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

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  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, 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]

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, 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]

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]

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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.

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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.

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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.

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Equations (1)

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M=S−βB/(1+exp[−k(S−ST)]),

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