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Quantitative High-Speed Imaging of Entire Developing Embryos with Simultaneous Multi-View Light Sheet Microscopy
Raju Tomer, Khaled Khairy, Fernando Amat and Philipp J. Keller*
Howard Hughes Medical Institute
Janelia Research Campus
19700 Helix Drive, Ashburn, VA 20147, USA
*To whom correspondence should be addressed. Email: kellerp(at)janelia.hhmi.org
Nature Methods, 3 June 2012 (Advance Online Publication)
Simultaneous multi-view imaging of mitotic cycles 10-13 in the Drosophila syncytial blastoderm (His2Av-GFPS65T transgenic stock). The entire embryo was recorded in 25-second intervals, using an image acquisition period of 10 seconds per time point. The data set consists of 111,840 high-resolution images (1.12 terabytes), which were acquired over a period of two hours. The video shows maximum-intensity projections of the fused three-dimensional image stacks.
Imaging framework: One-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 1 as a DivX AVI file (33 MB, DivX 6.9.2 codec).
Download Movie 1 as a QuickTime file (27 MB, H.264 codec).
Simultaneous multi-view imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock). The embryo was recorded in 30-second intervals over a period of 17 hours, using an image acquisition period of 15 seconds per time point. The data set consists of 1,066,520 high-resolution images (11 terabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused and background-corrected three-dimensional image stacks, providing dorsal and ventral views of the developing embryo. To reduce the file size of this video, frames were down-sampled by a factor of 2.
Imaging framework: One-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Technical note: The occurrence of subtle stripe patterns arises from column gain variability in first-generation sCMOS cameras, such as the Andor Neo cameras used in this recording.
Download Movie 2 as a DivX AVI file (176 MB, DivX 6.9.2 codec).
Download Movie 2 as a QuickTime file (91 MB, H.264 codec).
Simultaneous multi-view imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock). The embryo was recorded in 35-second intervals over a period of 19.5 hours, using an image acquisition period of 15 seconds per time point. The data set consists of 1,000,500 high-resolution images (10 terabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused and background-corrected three-dimensional image stacks, providing lateral views of the developing embryo. To reduce the file size of this video, frames were down-sampled by a factor of 2.
Imaging framework: One-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Technical note: Small patches of the chorion remained attached to the embryo during imaging, leading to a local reduction in image contrast for parts of the embryo (see e.g. Movies 1 and 2 for one-photon SiMView recordings of embryos without such patches).
Download Movie 3 as a DivX AVI file (189 MB, DivX 6.9.2 codec).
Download Movie 3 as a QuickTime file (97 MB, H.264 codec).
Simultaneous multi-view imaging of a live nuclei-labeled Drosophila embryo in stage 16 (His2Av-GFPS65T transgenic stock), using the two-photon SiMView framework. The video shows the three-dimensional image data plane per plane and illustrates excellent specimen coverage as well as the absence of spatio-temporal artifacts in simultaneous multi-view imaging. Note that comprehensive imaging of early stage 16 embryos is particularly challenging, owing to the low transparency and high complexity of the organism in this stage. To reduce the file size of this video, frames were down-sampled by a factor of 2.
Imaging framework: Two-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 4 as a DivX AVI file (44 MB, DivX 6.9.2 codec).
Download Movie 4 as a QuickTime file (7 MB, H.264 codec).
Simultaneous multi-view imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock), using the two-photon SiMView framework. The embryo was recorded in 30-second intervals over a period of 2 hours during germ band retraction, using an image acquisition period of 20 seconds per time point. The data set consists of 37,620 high-resolution images (387 gigabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused three-dimensional image stacks, providing dorsal and ventral views of the developing embryo. To reduce the file size of this video, frames were down-sampled by a factor of 2.
Imaging framework: Two-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 5 as a DivX AVI file (28 MB, DivX 6.9.2 codec).
Download Movie 5 as a QuickTime file (22 MB, H.264 codec).
