, and Y. M. Wang, “Precision analysis for standard deviation measurements of single fluorescent molecule images,” Opt. Express18, 6563–6576 (2010). [CrossRef] [PubMed]
  6. 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]
  7. 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]
  8. G. J. Schütz, V. P. Pastushenko, H. J. Gruber, H.-G. Knaus, B. Pragl, and H. Schindler, “3D imaging of individual ion channels in live cells at 40nm resolution,” Single Mol.1, 25–31 (2000). [CrossRef]
  9. L. Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett.90, 053902 (2007). [CrossRef]
  10. Y. Deng and J. W. Shaevitz, “Effect of aberration on height calibration in three-dimensional localization-based microscopy and particle tracking,” Appl. Opt.48, 1886–1890 (2009). [CrossRef] [PubMed]
  11. M. Speidel, A. Jonáš, and E.-L. Florin, “Three-dimensional tracking of fluorescent nanoparticles with sub-nanometer precision by use of off-focus imaging,” Opt. Lett.28, 69–71 (2003). [CrossRef] [PubMed]
  12. F. Aguet, D. van de Ville, and M. Unser, “A maximum-likelihood formalism for sub-resolution axial localization of fluorescent nanoparticles,” Opt. Express13, 10503–10522 (2005). [CrossRef] [PubMed]
  13. Z. Zhang and C.-H. Menq, “Three-dimensional particle tracking with subnanometer resolution using off-focus images,” Appl. Optics47, 2361–2370 (2008). [CrossRef]
  14. J. W. Shaevitz, “Bayesian estimation of the axial position in astigmatism-based three-dimensional particle tracking,” Int. J. Opt.2009, 896208 (2009).
  15. E. Toprak, H. Balci, B. H. Blehm, and P. R. Selvin, “Three-dimensional particle tracking via bifocal imaging,” Nano Lett.7, 2043–2045 (2007). [CrossRef] [PubMed]
  16. S. Ram, P. Prabhat, J. Chao, E. S. Ward, and R. J. Ober, “High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells,” Biophys. J.95, 6025–6043 (2008). [CrossRef] [PubMed]
  17. S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. USA106, 2995–2999 (2009). [CrossRef] [PubMed]
  18. B. Huang, S. A. Jones, B. Brandenburg, and X. Zhuang, “Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution,” Nat. Methods5, 1047–1052 (2008). [CrossRef] [PubMed]
  19. S. H. DeCenzo, M. C. DeSantis, and Y. M. Wang, “Single-image separation measurements of two unresolved fluorophores,” Opt. Express18, 16628–16639 (2010). [CrossRef] [PubMed]
  20. A. A. Arteni, G. Ajlani, and E. J. Boekema, “Structural organisation of phycobilisomes from Synechocystis sp. strain PCC6803 and their interaction with the membrane,” Biochim. Biophys. Acta1787, 272–279 (2009). [CrossRef] [PubMed]
  21. G. Ajlani, C. Vernotte, L. DiMagno, and R. Haselkorn, “Phycobilisome core mutants of Synechocystis PCC 6803,” Biochim. Biophys. Acta1231, 189–196 (1995). [CrossRef]
  22. N. Adir, Department of Chemistry, Technion, Israel Institute of Technology, Israel, is preparing a manuscript.
  23. H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: Use of cylindrical optics to encode particle position,” Biophys. J.67, 1291–1300 (1994). [CrossRef] [PubMed]
  24. S. K. Zareh, M. C. DeSantis, J. Kessler, J.-L. Li, and Y. M. Wang, “Single-image diffusion coefficient measurements of proteins in free solution,” Biophys. J. (in review).

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