Abstract

We report a simple optical setup to produce both axial and lateral structured illumination through a single objective lens. With a minimum of six full-field images obtained without moving either the sample or the microscope objective, 100nm diameter fluorescent beads can be localized axially with an accuracy of 50nm in a 1.76-μm-thick layer. We show that this axial localization improvement can easily be combined with classical lateral structured illumination, so that lateral resolution enhancement by a factor of 2 is maintained.

© 2008 Optical Society of America

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2007

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E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef] [PubMed]

F. Chasles, B. Dubertret, and A. C. Boccara, Opt. Lett. 31, 1274 (2006).
[CrossRef] [PubMed]

2003

2002

2000

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

M. G. L. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef] [PubMed]

1999

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

1997

1994

1993

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

1992

1988

M. L. Minsky, Scanning 10, 128 (1988).
[CrossRef]

Agard, D. A.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

Albrecht, B.

Bailey, B.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef] [PubMed]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Boccara, A. C.

Bonifacio, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Chasles, F.

Cremer, C.

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Dawson, M. D.

Dubertret, B.

Elson, D. S.

Failla, A. V.

Farkas, D. L.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Florin, E. L.

French, P. M. W.

Girirajan, T. P. K.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef] [PubMed]

Girkin, M.

Griffin, C.

Gu, E.

Gustafsson, M. G. L.

M. G. L. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef] [PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

Hell, S.

Hell, S. W.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

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

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef] [PubMed]

Jonas, A.

Juskaitis, R.

Kao, H. P.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

Kennedy, G. T.

Lanni, F.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Mason, M. D.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef] [PubMed]

Minsky, M. L.

M. L. Minsky, Scanning 10, 128 (1988).
[CrossRef]

Nagorni, M.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Neil, M. A.

Neil, M. A. A.

Oddos, S.

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Poher, V.

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef] [PubMed]

Schmidt, M.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Schweitzer, A.

Sedat, J. W.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

Speidel, M.

Stelzer, E. H. K.

Taylor, D. L.

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Verkman, A. S.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

Wichmann, J.

Wilson, T.

Zhang, H. X.

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef] [PubMed]

J. Microsc.

M. G. L. Gustafsson, J. Microsc. 198, 82 (2000).
[CrossRef] [PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Nat. Methods

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef] [PubMed]

Nature

B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, Nature 366, 44 (1993).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Rev. Sci. Instrum.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Scanning

M. L. Minsky, Scanning 10, 128 (1988).
[CrossRef]

Science

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacio, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, Science 313, 1642 (2006).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

Schematic of the illumination system. The excitation laser beam passes through a beam expander (not shown) and is reflected by a programmable diffractive optical element (DOE). The diffraction angles are adapted to the microscope pupil diameter with a 3.7 × telescope (TL1, TL2). A programmable diaphragm and a phase shifter (DOE) are placed in the Fourier plane of the first lens. The periscope (M) directs the selected beams through the microscope tube lens (ML1), and the beams are focused on the back focal plane (FP) of the microscope objective lens. The signal is recovered by epifluorescence and directed to a CCD camera using a dichroic filter (DF) and the camera tube lens (ML2).

Fig. 2
Fig. 2

Comparison of three images of 100 nm diameter fluorescent coated spheres obtained with different illumination setups: (a) non-SI, (b) two-beam classical SI, (c) three-beam SI.

Fig. 3
Fig. 3

Calculated axial position of a pair of 100 nm diameter fluorescent spheres for two different positions of the sample. Axial position is calculated independently for each illuminated pixel. The z = 0 plane is arbitrarily fixed at midpoint between the two objects. (a) The two spheres are within the objective depth of field; (c) same measurement after the sample was axially translated by 2 μ m ; (b), (d) corresponding histograms of the calculated positions. In (a) and (c) x and y are the measured pixel positions and the z axis codes the calculated axial position for each pixel.

Fig. 4
Fig. 4

Measured axial position of a 100 nm diameter fluorescent sphere with respect to the illumination structure. The z = 0 plane is arbitrarily fixed to one particular position. (a) Measured position versus position tuned with the piezo. (b) Absolute value of the difference between the axial position calculated using Eq. (2) and the position tuned using the piezo.

Equations (3)

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I ( x , z ) = I 0 [ α 2 + 4 cos ( 2 π x 2 p x + ϕ x ) 2 + 4 α cos ( 2 π x 2 p x + ϕ x ) cos ( 2 π z p z + ϕ z ) ] ,
2 p x = λ 0 n sin ( u ) , p z = λ 0 n ( 1 cos ( u ) ) .
I 1 I 1 2 I 0 ( I 1 + I 1 ) = sin ( β ) 1 cos ( β ) tan ( 2 π z p z ) .

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