Abstract

A new microscope combines optical sectioning by fluorophore excitation using a single light sheet with structured illumination. Several images with laterally intensity-modulated light sheets are recorded from scattering fluorescent specimens. By applying a simple data processing scheme, the nonmodulated volumes are identified. The blurred features become dark, and the resultant images are improved in terms of contrast and resolution. Hence, the instrument is capable of discriminating against contributions to the image that are induced by the optical properties of the specimen. The new microscope’s capabilities are demonstrated by imaging the internals of the head of an adult Drosophila melanogaster (fruit fly) expressing green fluorescent protein–labeled polycomb proteins.

© 2007 Optical Society of America

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  1. K. Greger, J. Swoger, and E. H. K. Stelzer, Rev. Sci. Instrum. 78, 023705 (2007).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  3. C. J. Engelbrecht and E. H. K. Stelzer, Opt. Lett. 31, 1477 (2006).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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2007 (2)

K. Greger, J. Swoger, and E. H. K. Stelzer, Rev. Sci. Instrum. 78, 023705 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

2006 (1)

2004 (2)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

L. H. Schaefer, D. Schuster, and J. Schaffer, J. Microsc. 216, 165 (2004).
[CrossRef] [PubMed]

2003 (1)

2001 (1)

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

1999 (1)

S. Dietzel, H. Niemann, B. Bruckner, C. Maurange, and R. Paro, Chromosoma 108, 83 (1999).
[CrossRef] [PubMed]

1997 (1)

Bruckner, B.

S. Dietzel, H. Niemann, B. Bruckner, C. Maurange, and R. Paro, Chromosoma 108, 83 (1999).
[CrossRef] [PubMed]

Cole, M. J.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Dayel, M. J.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

Dietzel, S.

S. Dietzel, H. Niemann, B. Bruckner, C. Maurange, and R. Paro, Chromosoma 108, 83 (1999).
[CrossRef] [PubMed]

Dowling, K.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Engelbrecht, C. J.

French, P. M. W.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Greger, K.

K. Greger, J. Swoger, and E. H. K. Stelzer, Rev. Sci. Instrum. 78, 023705 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

J. Swoger, J. Huisken, and E. H. K. Stelzer, Opt. Lett. 28, 1654 (2003).
[CrossRef] [PubMed]

Jones, R.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Juskaitis, R.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

M. A. Neil, R. Juskaitis, and T. Wilson, Opt. Lett. 22, 1905 (1997).
[CrossRef]

Lever, M. J.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Marcello, M.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

Maurange, C.

S. Dietzel, H. Niemann, B. Bruckner, C. Maurange, and R. Paro, Chromosoma 108, 83 (1999).
[CrossRef] [PubMed]

Neil, M. A.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

M. A. Neil, R. Juskaitis, and T. Wilson, Opt. Lett. 22, 1905 (1997).
[CrossRef]

Niemann, H.

S. Dietzel, H. Niemann, B. Bruckner, C. Maurange, and R. Paro, Chromosoma 108, 83 (1999).
[CrossRef] [PubMed]

Pampaloni, F.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

Paro, R.

S. Dietzel, H. Niemann, B. Bruckner, C. Maurange, and R. Paro, Chromosoma 108, 83 (1999).
[CrossRef] [PubMed]

Parsons-Karavassilis, D.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Schaefer, L. H.

L. H. Schaefer, D. Schuster, and J. Schaffer, J. Microsc. 216, 165 (2004).
[CrossRef] [PubMed]

Schaffer, J.

L. H. Schaefer, D. Schuster, and J. Schaffer, J. Microsc. 216, 165 (2004).
[CrossRef] [PubMed]

Schuster, D.

L. H. Schaefer, D. Schuster, and J. Schaffer, J. Microsc. 216, 165 (2004).
[CrossRef] [PubMed]

Siegel, J.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Stelzer, E. H. K.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, Rev. Sci. Instrum. 78, 023705 (2007).
[CrossRef] [PubMed]

C. J. Engelbrecht and E. H. K. Stelzer, Opt. Lett. 31, 1477 (2006).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

J. Swoger, J. Huisken, and E. H. K. Stelzer, Opt. Lett. 28, 1654 (2003).
[CrossRef] [PubMed]

Sucharov, L. O. D.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Swoger, J.

K. Greger, J. Swoger, and E. H. K. Stelzer, Rev. Sci. Instrum. 78, 023705 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

J. Swoger, J. Huisken, and E. H. K. Stelzer, Opt. Lett. 28, 1654 (2003).
[CrossRef] [PubMed]

Verveer, P. J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

Web, S. E. D.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

Wilson, T.

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

M. A. Neil, R. Juskaitis, and T. Wilson, Opt. Lett. 22, 1905 (1997).
[CrossRef]

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

Chromosoma (1)

S. Dietzel, H. Niemann, B. Bruckner, C. Maurange, and R. Paro, Chromosoma 108, 83 (1999).
[CrossRef] [PubMed]

J. Microsc. (2)

M. J. Cole, J. Siegel, S. E. D. Web, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. Neil, R. Juskaitis, and T. Wilson, J. Microsc. 203, 246 (2001).
[CrossRef] [PubMed]

L. H. Schaefer, D. Schuster, and J. Schaffer, J. Microsc. 216, 165 (2004).
[CrossRef] [PubMed]

Nat. Methods (1)

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, Nat. Methods 4, 311 (2007).
[PubMed]

Opt. Lett. (3)

Rev. Sci. Instrum. (1)

K. Greger, J. Swoger, and E. H. K. Stelzer, Rev. Sci. Instrum. 78, 023705 (2007).
[CrossRef] [PubMed]

Science (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

Main components in SPIM-SI. A, Top view: a cylindrical lens focuses the modulated laser beam in the specimen. The specimen is moved along the optical axis of the detection system, i.e., through the stationary light sheet, to create stacks of images. B, Front view through the specimen along the optical axis of the microscope objective lens. The collimated beam is modulated laterally and vertically by a grid. The grid’s microtranslation stage provides well-defined phases φ of the grid along the vertical axis.

Fig. 2
Fig. 2

Comparison of SPIM and SPIM-SI images of the front part of a Drosophila melanogaster trunk. A, Conventional SPIM image. B, SPIM-SI image. C, Details of the fruit fly trunk: a, labellum; b, pseudotracheal sensilla; c, left maxillary palpus; d, rostral membrane; e, Details seen in the SPIM-SI image (top right) but not in the SPIM image (top left). D, Normalized intensity values of two horizontal plots of images A and B, as indicated by the line in C. The size of all images is 364 μ m × 306 μ m . The direction of illumination is from the top. A and B are maximum projections of two stacks of images along the z-direction consisting of 181 planes with an axial pitch of 1.5 μ m . The 20 × objective lens was used in combination with a 10   line pair mm grid.

Fig. 3
Fig. 3

Comparison of SPIM and SPIM-SI images of the front part of a Drosophila melanogaster trunk. A, Conventional SPIM image; B, SPIM-SI image. The tip of the labellum was recorded with a 40 × objective lens and 150   line pair mm grid. The size of both images is 212 μ m × 157 μ m . Both are maximum intensity projections through 541 images with an axial pitch of 0.5 μ m .

Equations (1)

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I ( z ) = I 0 ° + I 120 ° e 2 π i 3 + I 240 ° e 4 π i 3 = ( ( I 0 ° I 120 ° ) 2 + ( I 120 ° I 240 ° ) 2 + ( I 240 ° I 0 ° ) 2 ) 2 .

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