Simultaneous multi-view imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock), using the two-photon SiMView framework. The embryo was recorded in 30-second intervals over a period of 3 hours during dorsal closure and ventral nerve cord (VNC) formation, using an image acquisition period of 20 seconds per time point. The data set consists of 68,460 high-resolution images (705 gigabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused three-dimensional image stacks, providing dorsal and ventral views of the developing embryo. To reduce the file size of this video, frames were down-sampled by a factor of 2.
Imaging framework: Two-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 6 as a DivX AVI file (31 MB, DivX 6.9.2 codec).
Download Movie 6 as a QuickTime file (43 MB, H.264 codec).
Recording of the hatched larva exiting the soft agarose cylinder after image acquisition of the C155-GAL4,UAS-mCD8::GFP embryo from Movie 12. The 0.4% agarose cylinder containing the specimen was slightly protruded from the glass capillary for simultaneous multi-view imaging in the light sheet microscope. Image acquisition in the light sheet microscope was stopped shortly after the onset of strong muscle contractions in the developing embryo. Following image acquisition, the agarose-embedded Drosophila embryo was transferred to the dissection microscope to control for normal hatching of the larva. The embryo’s previous location during light sheet-based imaging is visible as a dark ellipsoidal region inside the agarose cylinder. The video documenting the hatching process was recorded with an XM10 camera on an Olympus MVX10 macroscope.
Download Movie 7 as a DivX AVI file (17 MB, DivX 6.9.2 codec).
Download Movie 7 as a QuickTime file (21 MB, H.264 codec).
Superposition of image data of the histone-eGFP labeled embryo in Movie 1 and automated tracking results using a sequential Gaussian mixture model approach. The video shows the 12th and 13th mitotic cycles in the syncytial blastoderm and encompasses a total of 128 time points. Each object corresponds to a three-dimensional Gaussian rendered on top of the raw data using Vaa3D (Peng et al. 2010, Nature Biotechnology). The color scheme was selected in the first frame using a total of ten colors and trying to avoid collisions between neighbors. After this initial assignment, the color information was propagated in time using the tracking information. The segmentation and tracking pipeline achieves a segmentation efficiency of 95% and a tracking efficiency of 99%. Please see Fig. 5d,e and Supplementary Fig. 6 for the statistical analysis of the spatio-temporal lineaging information obtained from this reconstruction.
Download Movie 8 as a DivX AVI file (21 MB, DivX 6.9.2 codec).
Download Movie 8 as a QuickTime file (27 MB, H.264 codec).
This movie shows the same reconstruction results as Movie 8, but using a different color map for ellipsoid rendering, encoding information on mitotic wave propagation across the embryo: nuclei are shown in cyan as a default, except during nuclear division events. If a division is detected, the object is rendered in magenta and progressively fades back to cyan in the next 5 times points. The video reveals the speed and propagation pattern of the mitotic wave across the Drosophila embryo. The segmentation and tracking pipeline achieves 94% efficiency in the detection of nuclear divisions.
Download Movie 9 as a DivX AVI file (21 MB, DivX 6.9.2 codec).
Download Movie 9 as a QuickTime file (27 MB, H.264 codec).
Enlarged view of the reconstructed Drosophila embryo with nuclei tracking information on the left (see also Movies 8 and 9) and morphological nuclear segmentation on the right. The video shows the developmental period 63-84 minutes post fertilization in 35-second intervals. In this specific visualization, the tracking data was manually corrected for the errors quantified in the section “Quantitative Estimation of Segmentation and Tracking Accuracy” of the Online Methods. Please see Movies 8 and 9 for unfiltered segmentation, tracking and cell division detection results for the entire embryo.
Download Movie 10 as a DivX AVI file (8 MB, DivX 6.9.2 codec).
Download Movie 10 as a QuickTime file (5 MB, H.264 codec).
