Abstract

The resolution attainable with stimulated emission depletion (STED) microscopy greatly depends on the quality of the STED laser focus. So far, visual inspection of a measured STED focus has been the only convenient means of gauging the source of aberrations. Here we describe a method, requiring no instrument modifications, for obtaining an equivalent to the complex pupil function at the back aperture of the objective and show that it provides quantitative information about aberration sources (including aberrations induced by the objective or sample). We show the accuracy of this field representation to be sufficient for reconstructing the STED focus in three dimensions and determining corrective steps.

© 2012 Optical Society of America

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References

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  1. S. W. Hell, Nat. Meth. 6, 24 (2009).
    [CrossRef]
  2. D. Toomre and J. Bewersdorf, Annu. Rev. Cell Dev. Biol. 26, 285 (2010).
    [CrossRef]
  3. S. W. Hell and J. Wichmann, Opt. Lett. 19, 780 (1994).
    [CrossRef]
  4. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
    [CrossRef]
  5. S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, Opt. Express 17, 1714 (2009).
    [CrossRef]
  6. L. Kastrup, D. Wildanger, B. R. Rankin, and S. W. Hell, in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, A. Diaspro, ed. (Chapman & Hall/CRC, 2010), pp. 1–13.
  7. B. M. Hanser, M. G. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 216, 32 (2004).
    [CrossRef]
  8. T. J. Gould, J. R. Myers, and J. Bewersdorf, Opt. Express 19, 13351 (2011).
    [CrossRef]
  9. D. Débarre, T. Vieille, and E. Beaurepaire, J. Microsc. 244, 136 (2011).
    [CrossRef]
  10. T. Züchner, A. V. Failla, and A. J. Meixner, Angew. Chem. 50, 5274 (2011).
    [CrossRef]

2011

T. J. Gould, J. R. Myers, and J. Bewersdorf, Opt. Express 19, 13351 (2011).
[CrossRef]

D. Débarre, T. Vieille, and E. Beaurepaire, J. Microsc. 244, 136 (2011).
[CrossRef]

T. Züchner, A. V. Failla, and A. J. Meixner, Angew. Chem. 50, 5274 (2011).
[CrossRef]

2010

D. Toomre and J. Bewersdorf, Annu. Rev. Cell Dev. Biol. 26, 285 (2010).
[CrossRef]

2009

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
[CrossRef]

S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, Opt. Express 17, 1714 (2009).
[CrossRef]

S. W. Hell, Nat. Meth. 6, 24 (2009).
[CrossRef]

2004

B. M. Hanser, M. G. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 216, 32 (2004).
[CrossRef]

1994

Agard, D. A.

B. M. Hanser, M. G. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 216, 32 (2004).
[CrossRef]

Beaurepaire, E.

D. Débarre, T. Vieille, and E. Beaurepaire, J. Microsc. 244, 136 (2011).
[CrossRef]

Bewersdorf, J.

T. J. Gould, J. R. Myers, and J. Bewersdorf, Opt. Express 19, 13351 (2011).
[CrossRef]

D. Toomre and J. Bewersdorf, Annu. Rev. Cell Dev. Biol. 26, 285 (2010).
[CrossRef]

Cheng, Y.

Débarre, D.

D. Débarre, T. Vieille, and E. Beaurepaire, J. Microsc. 244, 136 (2011).
[CrossRef]

Deng, S.

Eggeling, C.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
[CrossRef]

Failla, A. V.

T. Züchner, A. V. Failla, and A. J. Meixner, Angew. Chem. 50, 5274 (2011).
[CrossRef]

Gould, T. J.

Gustafsson, M. G.

B. M. Hanser, M. G. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 216, 32 (2004).
[CrossRef]

Han, K. Y.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
[CrossRef]

Hanser, B. M.

B. M. Hanser, M. G. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 216, 32 (2004).
[CrossRef]

Hell, S. W.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
[CrossRef]

S. W. Hell, Nat. Meth. 6, 24 (2009).
[CrossRef]

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

L. Kastrup, D. Wildanger, B. R. Rankin, and S. W. Hell, in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, A. Diaspro, ed. (Chapman & Hall/CRC, 2010), pp. 1–13.

Irvine, S. E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
[CrossRef]

Kastrup, L.

