Abstract

A non-blind, shift-invariant image processing technique that fuses multi-view three-dimensional image data sets into a single, high quality three-dimensional image is presented. It is effective for 1) improving the resolution and isotropy in images of transparent specimens, and 2) improving the uniformity of the image quality of partially opaque samples. This is demonstrated with fluorescent samples such as Drosophila melanogaster and Medaka embryos and pollen grains imaged by Selective Plane Illumination Microscopy (SPIM). The application of the algorithm to SPIM data yields high-resolution images of organ structure and gene expression, in some cases at a sub-cellular level, throughout specimens ranging from several microns up to a millimeter in size.

© 2007 Optical Society of America

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References

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    [CrossRef]
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2007 (3)

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, "Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain," Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nat. Methods, 4, 311-313 (2007).
[PubMed]

K. Greger, J. Swoger, and E. H. K. Stelzer, "Basic building units and properties of a fluorescence single plane illumination microscope," Rev. Sci. Instrum. 78, 023705 (2007).
[CrossRef] [PubMed]

2006 (1)

2005 (1)

A. A. Gortchakov, et al. "Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster polytene chromosomes," Chromosoma 114, 54-65 (2005).
[CrossRef] [PubMed]

2004 (4)

R. Quiring, et al. "Large-scale expression screening by automated whole-mount in situ hybridization," Mech. Dev. 121, 971-976 (2004).
[CrossRef] [PubMed]

W. Drexler, "Ultrahigh-resolution optical coherence tomography," J. Biomed. Opt. 9, 47-74 (2004).
[CrossRef] [PubMed]

A. L. Wilke, S. A. Jordan, J. A. Sharpe, D. J. Price, and I. J. Jackson, "Widespread tangential dispersion and extensive cell death during early neurogenesis in the mouse neocortex," Dev. Biol. 267, 109-118 (2004).
[CrossRef]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K.Stelzer, "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 305, 1007-1009 (2004).
[CrossRef] [PubMed]

2003 (2)

2002 (3)

A. H. Voie, "Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy," Hearing Research 171, 119-128 (2002).
[CrossRef] [PubMed]

J. Sharpe, et al. "Optical projection tomography as a tool for 3D microscopy and gene expression studies," Science 296, 541-545 (2002).
[CrossRef] [PubMed]

M. Kozubek, et al. "Automated microaxial tomography of cell nuclei after specific labeling by fluorescence in situ hybridisation," Micron 33, 655-665 (2002).
[CrossRef] [PubMed]

2000 (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," PNAS 97, 8206-8210 (2000).
[CrossRef] [PubMed]

1999 (1)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "I5M: 3D widefield light microscopy with better than 100 nm axial resolution," J. Microsc. 195, 10-16 (1999).
[CrossRef] [PubMed]

1998 (1)

1997 (1)

K. Sätzler and R. Eils, "Resolution improvement by 3-D reconstructions from tilted views in axial tomography and confocal theta microscopy," Bioimaging 5, 171-182 (1997).
[CrossRef]

1996 (2)

S. Kikuchi, K. Sonobe, and N. Ohyama, "Three-dimensional microscopic computed tomography based on generalized Radon transform for optical imaging systems," Opt. Commun. 123, 725-733 (1996).
[CrossRef]

C. J. Cogswell, K. G. Larkin, and H. U. Klemm,   "Fluorescence microtomography: multi-angle image acquisition and 3D digital reconstruction," SPIE Proc. 2655, 109-115 (1996).
[CrossRef]

1994 (3)

J. Bradl, M. Hausmann, B. Schneider, B. Rinke, and C. Cremer, "A versatile 2π-tilting device for fluorescence microscopes," J. Microsc. 176, 211-221 (1994).
[CrossRef]

E. H. K. Stelzer and S. Lindek, "Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy," Opt. Commun. 111, 536-547 (1994).
[CrossRef]

S. Kikuchi, K. Sonobe, L. S. Sidharta, and N. Ohyama, "Three-dimensional computed tomography for optical microscopes," Opt. Commun. 107, 432-444 (1994).
[CrossRef]