SiMView recording of the histone-labeled Drosophila embryo shown in Movie 3 (lateral view), superimposed with the reconstructed cell tracks of three neuroblasts and one epidermoblast as well as their respective daughter cells. Four blastoderm cells were manually tracked through their divisions from time point 0 to 400 (120-353 minutes post fertilization), using Imaris (Bitplane) and ImageJ (http://rsbweb.nih.gov/ij/). At the beginning of the time sequence, the nuclei of cells differentiating into neuroblasts are highlighted by green arrows, whereas the nucleus of the cell differentiating into an epidermoblast is highlighted by an orange arrow. At the end of the time sequence, a rotation view of the last time point is shown together with the full cell tracks to visualize their three-dimensional geometry within the embryo. Optical slices and lineage trees for these lineage reconstructions are shown in Supplementary Fig. 10. To reduce the file size of this video, frames were down-sampled by a factor of 1.4.
Download Movie 11 as a DivX AVI file (95 MB, DivX 6.9.2 codec).
Download Movie 11 as a QuickTime file (61 MB, H.264 codec).
Simultaneous multi-view imaging of Drosophila nervous system development (C155-GAL4,UAS-mCD8::GFP transgenic embryo). The entire embryo was recorded in 30-second intervals over a period of 6 hours, using an image acquisition period of 15 seconds per time point. The data set consists of 392,560 high-resolution images (4 terabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused three-dimensional image stacks, providing dorsal and ventral views of the developing embryo. See Supplementary Fig. 12 for optical slices of the Drosophila VNC and brain lobes, which demonstrate that cellular resolution is achieved for deep structures in these global recordings. To reduce the file size of this video, frames were down-sampled by a factor of 2.
Imaging framework: One-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 12 as a DivX AVI file (64 MB, DivX 6.9.2 codec).
Download Movie 12 as a QuickTime file (37 MB, H.264 codec).
Simultaneous multi-view imaging of Drosophila nervous system development (C155-GAL4,UAS-mCD8::GFP transgenic embryo). The entire embryo was recorded in 25-second intervals over a period of 5.5 hours, using an image acquisition period of 15 seconds per time point. The data set consists of 409,760 high-resolution images (4 terabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused three-dimensional image stacks, providing dorsal and ventral views of the developing embryo. See Supplementary Fig. 12 for optical slices of the Drosophila VNC and brain lobes, which demonstrate that cellular resolution is achieved for deep structures in these global recordings. To reduce the file size of this video, frames were down-sampled by a factor of 2.
Imaging framework: One-photon SiMView.
Detection objectives: 2x Nikon CFI75 LWD 16x/0.80 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 13 as a DivX AVI file (73 MB, DivX 6.9.2 codec).
Download Movie 13 as a QuickTime file (45 MB, H.264 codec).
Rotating Amira volume rendering of the SiMView time-lapse microscopy data set visualized in Movie 13. The visualization by volume rendering highlights spatial relationships and simplifies following global morphogenetic processes. However, small structures are lost and spatial resolution is substantially reduced in the rendering process. For structural details, please see the complementary visualization by maximum-intensity projections in Movie 13. To reduce the file size of this video, frames were down-sampled by a factor of 3.
Download Movie 14 as a DivX AVI file (110 MB, DivX 6.9.2 codec).
Download Movie 14 as a QuickTime file (31 MB, H.264 codec).
The video shows a sub-region visualization of only the dorsal posterior part of the whole-embryo recording in Movie 12. The vitelline membrane was computationally removed to highlight the fine spatio-temporal dynamics of axonal outgrowth captured with simultaneous multi-view light sheet microscopy.
Download Movie 15 as a DivX AVI file (30 MB, DivX 6.9.2 codec).
Download Movie 15 as a QuickTime file (15 MB, H.264 codec).