L. Kastrup, D. Wildanger, B. R. Rankin, and S. W. Hell, in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, A. Diaspro, ed. (Chapman & Hall/CRC, 2010), pp. 1–13.

Li, R.

Liu, L.

Meixner, A. J.

T. Züchner, A. V. Failla, and A. J. Meixner, Angew. Chem. 50, 5274 (2011).
[CrossRef]

Myers, J. R.

Rankin, B. R.

L. Kastrup, D. Wildanger, B. R. Rankin, and S. W. Hell, in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, A. Diaspro, ed. (Chapman & Hall/CRC, 2010), pp. 1–13.

Rittweger, E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
[CrossRef]

Sedat, J. W.

B. M. Hanser, M. G. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 216, 32 (2004).
[CrossRef]

Toomre, D.

D. Toomre and J. Bewersdorf, Annu. Rev. Cell Dev. Biol. 26, 285 (2010).
[CrossRef]

Vieille, T.

D. Débarre, T. Vieille, and E. Beaurepaire, J. Microsc. 244, 136 (2011).
[CrossRef]

Wichmann, J.

Wildanger, D.

L. Kastrup, D. Wildanger, B. R. Rankin, and S. W. Hell, in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, A. Diaspro, ed. (Chapman & Hall/CRC, 2010), pp. 1–13.

Xu, Z.

Züchner, T.

T. Züchner, A. V. Failla, and A. J. Meixner, Angew. Chem. 50, 5274 (2011).
[CrossRef]

Angew. Chem.

T. Züchner, A. V. Failla, and A. J. Meixner, Angew. Chem. 50, 5274 (2011).
[CrossRef]

Annu. Rev. Cell Dev. Biol.

D. Toomre and J. Bewersdorf, Annu. Rev. Cell Dev. Biol. 26, 285 (2010).
[CrossRef]

J. Microsc.

B. M. Hanser, M. G. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 216, 32 (2004).
[CrossRef]

D. Débarre, T. Vieille, and E. Beaurepaire, J. Microsc. 244, 136 (2011).
[CrossRef]

Nat. Meth.

S. W. Hell, Nat. Meth. 6, 24 (2009).
[CrossRef]

Nat. Photon.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photon. 3, 144 (2009).
[CrossRef]

Opt. Express

Opt. Lett.

Other

L. Kastrup, D. Wildanger, B. R. Rankin, and S. W. Hell, in Nanoscopy and Multidimensional Optical Fluorescence Microscopy, A. Diaspro, ed. (Chapman & Hall/CRC, 2010), pp. 1–13.

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Figures (3)

Fig. 1.
Fig. 1.

(a) Raw image after subtraction of the noise floor (as determined by area outside the dashed green circle) and multiplication with the cleanup filter shown in (d). (b) Magnified region shown in light blue box in (a). (c) Same region after 100 iterations of the noise-suppression scheme using the filter kernel shown in (e). (f) Mask showing pixels with negative values in (b) in white. The solid red circle in (d) encloses values >98% of the maximum. The solid magenta curve shows a central line profile through (d). White scale bars: 1 μm.

Fig. 2.
Fig. 2.

Pupil magnitudes (left) and phases (right) retrieved from two z-stacks (top and bottom). Magnitude images are normalized to their maximum values. Dynamic range of phase images: [π,π].

Fig. 3.
Fig. 3.

First and third rows: z-stacks with measured (preprocessed) STED foci. Second and fourth rows: STED foci reconstructed from the retrieved pupil functions (based on images marked by white asterisks). In each row, the intensities are normalized to the highest pixel intensity in that row after applying a gain to the images as indicated above each column. Only the central regions of the images are displayed (the surrounding low-intensity regions were reconstructed equally well). White scale bar: 1 μm.

Equations (2)

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h(x,y,z)=t=02|FT2D1{[T2t+1(kx,ky)iT2t+2(kx,ky)]P(kx,ky)e2πikz(kx,ky)z}|2.
T1(kx,ky)=cos(θ)cos2(φ)+sin2(φ),T2(kx,ky)=(cos(θ)1)sin(φ)cos(φ),T3(kx,ky)=(cos(θ)1)sin(φ)cos(φ),T4(kx,ky)=cos(θ)sin2(φ)+cos2(φ),T5(kx,ky)=sin(θ)cos(φ),T6(kx,ky)=sin(θ)sin(φ),

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