1992 (3)

S. Hell, and E. H. K. Stelzer, "Properties of a 4Pi confocal fluorescence microscope," J. Opt. Soc. Am. A 9, 2159-2166 (1992).
[CrossRef]

J. Bradl, M. Hausmann, V. Ehemann, D. Komitowski, and C. Cremer, "A tilting device for three-dimensional microscopy: application to in situ imaging of interphase cell nuclei," J. Microsc. 168, 47-57 (1992).
[CrossRef] [PubMed]

L. G. Brown, "A survey of image registration techniques," ACM Computing Surveys 24, 325-376 (1992).
[CrossRef]

1991 (1)

M. Frasch, "The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene Regulator of Chromatin Condensation, RCC1," EMBO J. 10, 1225-1236 (1991).
[PubMed]

1989 (1)

P. J. Shaw, D. A. Agard, Y. Hiraoka, and J. W. Sedat,   "Tilted view reconstruction in optical microscopy," Biophys. J. 55, 101-110 (1989).
[CrossRef] [PubMed]

1975 (1)

R. A. Brooks and G. Di Chiro, "Theory of image reconstruction in computed tomography," Radiology 117, 561-572 (1975).
[PubMed]

ACM Computing Surveys (1)

L. G. Brown, "A survey of image registration techniques," ACM Computing Surveys 24, 325-376 (1992).
[CrossRef]

Appl. Opt. (1)

Bioimaging (1)

K. Sätzler and R. Eils, "Resolution improvement by 3-D reconstructions from tilted views in axial tomography and confocal theta microscopy," Bioimaging 5, 171-182 (1997).
[CrossRef]

Biophys. J. (1)

P. J. Shaw, D. A. Agard, Y. Hiraoka, and J. W. Sedat,   "Tilted view reconstruction in optical microscopy," Biophys. J. 55, 101-110 (1989).
[CrossRef] [PubMed]

Chromosoma (1)

A. A. Gortchakov, et al. "Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster polytene chromosomes," Chromosoma 114, 54-65 (2005).
[CrossRef] [PubMed]

Dev. Biol. (1)

A. L. Wilke, S. A. Jordan, J. A. Sharpe, D. J. Price, and I. J. Jackson, "Widespread tangential dispersion and extensive cell death during early neurogenesis in the mouse neocortex," Dev. Biol. 267, 109-118 (2004).
[CrossRef]

EMBO J. (1)

M. Frasch, "The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene Regulator of Chromatin Condensation, RCC1," EMBO J. 10, 1225-1236 (1991).
[PubMed]

Hearing Research (1)

A. H. Voie, "Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy," Hearing Research 171, 119-128 (2002).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

W. Drexler, "Ultrahigh-resolution optical coherence tomography," J. Biomed. Opt. 9, 47-74 (2004).
[CrossRef] [PubMed]

J. Microsc. (3)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "I5M: 3D widefield light microscopy with better than 100 nm axial resolution," J. Microsc. 195, 10-16 (1999).
[CrossRef] [PubMed]

J. Bradl, M. Hausmann, V. Ehemann, D. Komitowski, and C. Cremer, "A tilting device for three-dimensional microscopy: application to in situ imaging of interphase cell nuclei," J. Microsc. 168, 47-57 (1992).
[CrossRef] [PubMed]

J. Bradl, M. Hausmann, B. Schneider, B. Rinke, and C. Cremer, "A versatile 2π-tilting device for fluorescence microscopes," J. Microsc. 176, 211-221 (1994).
[CrossRef]

J. Opt. Soc. Am. A (1)

Mech. Dev. (1)

R. Quiring, et al. "Large-scale expression screening by automated whole-mount in situ hybridization," Mech. Dev. 121, 971-976 (2004).
[CrossRef] [PubMed]

Micron (1)

M. Kozubek, et al. "Automated microaxial tomography of cell nuclei after specific labeling by fluorescence in situ hybridisation," Micron 33, 655-665 (2002).
[CrossRef] [PubMed]

Nat. Methods (2)

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, "Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain," Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nat. Methods, 4, 311-313 (2007).
[PubMed]