High-magnification simultaneous multi-view imaging of Drosophila nervous system development (Ftz-ng-GAL4,10XUAS-IVS-myr::GFP transgenic embryo). A 350-µm-long anterior-posterior section of the embryo (approximately 2/3 of the embryo) was recorded in 30-second intervals over a period of 1.5 hours, using an image acquisition period of 15 seconds per time point. The data set consists of 72,600 high-resolution images (748 gigabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused three-dimensional image stacks, providing dorsal and ventral views of the developing embryo. To reduce the file size of this video, frames were down-sampled by a factor of 4.5. The vitelline membrane was computationally removed to highlight the fine spatio-temporal dynamics of axonal outgrowth captured with simultaneous multi-view light sheet microscopy. See Movie 19 for sub-regions of the optical slices recorded in this experiment.
Imaging framework: One-photon SiMView.
Detection objectives: 2x Carl Zeiss Plan-Apochromat 40x/1.0 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 16 as a DivX AVI file (25 MB, DivX 6.9.2 codec).
Download Movie 16 as a QuickTime file (14 MB, H.264 codec).
Rotating Amira volume rendering of the SiMView time-lapse microscopy data set visualized in Movie 16. The microscopy data set was down-sampled for this volume rendering, which focuses on dynamic process on the ventral side. The visualization by volume rendering highlights spatial relationships and simplifies following global morphogenetic processes. However, small structures are lost and spatial resolution is substantially reduced in the rendering process. For structural details, please see the complementary visualization by maximum-intensity projections in Movie 16. To reduce the file size of this video, frames were down-sampled by a factor of 8.
Download Movie 17 as a DivX AVI file (137 MB, DivX 6.9.2 codec).
Download Movie 17 as a QuickTime file (50 MB, H.264 codec).
High-magnification simultaneous multi-view imaging of Drosophila nervous system development (Ftz-ng-GAL4,10XUAS-IVS-myr::GFP transgenic embryo). A 350-µm-long anterior-posterior section of the embryo (approximately 2/3 of the embryo) was recorded in 30-second intervals over a period of 8.5 hours, using an image acquisition period of 15 seconds per time point. The data set consists of 460,460 high-resolution images (4.63 terabytes). The video shows separate maximum-intensity projections of the first and second halves of the fused three-dimensional image stacks, providing dorsal and ventral views of the developing embryo. To reduce the file size of this video, frames were down-sampled by a factor of 6. See Movies 19 and 20 for high-resolution sub-regions of the optical slices recorded in this series of experiments.
Imaging framework: One-photon SiMView.
Detection objectives: 2x Carl Zeiss Plan-Apochromat 40x/1.0 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 18 as a DivX AVI file (249 MB, DivX 6.9.2 codec).
Download Movie 18 as a QuickTime file (67 MB, H.264 codec).
The video shows full-resolution maximum-intensity projections of a sub-region of the recording visualized in Movie 16. The sub-region represents 2.5% of the volume covered by the complete recording (cropped by 75% laterally and 90% axially).
Download Movie 19 as a DivX AVI file (15 MB, DivX 6.9.2 codec).
Download Movie 19 as a QuickTime file (14 MB, H.264 codec).
The video shows full-resolution maximum-intensity projections of a sub-region of the ventral nerve cord (VNC), recorded with one-photon SiMView. The sub-region represents 0.7% of the volume covered by the complete recording (cropped by 88% laterally and 94% axially). A 350-µm-long anterior-posterior section of the embryo (approximately 2/3 of the embryo) was recorded in 30-second intervals over a period of 2 hours, using an image acquisition period of 15 seconds per time point. The complete data set consists of 107,520 high-resolution images (1.08 terabytes).
Imaging framework: One-photon SiMView.
Detection objectives: 2x Carl Zeiss Plan-Apochromat 40x/1.0 W.
Cameras: 2x Andor Neo sCMOS.
Download Movie 20 as a DivX AVI file (31 MB, DivX 6.9.2 codec).
Download Movie 20 as a QuickTime file (33 MB, H.264 codec).
