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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

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

Two-photon excitation STED microscopy

Gael Moneron and Stefan W. Hell  »View Author Affiliations


Optics Express, Vol. 17, Issue 17, pp. 14567-14573 (2009)
http://dx.doi.org/10.1364/OE.17.014567


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Abstract

We report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with stimulated-emission depletion (STED). We demonstrate an easy-to-implement and promising laser combination based on a short-pulse laser source for two-photon excitation and a continuous-wave (CW) laser source for resolution enhancement. Images of fluorescent nanoparticles and the immunostained transcription regulator NFκB in mammalian cell nuclei exhibit resolutions of <50 nm and ~70 nm in the focal plane, respectively, corresponding to a 4–5.4-fold improvement over the diffraction barrier.

© 2009 OSA

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(180.5810) Microscopy : Scanning microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: June 26, 2009
Revised Manuscript: July 29, 2009
Manuscript Accepted: July 31, 2009
Published: August 3, 2009

Virtual Issues
Vol. 4, Iss. 10 Virtual Journal for Biomedical Optics

Citation
Gael Moneron and Stefan W. Hell, "Two-photon excitation STED microscopy," Opt. Express 17, 14567-14573 (2009)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-17-17-14567


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References

  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]
  2. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy,” Opt. Lett. 19(11), 780–782 (1994). [CrossRef] [PubMed]
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  11. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009). [CrossRef]
  12. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006). [CrossRef] [PubMed]
  13. R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007). [CrossRef]
  14. U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (2008). [CrossRef] [PubMed]
  15. V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008). [CrossRef] [PubMed]
  16. List of fluorescent dyes used in STED microscopy: http://www.mpibpc.mpg.de/abteilungen/200/STED_Dyes.htm
  17. D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008). [CrossRef] [PubMed]
  18. B. R. Rankin, R. R. Kellner, and S. W. Hell, “Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source,” Opt. Lett. 33(21), 2491–2493 (2008). [CrossRef] [PubMed]
  19. K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007). [CrossRef] [PubMed]
  20. S. W. Hell, M. Booth, S. Wilms, C. M. Schnetter, A. K. Kirsch, D. J. Arndt-Jovin, and T. M. Jovin, “Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation,” Opt. Lett. 23(15), 1238–1240 (1998). [CrossRef]
  21. M. J. Booth and S. W. Hell, “Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line,” J. Microsc. 190(3), 298–304 (1998). [CrossRef] [PubMed]
  22. T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, and S. W. Hell, “2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy,” Microsc. Res. Tech. 70(1), 1–9 (2007). [CrossRef]

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