Nature (1)

A. Abbott, "Biology’s new dimension," Nature 424, 870-872 (2003).
[CrossRef] [PubMed]

Opt. Commun. (3)

S. Kikuchi, K. Sonobe, and N. Ohyama, "Three-dimensional microscopic computed tomography based on generalized Radon transform for optical imaging systems," Opt. Commun. 123, 725-733 (1996).
[CrossRef]

E. H. K. Stelzer and S. Lindek, "Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy," Opt. Commun. 111, 536-547 (1994).
[CrossRef]

S. Kikuchi, K. Sonobe, L. S. Sidharta, and N. Ohyama, "Three-dimensional computed tomography for optical microscopes," Opt. Commun. 107, 432-444 (1994).
[CrossRef]

Opt. Lett. (2)

PNAS (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," PNAS 97, 8206-8210 (2000).
[CrossRef] [PubMed]

Radiology (1)

R. A. Brooks and G. Di Chiro, "Theory of image reconstruction in computed tomography," Radiology 117, 561-572 (1975).
[PubMed]

Rev. Sci. Instrum. (1)

K. Greger, J. Swoger, and E. H. K. Stelzer, "Basic building units and properties of a fluorescence single plane illumination microscope," Rev. Sci. Instrum. 78, 023705 (2007).
[CrossRef] [PubMed]

Science (2)

J. Sharpe, et al. "Optical projection tomography as a tool for 3D microscopy and gene expression studies," Science 296, 541-545 (2002).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K.Stelzer, "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 305, 1007-1009 (2004).
[CrossRef] [PubMed]

SPIE Proc. (1)

C. J. Cogswell, K. G. Larkin, and H. U. Klemm,   "Fluorescence microtomography: multi-angle image acquisition and 3D digital reconstruction," SPIE Proc. 2655, 109-115 (1996).
[CrossRef]

Other (4)

S. Kawata, "The optical computed tomography microscope," in Advances in Optical and Electron Microscopy, T. Mulvey and C. R. J. Sheppard, eds., (Academic Press Limited, San Diego, 1994) Vol. 14.

M. Born and E. Wolf, Principles of Optics 7th ed., (Cambridge University Press, Cambridge, CB2 2RU, U. K., 1999).

See the Medaka Expression Pattern Database (MEPD), http://pubservl.embl.de:8280/pubserv/servlet/de.embl.th.mepd.servlets.MdbShowClone01?cloneID=1251>.

W. H. Press, S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery, Numerical Recipes in C++, 2nd Ed. (Cambridge University Press, Cambridge, U.K., 2002).

Supplementary Material (4)

» Media 1: MOV (1550 KB)     
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» Media 4: MOV (99 KB)     

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Figures (8)

Fig. 1.
Fig. 1.

SPIM schematic. The sample (S) is illuminated by a thin light sheet generated by passing a collimated laser beam through a cylindrical lens (CL). The region of the sample that is imaged onto the CCD camera by the objective (OL) and tube (TL) lenses is illuminated by the light sheet. The emission filter (EM) blocks scattered illumination light. The sample is scanned along the detection axis to create a 3D image stack. It can then be physically rotated to different orientations and re-scanned, generating sets of 3D stacks along different viewing angles. An immersion-medium-filled chamber (IC) encloses the specimen and reduces the negative effects of aberration-inducing interfaces.

Fig. 2.
Fig. 2.

Illustration of image distortions caused by anisotropic and spatially varying image quality, and the compensation thereof by fusing two images. a) The undistorted sample fluorophore distribution. Top row: Images distorted by reduced resolution horizontally (b) or vertically (c). d) Fusion of b) and c) shows improved resolution isotropy. Bottom row: Images distorted by absorption while detecting from the left (f) and from the right (g). h) Fusion of f) and g) shows improved spatial coverage of the sample. Arrows indicate the directions along which the light is detected.

Fig. 3.
Fig. 3.