Figures included in the PDF documents (Adobe Acrobat Reader 6.0 or higher required):
- Figure 1: Technology framework for light sheet-based simultaneous multi-view imaging
- Figure 2: Quantitative live imaging of entire early Drosophila embryos
- Figure 3: Simultaneous multi-view imaging of fast cellular dynamics
- Figure 4: Simultaneous multi-view two-photon imaging of entire embryos
- Figure 5: Quantitative reconstruction of nuclei dynamics in the syncytial blastoderm
- Figure 6: Simultaneous multi-view imaging of Drosophila neural development
- Supplementary Figure 1: Technology framework for simultaneous multi-view imaging
- Supplementary Figure 2: Optical implementation of the SiMView imaging platform
- Supplementary Figure 3: SiMView real-time electronics and computational hardware
- Supplementary Figure 4: Point-spread-functions in one- and two-photon SiMView
- Supplementary Figure 5: Spatio-temporal artifacts in sequential multi-view imaging
- Supplementary Figure 6: Quantifying Drosophila nuclei dynamics in the syncytial blastoderm
- Supplementary Figure 7: Simultaneous multi-view imaging of Drosophila embryogenesis
- Supplementary Figure 8: Post-acquisition larval hatching in sequential multi-view imaging
- Supplementary Figure 9: Manual cell tracking in the retracting germ band
- Supplementary Figure 10: Reconstruction of neuroblast and epidermoblast lineages
- Supplementary Figure 11: Time-course of C155-GAL4,UAS-mCD8::eGFP signal intensity
- Supplementary Figure 12: SiMView optical slices of the Drosophila VNC and brain lobes
Download high-quality Figures 1-6 (50 MB, ZIP archive).
Download high-quality Figures S1-S12 (16 MB, ZIP archive).
Fast, high-contrast imaging of animal development by scanned light sheet-based structured illumination microscopy
Philipp J. Keller1,2*, Annette D. Schmidt3, Anthony Santella4, Khaled Khairy1, Zhirong Bao4, Joachim Wittbrodt3,5 and Ernst H.K. Stelzer1,6*
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Germany
2Janelia Research Campus, Howard Hughes Medical Institute, USA
3Institute of Zoology, Department for Developmental Physiology, University of Heidelberg, Germany
4Developmental Biology Program, Sloan-Kettering Institute, USA
5Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Germany
6Frankfurt Institute for Molecular Life Sciences, Goethe University, Germany
*To whom correspondence should be addressed. Email: kellerp(at)janelia.hhmi.org, stelzer(at)embl.de
Nature Methods, 30 July 2010, vol. 7, no. 8, pp. 637-642
Note: If you encounter problems during playback of the DivX movies, you most likely require the latest version of the DivX codec for your media player. The codec is freely available for download on the following websites:
Apple Mac: http://www.divx.com/en/software/mac
Microsoft Windows: http://www.divx.com/en/software/divx-plus
A schematic illustration of the DSLM with an intensity-modulated laser illumination pattern. The sinusoidal intensity profile was generated by scanning the beam through the specimen at a constant speed while synchronously modulating the laser intensity with an acousto-optical tunable filter (AOTF). Inset, close-up of the illuminated specimen inside the specimen chamber.
Download Movie 1 as a DivX AVI file (2.0 MB, DivX 6.8.5 codec).
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Maximum-intensity projections of a DSLM time-lapse recording of a membrane- and nuclei-labeled zebrafish embryo. Membranes were imaged using structured illumination (SI-25, top row), nuclei using standard light sheet illumination (bottom left). The top-most cell layer (the enveloping layer, EVL) was removed computationally in the ras-eGFP channel and is shown on the right, separate from the deeper cell layers (left). The egg diameter is approximately 720 mm.
Download Movie 2 as a DivX AVI file (26.3 MB, DivX 6.8.5 codec).
Download Movie 2 as a QuickTime file (38.9 MB, H.264 codec).
