Abstract

Multidirectional selective plane illumination microscopy (mSPIM) reduces absorption and scattering artifacts and provides an evenly illuminated focal plane. mSPIM solves two common problems in light-sheet-based imaging techniques: The shadowing in the excitation path due to absorption in the specimen is eliminated by pivoting the light sheet; the spread of the light sheet by scattering in the sample is compensated by illuminating the sample consecutively from opposing directions. The resulting two images are computationally fused yielding a superior image. The effective light sheet is thinner, and the axial resolution is increased by 2 over single-directional SPIM. The multidirectional illumination proves essential in biological specimens such as millimeter-sized embryos. The performance of mSPIM is demonstrated by the imaging of live zebrafish embryos.

© 2007 Optical Society of America

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References

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2007

J. A. N. Buytaert and J. J. J. Dirckx, J. Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

J. Swoger, P. Verveer, K. Greger, J. Huisken, and E. H. K. Stelzer, Opt. Express 15, 8029 (2007).
[CrossRef] [PubMed]

2006

2004

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

2003

2001

S. Pauls, B. Geldmacher-Voss, and J. A. Campus-Ortega, Dev. Genes Evol. 211, 603 (2001).
[CrossRef]

1993

A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
[CrossRef] [PubMed]

Dev. Genes Evol.

S. Pauls, B. Geldmacher-Voss, and J. A. Campus-Ortega, Dev. Genes Evol. 211, 603 (2001).
[CrossRef]

J. Biomed. Opt.

J. A. N. Buytaert and J. J. J. Dirckx, J. Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

J. Microsc.

A. H. Voie, D. H. Burns, and F. A. Spelman, J. Microsc. 170, 229 (1993).
[CrossRef] [PubMed]

Nat. Methods

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Science

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

Trends Cell Biol.

D. Beis and D. Y. R. Stainier, Trends Cell Biol. 16, 105 (2006).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

(a) Schematic of mSPIM. (b) Parallel illumination of the sample in SPIM results in shadows and attenuation across the FOV. (c) In mSPIM these artifacts are severely reduced by sequentially illuminating the sample from two sides with a thinner pivoting light sheet. IL, illumination; DL, detection lens; S, sample.

Fig. 2
Fig. 2

Setup for mSPIM: (a) Top view of the instrument, (b) top and side view of the beams in the scan and illumination unit (not to scale). The laser unit provides the light from one or multiple lasers (L1/2). The beam is scanned in the vertical direction by a resonant mirror (RM) and expanded by a telescope (X). A vertical slit (VS) is used to control the numerical aperture NA z and the thickness of the light sheet. A flip mirror (FM) directs the light into illumination unit 1 or 2. In these identical units a cylindrical lens (C) focuses the light to a horizontal line that is imaged into the back focal plane (BFP) of the illumination lens (IL1/2) by a telescope (T). A horizontal slit (HS) is used to confine the excitation light along y to the FOV in the sample (S). The resulting light sheet exiting the IL is collimated in y and focused in z. It is aligned in z to match the focal plane of the detection lens (DL) by tilting one mirror (M; red rays in the top view). The scanning of the RM results in the pivoting of the light sheet (red rays in side view). The sample can be translated in x , y , z and rotated about y with micromotors (not shown). The detection unit consists of the DL and one or multiple cameras (CCD1/2) with dichroic (DC), filters (F), and tube lenses (TL). Planes conjugate to the illumination focal plane (FP) are marked with asterisks.

Fig. 3
Fig. 3

Head of a live 35 h old zebrafish embryo imaged with (a) SPIM and (b) mSPIM. In mSPIM stripes are eliminated. (c)–(e) 10 h old embryo imaged with mSPIM and illuminated from (c) left and (d) right. In the merge of the two images (e) no attenuation is visible. (f)–(i) Magnified view of the area delineated in (c). Scale bars 50 μ m . Sample mounted as described in [1].

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