Slices perpendicular to the rotation axis, through 3D auto fluorescence data stacks of a paper mulberry pollen grain. a) 0°-view. b) 90°-view. c-f) Fusions of 36 views by: c) arithmetic mean; d) weighted spectral mean; e) MVD-Wiener; f) MVD-MAPGG. Arrowheads in a) and b) indicate the directions of the respective detection axes. Scale bar = 3 μm. See also Movie 1 (1.55MB), slices rotating through the grain of paper mulberry pollen, for which the left image is the 0° view, and the right is the MVD-MAPGG fusion. [Media 1]

Fig. 4.
Fig. 4.

Effects of the number of views used in the MVD-MAPGG fusion of paper mulberry pollen autofluorescence images. Top: slices perpendicular to SPIM rotation axis, for different numbers of fused images. Bottom: corresponding power spectra (plotted with a non-linear look-up table to emphasize the high-frequency components). The detection axes are indicated by the white arrows in a-d. Scale bar = 3 μm.

Fig. 5.
Fig. 5.

Drosophila melanogaster embryo. Green: uncondensed DNA; red: condensed DNA. a) Maximum-value projections of a single-view data set, through the ventral (top) and dorsal (bottom) halves of the embryo. b) as a), but for an 8-view MVD-Wiener fusion. c) Expanded image of the 100 μm wide region indicated in b). Scale bar = 100 μm. See also Movie 2, maximum-value projections through the MVD-Wiener fusion of the Drosophila embryo. [Media 2]

Fig. 6.
Fig. 6.

Medaka embryo, stage 32, nuclear label (green) and McF0001MGR-1G19bd1 in situ hybridization (red). Left: single-view images; right: 6-view MAPGG fusion. a,b) Maximum-value projections along orthogonal axes. The regions of expression of McF0001MGR-1G19bd1 in the tectum proliferative zone (TPZ), pineal gland (PG), and telencephalon (tel) are clearly visible. For comparison, the inset shows the traditional blue-labeled transmission image. c) Slice at the depth indicated by the dashed line in a). Internal structures such as the lens (L), pigmented epithelium (PE), ganglion cell layer (GCL), outer (ONL) and inner nuclear layers (INL), and the inner plexiform layer (IPL) of the retina are well-defined in the fusion. The illumination (ill), detection (det), and rotation (rot) axes are indicated for the single-view images. Scale bar = 200 μm. See also Movie 3, an animation of the MVD-MAPGG fusion showing maximum-value projections at the top and slices at the position indicated by the white line at the bottom. [Media 3]

Fig. 7.
Fig. 7.

Image processing algorithm. Inputs: N, number of views; ij , j th image; ϕj , j th viewing angle; M, optical magnification; ∆x, pixel pitch (lateral); ∆z, slice spacing (axial); f HP, high-pass spatial frequency filter; r max, maximum registration error; pj , j th PSF; μ, MVD-Wiener regularization parameter; k max, MVD-Wiener termination parameter; g σ, MVD-MAPGG Gaussian filter; k max, MVD-MAPGG maximum number of iterations. Output: e, sample distribution estimate. Internal: r⃑ = (x,y,z), position; s⃑, spatial frequency; I⃑ , mean image integral; e 0, registration target; xj , j th cross-correlation; r⃑, position of peak of xj ; r, gradient vector; d, search direction; α, step size along d. Functional: Σ w j , weighted average (see text); CGA, conjugate gradient algorithm [3]; MAPGG, maximum a posteriori with Gaussian noise and prior algorithm [15]; *, convolution operator; ⊗, correlation operator; ã , Fourier transform of a.

Fig. 8.
Fig. 8.

Slices perpendicular to the anterior/posterior axis of the Drosophila melanogaster embryo shown in Fig. 5. Slices from a) a view showing the upper left portion of the embryo clearly; b) a view taken at 180° with respect to the orientation in a); c) a view taken at 45° with respect to a). d) Overlay of the slices in a-c). The views in a) and b) contain very little overlapping information, which would make them difficult to register in isolation. Scale bar = 100 μm. See also Movie 4, an animation of progressive construction of a running-mean registration target. Individual cross-sections are shown in the red channel, and the green channel shows the running mean registration target as each view is successively added. [Media 4]

Equations (2)

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