DSLM-SI long-term imaging of a membrane-labeled zebrafish embryo. Maximum-intensity projections of a DSLM-SI time-lapse recording of a zebrafish embryo (ras-eGFP transgenic line), during the period 9–67 h.p.f.. To provide an unobstructed view of the embryo, the top-most cell layer (the enveloping layer, EVL) was removed computationally. The egg diameter is approximately 720 mm. Images were deconvolved with the Lucy-Richardson algorithm (10 iterations). Fluorescence was detected with a Carl Zeiss C-Apochromat 10´/0.45 W objective.
Download Movie 3 as a DivX AVI file (136 MB, DivX 6.8.5 codec).
Download Movie 3 as a QuickTime file (102 MB, H.264 codec).
Maximum-intensity projections of a DSLM-SI multiple-view time-lapse recording of a nuclei-labeled Drosophila embryo. The embryo is approximately 520 mm long. In total 137,520 images were recorded for 191 time points (3 min sampling intervals). Temperature: 20 °C during preparation (0-2 h.p.f.), 24 °C during imaging (> 2 h.p.f.). Images were deconvolved with the Lucy-Richardson algorithm (5 iterations). Carl Zeiss Plan-Apochromat 20´/1.0 W.
Download Movie 4 as a DivX AVI file (16.7 MB, DivX 6.8.5 codec).
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Computational alignment of the four point clouds representing the nuclei detected in the four DSLM-SI views of the developing Drosophila embryo. Nuclei shown in different colors originate from different microscopic views.
Download Movie 5 as a DivX AVI file (20.9 MB, DivX 6.8.5 codec).
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Different perspectives of the Drosophila Digital Embryo, obtained by multiple-view fusion of the four nuclear point clouds and color-coding of directed regional nuclei movement speeds over 10-min intervals (0–0.8 mm min–1, cyan to orange).
Download Movie 6 as a DivX AVI file (22.0 MB, DivX 6.8.5 codec).
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Figures included in the PDF documents (Adobe Acrobat Reader 6.0 or higher required):
- Figure 1: Light sheet-based structured illumination with digitally adjustable frequency
- Figure 2: Enhancing image contrast with DSLM structured illumination
- Figure 3: Multiple-view imaging of Drosophila embryogenesis with DSLM-SI
- Figure 4: Spatiotemporal image contrast optimization by DSLM-SI frequency chirping
- Figure S1: DSLM-SI implementation
- Figure S2: DSLM-SI illumination patterns for the typical range of SI frequencies
- Figure S3: DSLM-SI illumination patterns for high SI frequencies
- Figure S4: Mirror surface reconstruction with DSLM-SI
- Figure S5: Imaging tissue phantoms with DSLM-SI
- Figure S6: DSLM-SI performance at low to intermediate scattering coefficients
- Figure S7: DSLM-SI performance at high scattering coefficients
- Figure S8: Contrast enhancement in Medaka DSLM-SI recordings
- Figure S9: DSLM-SI coverage in zebrafish multi-channel recordings
- Figure S10: DSLM-SI coverage at advanced zebrafish embryonic development
- Figure S11: DSLM-SI coverage at late zebrafish embryonic development
- Figure S12: Removal of scattered light in zebrafish DSLM-SI recordings
- Figure S13: Removal of scattered light in Drosophila DSLM-SI recordings
- Figure S14: DSLM-SI penetration depth in early Drosophila embryogenesis
- Figure S15: Workflow for processing and visualization of DSLM-SI recordings
- Figure S16: Multiple-view fusion of the Drosophila Digital Embryo
Download high-quality Figures 1-4 (134 MB, ZIP archive).
Download high-quality Figures S1-S16 (235 MB, ZIP archive).
Each digital embryo database is provided as a Matlab MAT-file, containing a multi-cell array (with each cell corresponding to one time point) and a two-dimensional parameter table in each cell characterizing the nuclei segmented at the respective time point. Each table has n rows (n being the number of nuclei at the respective time point) and 6 columns listing the following parameters:
Columns 1-3: x-y-z coordinates of the center-of-mass of the nucleus (length unit = 370 nm).
Column 4: Diameter of the sphere matching the volume occupied by the nucleus.
