Abstract

Stimulated emission depletion (STED) microscopy provides diffraction-unlimited resolution in fluorescence microscopy. Imaging at the nanoscale, however, requires precise alignment of the depletion and excitation laser foci of the STED microscope. We demonstrate here that adaptive optics can be implemented to automatically align STED and confocal images with a precision of 4.3±2.3  nm.

© 2013 Optical Society of America

Full Article  |  PDF Article
OSA Recommended Articles
Adaptive optics enables 3D STED microscopy in aberrating specimens

Travis J. Gould, Daniel Burke, Joerg Bewersdorf, and Martin J. Booth
Opt. Express 20(19) 20998-21009 (2012)

Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics

Brian R. Patton, Daniel Burke, David Owald, Travis J. Gould, Joerg Bewersdorf, and Martin J. Booth
Opt. Express 24(8) 8862-8876 (2016)

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

Egidijus Auksorius, Bosanta R. Boruah, Christopher Dunsby, Peter M. P. Lanigan, Gordon Kennedy, Mark A. A. Neil, and Paul M. W. French
Opt. Lett. 33(2) 113-115 (2008)

More Recommended Articles

References

  • View by:
  • Article Order
  • |
  • Year
  • |
  • Author
  • |
  • Publication
  1. T. J. Gould, S. T. Hess, and J. Bewersdorf, Annu. Rev. Biomed. Eng. 14, 231 (2012).
    [Crossref]
  2. S. W. Hell, Nat. Methods 6, 24 (2009).
    [Crossref]
  3. S. W. Hell and J. Wichmann, Opt. Lett. 19, 780 (1994).
    [Crossref]
  4. T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, Opt. Express 20, 20998 (2012).
    [Crossref]
  5. E. Auksorius, B. R. Boruah, C. Dunsby, P. M. Lanigan, G. Kennedy, M. A. Neil, and P. M. French, Opt. Lett. 33, 113 (2008).
    [Crossref]
  6. G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
    [Crossref]
  7. D. Wildanger, J. Buckers, V. Westphal, S. W. Hell, and L. Kastrup, Opt. Express 17, 16100 (2009).
    [Crossref]
  8. P. Torok and P. Munro, Opt. Express 12, 3605 (2004).
    [Crossref]
  9. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
    [Crossref]
  10. G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
    [Crossref]

2012 (2)

T. J. Gould, S. T. Hess, and J. Bewersdorf, Annu. Rev. Biomed. Eng. 14, 231 (2012).
[Crossref]

T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, Opt. Express 20, 20998 (2012).
[Crossref]

2009 (2)

2008 (1)

2007 (1)

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

2006 (1)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

2004 (1)

2000 (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[Crossref]

1994 (1)

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Auksorius, E.

Bewersdorf, J.

T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, Opt. Express 20, 20998 (2012).
[Crossref]

T. J. Gould, S. T. Hess, and J. Bewersdorf, Annu. Rev. Biomed. Eng. 14, 231 (2012).
[Crossref]

Booth, M. J.

Boruah, B. R.

Buckers, J.

Burke, D.

Donnert, G.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Dunsby, C.

Dyba, M.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[Crossref]

Eggeling, C.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Egner, A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[Crossref]

French, P. M.

Gould, T. J.

T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, Opt. Express 20, 20998 (2012).
[Crossref]

T. J. Gould, S. T. Hess, and J. Bewersdorf, Annu. Rev. Biomed. Eng. 14, 231 (2012).
[Crossref]

Hell, S. W.

S. W. Hell, Nat. Methods 6, 24 (2009).
[Crossref]

D. Wildanger, J. Buckers, V. Westphal, S. W. Hell, and L. Kastrup, Opt. Express 17, 16100 (2009).
[Crossref]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[Crossref]

S. W. Hell and J. Wichmann, Opt. Lett. 19, 780 (1994).
[Crossref]

Hess, S. T.

T. J. Gould, S. T. Hess, and J. Bewersdorf, Annu. Rev. Biomed. Eng. 14, 231 (2012).
[Crossref]

Jahn, R.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Jakobs, S.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[Crossref]

Kastrup, L.

Keller, J.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Kennedy, G.

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[Crossref]

Lanigan, P. M.

Luhrmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Medda, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Munro, P.

Neil, M. A.

Rizzoli, S. O.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Schonle, A.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

Torok, P.

Westphal, V.

D. Wildanger, J. Buckers, V. Westphal, S. W. Hell, and L. Kastrup, Opt. Express 17, 16100 (2009).
[Crossref]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

Wichmann, J.

Wildanger, D.

Wurm, C. A.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

Annu. Rev. Biomed. Eng. (1)

T. J. Gould, S. T. Hess, and J. Bewersdorf, Annu. Rev. Biomed. Eng. 14, 231 (2012).
[Crossref]

Biophys. J. (1)

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, Biophys. J. 92, L67 (2007).
[Crossref]

Nat. Methods (1)

S. W. Hell, Nat. Methods 6, 24 (2009).
[Crossref]

Opt. Express (3)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U.S.A. (1)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[Crossref]

Proc. Natl. Acad. Sci. USA (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. USA 97, 8206 (2000).
[Crossref]

Cited By

OSA participates in Crossref's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (3)

Fig. 1.
Fig. 1.

Simplified schematic of the STED setup. The SLM displays a helicoidal phase ramp plus a sum of the Zernike polynomials tip and tilt weighted by the bias amplitudes btip and btilt. This phase pattern is imposed on the reflected STED laser and imaged into the pupil plane of the objective (obj.) Dichroic mirrors direct excitation light (exc.) onto the objective and emitted light (em.) onto the detector (det.). For 3D alignment, a circular λ/2 phase mask replaced the helicoidal phase ramp on the SLM, and a defocus term was included.

Download Full Size | PPT Slide | PDF

Fig. 2.
Fig. 2.

Alignment of toroidal STED focus. (a) Merger of the excitation focus (green) and a poorly aligned depletion focus (red). (b) Merger of corresponding confocal (green) and STED (red) images of fluorescent beads imaged with the foci shown in (a). (c) Line profiles as indicated in (b): confocal (green) and STED (red). (d), (e) Metric curves for alignment procedure: (d) initial coarse alignment using metric defined in Eq. 1 and (e) subsequent fine adjustment optimized STED image brightness. (f)–(h) Same as (a)–(c) after running the auto-alignment routine. To demonstrate the increased STED image intensity in (h) relative to (c), the confocal and STED curves are normalized to their respective maximal values in (f). All images are smoothed using a Gaussian filter with a full width half-maximum of 2.35 pixels. Pixel sizes are (a), (f) 20 nm and (b), (g) 40 nm.

Download Full Size | PPT Slide | PDF

Fig. 3.
Fig. 3.

Axial alignment of 3D STED focus. (a) Merge of confocal (green) and STED (red) images of fluorescent beads. (b) As in (a) after running the auto-aligning routine. (c) Line profiles as indicated in (a) and (b): confocal (green) and STED (red). All curves are normalized to their respective maximal values. The optimized STED peak was ∼18% brighter than the nonaligned peak. Images shown in (a) and (b) were smoothed with a Gaussian filter with a 2 pixel (40 nm pixel size) full width half-maximum.

Download Full Size | PPT Slide | PDF

Equations (1)

Equations on this page are rendered with MathJax. Learn more.

M=S−βB/(1+exp[−k(S−ST)]),

Metrics