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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editor: Gregory W. Faris
  • Vol. 4, Iss. 2 — Feb. 10, 2009

Isotropic 3D Nanoscopy based on single emitter switching

Claas v. Middendorff, Alexander Egner, Claudia Geisler, StefanW. Hell, and Andreas Schönle  »View Author Affiliations

Optics Express, Vol. 16, Issue 25, pp. 20774-20788 (2008)

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We propose and analyze a method for isotropic resolution in far-field fluorescence nanoscopy based on switching and mathematically localizing individual emitters. Under typical imaging conditions, the coherent detection of fluorescence light through two opposing high angle lenses strongly improves the 3D-resolution down to 5–10nm in all directions. Furthermore, we give a detailed analysis of the resolution of this and other single molecule switching based approaches using the Fisher information matrix. We verify the results by Monte-Carlo simulations of the imaging process and by applying a simple maximum-likelihood estimator for position determination.

© 2008 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(110.2990) Imaging systems : Image formation theory
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:

Original Manuscript: October 15, 2008
Revised Manuscript: November 21, 2008
Manuscript Accepted: November 24, 2008
Published: December 1, 2008

Virtual Issues
Vol. 4, Iss. 2 Virtual Journal for Biomedical Optics

Claas v. Middendorff, Alexander Egner, Claudia Geisler, Stefan W. Hell, and Andreas Schönle, "Isotropic 3D Nanoscopy based on single emitter switching," Opt. Express 16, 20774-20788 (2008)

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  1. E. Abbe, "Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung," Arch. f. Mikr. Anat. 9, 413-420 (1873). [CrossRef]
  2. 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]
  3. S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 1347-1355 (2003). [CrossRef] [PubMed]
  4. S. W. Hell, "Far-field optical nanoscopy," Science 316, 1153-1158 (2007). [CrossRef] [PubMed]
  5. G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. USA 103, 440-445 (2006). [CrossRef]
  6. B. Harke, C. Ullal, J. Keller, and S. W. Hell, "Three-dimensional nanoscopy of colloidal crystals," Nano. Lett. 8, 1309-1313 (2008). [CrossRef] [PubMed]
  7. S. Hell and E. H. K. Stelzer, "Properties of a 4Pi-confocal fluorescence microscope," J. Opt. Soc. Am. A 9, 2159-2166 (1992). [CrossRef]
  8. S. W. Hell, E. H. K. Stelzer, S. Lindek, and C. Cremer, "Confocal microscopy with enhanced detection aperture: type B 4Pi-confocal microscopy," Opt. Lett. 19, 222-224 (1994). [CrossRef] [PubMed]
  9. M. Nagorni and S. W. Hell, "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts," J. Opt. Soc. Am. A 18, 36-48 (2001). [CrossRef]
  10. M. Dyba and S. W. Hell, "Focal spots of size ⌊ /23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163, 901 (2002). [CrossRef]
  11. R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, "Spherical nanosized focal spot unravels the interior of cells," Nature Methods 5, 539 - 544 (2008). [CrossRef] [PubMed]
  12. M. Hofmann, C. Eggeling, S. Jakobs, and S.W. Hell, "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. USA 102, 565-569 (2005). [CrossRef]
  13. W. Heisenberg, The Physical Principles of Quantum Theory (Chicago Univ. Press, Chicago, 1930).
  14. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging intracellular fluorescent proteins at nanometer resolution," Science 313, 1642-1645 (2006). [CrossRef] [PubMed]
  15. M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nature Methods 3, 793-796 (2006). [CrossRef] [PubMed]
  16. S. T. Hess, T. P. Girirajan, and M. D. Mason, "Ultra-high resolution imaging by fluorescent photoactivation localization microscopy (FPALM)," Biophys. J. 91, 4258-4272 (2006). [CrossRef] [PubMed]
  17. A. Egner, C. Geisler, C. v. Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007). [CrossRef] [PubMed]
  18. H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig "Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes," Proc. Natl. Acad. Sci. USA 104, 20308-20313 (2007). [CrossRef] [PubMed]
  19. H. Bock, C. Geisler, C. A. Wurm, C. v. Middendorff, S. Jakobs, A. Schönle, A. Egner, S.W. Hell, and C. Eggeling, "Two-color far-field fluorescence nanoscopy based on photoswitchable emitters," Appl. Phys. B 88, 161-165 (2007). [CrossRef]
  20. M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, "Multicolor super-resolution imaging with photo-switchable fluorescent probes," Science 317, 1749-1752 (2007). [CrossRef] [PubMed]
  21. M. Bossi, J. F¨olling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano. Lett. 8, 2463-2468 (2008). [CrossRef] [PubMed]
  22. J. F¨olling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic rhodamines provide nanoscopy with optical sectioning," Angew. Chem. Int. Ed. 46, 6266 - 6270 (2007). [CrossRef]
  23. B. Huang, W. Wang, M. Bates, and X. Zhuang, "Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy," Science 319, 810-813 (2008). [CrossRef] [PubMed]
  24. M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. Bennett, S. T. Hess, and J. Bewersdorf, "Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples," Nature Methods 5, 527-529 (2008). [CrossRef] [PubMed]
  25. J. Nowakowski and M. Elbaum, "Fundamental limits in estimating light pattern position," J. Opt. Soc. Am. 73, 1744-1758 (1983). [CrossRef]
  26. K. Winick, "Cramer-Rao lower bounds on the performance of charge-coupled-device optical position estimators," J. Opt. Soc. Am. A 3, 1809-1815 (1986). [CrossRef]
  27. 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]
  28. M. K. Cheezum, W. F. Walker, and W. H. Guilford, "Quantitative comparison of algorithms for tracking single fluorescent particles," Biophys. J. 81, 2378-2388 (2001). [CrossRef] [PubMed]
  29. R. J. Ober, S. Ram, and E. S. Ward, "Localization accuracy in single-molecule microscopy," Biophys. J. 86, 1185-1200 (2004). [CrossRef] [PubMed]
  30. A. d. Dekker, S. v. Art, A. v. Bos, and D. v. Dyck, "Maximum likelihood estimation of structure parameters from high resolution electron microscopy images : A theoretical framework," Ultramicroscopy 104, 83-106 (2005). [CrossRef]
  31. R. A. Fisher, "Theory of statistical estimation," Proc. Cambridge Philos. Soc. 22, 700-725 (1925). [CrossRef]
  32. C. Rao, "Information and the accuracy attainable in the estimation of statistical parameters," Bull. Calcutta Math. Soc. 37, 81-89 (1945).
  33. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. R. Soc. Lond. A 253, 358-379 (1959). [CrossRef]
  34. D. R. Cox and D. V. Hinkley, Theoretical Statistics (Chapman and Hall, London, 1974).
  35. H. Cramer, Mathematical Methods of Statistics (Princeton Univ. Press, Princeton, 1946).
  36. A. G. Basden, C. A. Haniff, and C. D. Mackay, "Photon counting strategies with low-light-level CCDs," Monthly Notices RAS 345(3), 985-991 (2003). [CrossRef]
  37. H. P. Kao and A. 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]
  38. M. Born and E. Wolf, Principles of Optics (Cambridge Univ. Press, Cambridge, 2002).
  39. F. Aguet, D. V. D. Ville, and M. Unser, "A maximum-likelihood formalism for sub-resolution axial localization of fluorescent nanoparticles," Opt. Express 13,10503-10522 (2005). [CrossRef] [PubMed]
  40. M. G. Gustafsson, D. A. Agard, and J. W. Sedat, "I5M: 3D widefield light microscopy with better than 100 nm axial resolution," J. Microsc. 195, 10-16 (1999). [CrossRef] [PubMed]
  41. A. Egner, S. Jakobs, and S. W. Hell, "Fast 100-nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast," Proc. Natl. Acad. Sci. USA 99, 3370-3375 (2002). [CrossRef] [PubMed]
  42. H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, "Cooperative 4Pi excitation and detection yields 7-fold sharper optical sections in live cell microscopy," Biophys. J. 87, 4146-4152 (2004). [CrossRef] [PubMed]
  43. D. Khimich, R. Nouvian, R. Pujol, S. T. Dieck, A. Egner, E. D. Gundelfinger, and T. Moser, "Hair cell synaptic ribbons are essential for synchronous auditory signalling," Nature 434(7035), 889-894 (2005). [CrossRef]
  44. A. Egner, S. Verrier, A. Goroshkov, H.-D. Sling, and S. W. Hell, "4Pi-microscopy of the Golgi apparatus in live mammalian cells," J. Struct. Biol. 147(1), 70-76 (2004). [CrossRef]
  45. V. Westphal and S. W. Hell, "Nanoscale resolution in the focal plane of an optical microscope," Phys. Rev. Lett. 94,143,903 (2005). [CrossRef]

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