Column 5: Average fluorescence intensity of the nucleus (arbitrary units).
Column 6: Index of the microscopic view the nucleus originates from (only applicable to the raw reconstruction of the multiple-view data set; indices 1-4 correspond to views 0°/90°/180°/270°).
Specification of data set 1:
Filename: "drosophila_raw.mat"
Number of time points and temporal sampling:
191 time points (120-690 m.p.f.), 180 sec intervals
Total number of nucleus entries: 3,624,857
Database size: 49.5 MB
Related database items: Figures 3a-b, S13-S16; Movie 4, Movie 5
Download the Matlab database for data set 1.
Specification of data set 2:
Filename: "drosophila_fused.mat"
Number of time points and temporal sampling:
191 time points (120-690 m.p.f.), 180 sec intervals
Total number of nucleus entries: 1,486,971
Database size: 20.5 MB
Related database items: Figures 3a-c, S13-S16; Movie 4, Movie 6
Download the Matlab database for data set 2.
Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy
Philipp J. Keller1,2*, Annette D. Schmidt2, Joachim Wittbrodt1,2,3,4* and Ernst H.K. Stelzer1
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Germany
2Developmental Biology Unit, European Molecular Biology Laboratory, Germany
3Institute of Zoology, Department for Developmental Physiology University of Heidelberg, Germany
4Institute of Toxicology and Genetics Karlsruhe Institute of Technology, Germany
*To whom correspondence should be addressed. Email: keller(at)embl.de, wittbrod(at)embl.de
Science, 14 November 2008, vol. 322, no. 5904, pp. 1065-1069
Note: If you encounter problems during playback of the DivX movies, you most likely require the latest version of the DivX codec for your media player. The codec is freely available for download on the following websites:
Apple Mac: http://www.divx.com/en/software/mac
Microsoft Windows: http://www.divx.com/en/software/divx-plus
The movie shows a schematic illustration of specimen illumination and fluorescence light detection in the standard DSLM imaging mode. The laser beam illuminates the sample from the side and excites fluorophores along a single line inside the specimen. A pair of laser scanners moves the excitation laser line vertically and horizontally. An optically sectioned image is recorded by rapidly scanning an entire plane in the specimen and detecting the fluorescence at a right angle to the illumination axis. The top left inset shows a zoom-in on the illuminated fraction of the specimen, which is embedded in an agarose cylinder and placed inside the specimen chamber.
Download Movie 1 as a DivX AVI file (2 MB, DivX 6.8.2 codec).
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The movie shows maximum-intensity projections of a DSLM time-lapse multi-view recoding of zebrafish embryonic development, with a view on both the animal and vegetal hemispheres. The wild-type zebrafish embryo was injected with H2B-eGFP mRNA at the one cell stage. Imaging was started in the 64-cell stage. The entire data set consists of 1,226 time points, recorded in 90 s intervals. At each time point, 370 images with 2048 x 2048 pixels each were recorded with a z-spacing of 2.96 µm. The entire data set comprises 453,620 images (3.5 TBytes). The images were deconvolved with the Lucy-Richardson-algorithm (10 iterations) and normalized for constant overall intensity, thus compensating for the increasing intensity over time due to GFP expression. Detection lens: Carl Zeiss C-Apochromat 10x/0.45 W.
Download Movie 2 as a DivX AVI file (30 MB, DivX 6.8.2 codec).
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The movie shows an orthographic rendering of the digital embryo obtained from the microscopy data shown in Movie 2. In each frame, the reconstructions at ten time points, corresponding to a time interval of 15 min, are superimposed to indicate the nuclear movement directions. The darker shading corresponds to older time points. The colour-code indicates the current movement speed of each nucleus. A bright orange corresponds to an average movement speed of 1.2 µm/min or more, while a bright cyan highlights effectively non-migratory nuclei. Intermediate speeds are shown by linear interpolation along the colour gradient.
Download Movie 3 as a DivX AVI file (28 MB, DivX 6.8.2 codec).
