OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 25 — Dec. 6, 2010
  • pp: 26417–26429

Analytical description of STED microscopy performance

Marcel Leutenegger, Christian Eggeling, and Stefan W. Hell  »View Author Affiliations


Optics Express, Vol. 18, Issue 25, pp. 26417-26429 (2010)
http://dx.doi.org/10.1364/OE.18.026417


View Full Text Article

Enhanced HTML    Acrobat PDF (1219 KB)





Browse Journals / Lookup Meetings

Browse by Journal and Year


   


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools

Share
Citations

Abstract

Stimulated emission depletion (STED) resolves fluorescent features that are closer than the far-field optical diffraction limit by applying a spatially modulated light field keeping all but one of these features dark consecutively. For estimating the efficiency of transient fluorophore darkening, we developed analytical equations considering the spatio-temporal intensity profile of the STED beam. These equations provide a quick analysis and optimization of the resolution and contrast to be gained under various conditions, such as continuous wave or pulsed STED beams having different pulse durations. Particular emphasis is placed on fluorescence fluctuation methods such as correlation spectroscopy (FCS) using STED.

© 2010 OSA

OCIS Codes
(050.1940) Diffraction and gratings : Diffraction
(180.2520) Microscopy : Fluorescence microscopy
(260.2510) Physical optics : Fluorescence

ToC Category:
Microscopy

History
Original Manuscript: October 19, 2010
Revised Manuscript: November 22, 2010
Manuscript Accepted: November 25, 2010
Published: December 1, 2010

Citation
Marcel Leutenegger, Christian Eggeling, and Stefan W. Hell, "Analytical description of STED microscopy performance," Opt. Express 18, 26417-26429 (2010)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-18-25-26417


Sort:  Author  |  Year  |  Journal  |  Reset  

References

  1. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994). [CrossRef] [PubMed]
  2. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000). [CrossRef] [PubMed]
  3. S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007). [CrossRef] [PubMed]
  4. A. Thomas, Progress in Stimulated Emission Depletion Microscopy (Shaker, Achen 2001); Ph.D. thesis, Heidelberg (2001).
  5. S. W. Hell, “Increasing the Resolution of Far-Field Fluorescence Microscopy by Point-Spread-Function Engineering,” in Topics In Fluorescence Spectroscopy 5: Nonlinear and Two-Photon-Induced Fluorescence, J. Lakowicz, ed. (Plenum Press, New York, 1997).
  6. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008). [CrossRef] [PubMed]
  7. The actual number of de-excitation events is limited by the number of excitations per pulse.
  8. C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” N. J. Phys. 11(10), 103054 (2009). [CrossRef]
  9. V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005). [CrossRef] [PubMed]
  10. V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003). [CrossRef]
  11. M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003). [CrossRef] [PubMed]
  12. C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998). [CrossRef] [PubMed]
  13. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]
  14. K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” N. J. Phys. 8(6), 106 (2006). [CrossRef]
  15. M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, “Fast focus field calculations,” Opt. Express 14(23), 11277–11291 (2006). [CrossRef] [PubMed]
  16. M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express 16(12), 8519–8531 (2008). [CrossRef] [PubMed]
  17. 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]
  18. S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003). [CrossRef] [PubMed]
  19. X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010). [CrossRef]
  20. D. Magde, E. Elson, and W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972). [CrossRef]
  21. R. Rigler, and E. S. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications, Springer Ser. Chem. Phys. 65, Berlin Heidelberg, Germany (2001).
  22. L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005). [CrossRef] [PubMed]
  23. D. E. Koppel, “Statistical Accuracy in Fluorescence Correlation Spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974). [CrossRef]
  24. In case of a long-lived excited state, i.e. ≈12ns in a nano-diamond nitrogen vacancy center [25], the required CW power is only about twice the average power for pulsed operation.
  25. K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009). [CrossRef] [PubMed]
  26. E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008). [CrossRef] [PubMed]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.


« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited