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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 2 — Jan. 18, 2010
  • pp: 1302–1309

Fast STED microscopy
with continuous wave fiber lasers

Gael Moneron, Rebecca Medda, Birka Hein, Arnold Giske, Volker Westphal, and Stefan W. Hell  »View Author Affiliations


Optics Express, Vol. 18, Issue 2, pp. 1302-1309 (2010)
http://dx.doi.org/10.1364/OE.18.001302


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Abstract

We report on fast beam-scanning stimulated-emission-depletion (STED) microscopy in the visible range using for resolution enhancement compact, low cost and turn-key continuous wave (CW) fiber lasers emitting at 592 nm. Spatial resolutions of 35 to 65 nm in the focal plane are shown for various samples including fluorescent nanoparticles, immuno-stained cells with a non-exhaustive selection of 5 commonly used organic fluorescent markers, and living cells expressing the yellow fluorescent protein Citrine. The potential of the straightforward combination of CW-STED and fast beam scanning is illustrated in a movie of the endoplasmic reticulum (ER) of a living cell, composed of 100 frames (6 µm × 12 µm), each of them acquired in a time shorter than 0.2 s.

© 2010 OSA

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(180.5810) Microscopy : Scanning microscopy

ToC Category:
Microscopy

History
Original Manuscript: November 18, 2009
Revised Manuscript: January 3, 2010
Manuscript Accepted: January 7, 2010
Published: January 12, 2010

Virtual Issues
Vol. 5, Iss. 3 Virtual Journal for Biomedical Optics

Citation
Gael Moneron, Rebecca Medda, Birka Hein, Arnold Giske, Volker Westphal, and Stefan W. Hell, "Fast STED microscopy
with continuous wave fiber lasers," Opt. Express 18, 1302-1309 (2010)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-18-2-1302


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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. S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009). [CrossRef] [PubMed]
  3. T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999). [CrossRef]
  4. 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]
  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. U.S.A. 103(31), 11440–11445 (2006). [CrossRef] [PubMed]
  6. 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]
  7. 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]
  8. 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]
  9. A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009). [CrossRef] [PubMed]
  10. C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009). [CrossRef]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. A. Schönle, Imspector Image Acquisition & Analysis Software, v0.1, (2006): http://www.imspector.de
  16. O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001). [CrossRef]
  17. B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008). [CrossRef] [PubMed]
  18. 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]
  19. D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009). [CrossRef] [PubMed]
  20. G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007). [CrossRef]
  21. G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009). [CrossRef] [PubMed]
  22. List of fluorescent dyes used in STED microscopy: http://www.mpibpc.mpg.de/abteilungen/200/STED_Dyes.html

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