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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 16, Iss. 25 — Dec. 8, 2008
  • pp: 21093–21104

Nanoscale separation of molecular species based on their rotational mobility

Ilaria Testa, Andreas Schönle, Claas v. Middendorff, Claudia Geisler, Rebecca Medda, Christian A. Wurm, Andre C. Stiel, Stefan Jakobs, Mariano Bossi, Christian Eggeling, Stefan W. Hell, and Alexander Egner  »View Author Affiliations

Optics Express, Vol. 16, Issue 25, pp. 21093-21104 (2008)

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We combine far-field fluorescence nanoscopy through serialized recording of switchable emitters with polarization-sensitive fluorescence detection. In addition to imaging with nanoscale spatial resolution, this technique allows determination of the fluorescence anisotropy of each detected dipole emitter and thus an estimate of its rotational mobility. Sub-populations of fluorescent markers can thus be separated based on their interaction with the sample. We applied this new functional nanoscopy to imaging of living mammalian cells.

© 2008 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:

Original Manuscript: November 12, 2008
Revised Manuscript: December 3, 2008
Manuscript Accepted: December 3, 2008
Published: December 4, 2008

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

Ilaria Testa, Andreas Schönle, Claas v. Middendorff, Claudia Geisler, Rebecca Medda, Christian A. Wurm, Andre C. Stiel, Stefan Jakobs, Mariano Bossi, Christian Eggeling, Stefan W. Hell, and Alexander Egner, "Nanoscale separation of molecular species based on their rotational mobility," Opt. Express 16, 21093-21104 (2008)

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  1. E. Abbe, "Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung," Arch. Mikr. Anat. 9, 413-468 (1873). [CrossRef]
  2. S. W. Hell, "Improvement of lateral resolution in far-field light microscopy using two-photon excitation with offset beams," Opt. Commun. 106, 19-24 (1994). [CrossRef]
  3. 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]
  4. S. W. Hell, "Toward fluorescence nanoscopy," Nature Biotechnol. 21, 1347-1355 (2003). [CrossRef]
  5. S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007). [CrossRef] [PubMed]
  6. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A 97, 8206-8210 (2000). [CrossRef]
  7. 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]
  8. M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat. Methods 3, 793-796 (2006). [CrossRef] [PubMed]
  9. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006). [CrossRef] [PubMed]
  10. A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A.-C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007). [CrossRef] [PubMed]
  11. W. Heisenberg, The Physical Principles of the Quantum Theory (Chicago Univ Press, Chicago, 1930).
  12. N. Bobroff, "Position measurement with a resolution and noise-limited instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986). [CrossRef]
  13. E. Betzig, "Proposed method for molecular optical imaging," Opt. Lett. 20, 237-239 (1995). [CrossRef] [PubMed]
  14. S. W. Hell, J. Soukka, and P. E. Hänninen, "Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy," Bioimaging 3, 65-69 (1995). [CrossRef]
  15. T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996). [CrossRef] [PubMed]
  16. T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000). [CrossRef] [PubMed]
  17. 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]
  18. W. E. Moerner and L. Kador, "Optical detection and spectroscopy of single molecules in a solid," Phys. Rev. Lett. 62, 2535-2538 (1989). [CrossRef] [PubMed]
  19. M. Orrit and J. Bernard, "Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal," Phys. Rev. Lett. 65, 2716-2719 (1990). [CrossRef] [PubMed]
  20. E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990). [CrossRef]
  21. T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996). [CrossRef] [PubMed]
  22. A. Schönle and S. W. Hell "Fluorescence nanoscopy goes multicolor," Nature Biotechnol. 25, 1234-1235 (2007). [CrossRef]
  23. M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008). [CrossRef] [PubMed]
  24. M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008). [CrossRef] [PubMed]
  25. T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77, 3979-3982 (1996). [CrossRef] [PubMed]
  26. J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999). [CrossRef]
  27. J. Fölling, 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]
  28. A. van Blaaderen and A. Vrij, "Synthesis and Characterization of Colloidal Dispersions of Fluorescent, Monodisperse Silica Spheres," Langmuir 8, 2921-2931 (1992). [CrossRef]
  29. J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008). [CrossRef] [PubMed]
  30. A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008). [CrossRef] [PubMed]
  31. J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004). [CrossRef] [PubMed]
  32. P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007). [CrossRef] [PubMed]
  33. C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007). [CrossRef]
  34. J. Enderlein, "Theoretical study of detection of a dipole emitter through an objectiv with high numerical aperture," Opt. Lett. 25, 634-636 (2000). [CrossRef]
  35. K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy," Methods 29, 74-85 (2003). [CrossRef] [PubMed]
  36. T. L. Hill and M. W. Kischner, "Subunit treadmilling of microtubules or actin in the presence of cellular barriers: possible conversion of chemical free energy into mechanical work," Proc. Natl. Acad. Sci. USA 79, 490-494 (1982). [CrossRef] [PubMed]
  37. C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).
  38. J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008). [CrossRef]

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