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The movie shows maximum-intensity projections of a DSLM time-lapse multi-view recording of zebrafish MZoep mutant embryonic development, with a view on the dorsal and ventral hemispheres. Preparation and microscopy settings as in Movie 2.
Download Movie 4 as a DivX AVI file (32 MB, DivX 6.8.2 codec).
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The movie shows an orthographic rendering of the MZoep mutant digital embryo obtained from the microscopy data shown in Movie 4, with a dorsal view on the left and a lateral view on the right. Visualization settings as in Movie 3.
Download Movie 5 as a DivX AVI file (29 MB, DivX 6.8.2 codec).
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The movie shows cell tracking in a slice through the shield for the microscopy time-lapse data set presented in Movie 4 (left: speed colour-code, right: directional colour-code). The 100-µm-thick slices are shown from a lateral view, with the dorsal side to the right. Speed colour-code as in Movie 3. The nuclear movements were analyzed for 30 min intervals and colour-encoded as follows (see also sphere at the bottom): anterior movements (green), posterior movements (cyan), movements away from the body axis (yellow), movements towards the body axis (red) and movements toward the yolk cell (pink). The corresponding colours were assigned, if a movement of at least 20 µm along the respective direction was determined within a time interval of 30 min.
Download Movie 6 as a DivX AVI file (13 MB, DivX 6.8.2 codec).
Download Movie 6 as a QuickTime file (14 MB, H.264 codec).
The movie shows maximum-intensity projections of a DSLM time-lapse recoding of zebrafish embryonic development, with a view on the animal hemisphere. The wild-type zebrafish embryo was injected with H2B-eGFP mRNA at the one cell stage. Imaging was started at 1.6 hours post fertilization, during the transition from the 32- to the 64-cell stage. The entire data set consists of 1,470 time points, recorded in 60 s intervals. For each time point, 205 images with 2048 x 2048 pixels each were recorded within 30 s with a z-spacing of 2.96 µm (i.e. a 600 µm deep volume). The entire data set comprises 301,350 images (2.3 TBytes). The images were deconvolved with the Lucy-Richardson-algorithm (20 iterations) and normalized for constant overall intensity, thus compensating for the increasing intensity over time due to GFP-production. Since the illumination laser beam has to travel through the yolk in order to excite fluorophores on the far left side of the animal hemisphere, the average nuclear brightness is lower in this part of the embryo. However, because of the excellent signal-to-noise ratio and the dynamic range of 14 bit of the DSLM images, the corresponding nuclei can also be robustly identified (see Figure S5). Detection lens: Carl Zeiss C-Apochromat 10x/0.45 W.
Download Movie 7 as a DivX AVI file (30 MB, DivX 6.8.2 codec).
Download Movie 7 as a QuickTime file (39 MB, H.264 codec).
The movie shows a perspective rendering of the reconstruction as well as nuclei population statistics for the zebrafish wild-type embryo in Movie 7. Left: view on the animal pole from the centre of the yolk cell. Right: lateral view. In each frame, the reconstructions at ten time points, corresponding to a time interval of 10 min, are superimposed to indicate the nuclear movement directions. The darker shading corresponds to older time points.
Download Movie 8 as a DivX AVI file (30 MB, DivX 6.8.2 codec).
Download Movie 8 as a QuickTime file (32 MB, H.264 codec).
We tracked all nuclei reconstructed from the microscopy time-lapse data set shown in Movie 7. The digital embryo is visualized as a perspective rendering with a view on the animal pole from the centre of the yolk cell. The nuclear movements were analyzed for 30 min intervals and colour-encoded as follows (see also sphere to bottom left): anterior movements (green), posterior movements (cyan), movements away from the body axis (yellow), movements towards the body axis (red) and movements toward the yolk cell (pink). The corresponding colours were assigned, if a movement of at least 20 µm along the respective direction was determined within a time interval of 30 min.
Download Movie 9 as a DivX AVI file (26 MB, DivX 6.8.2 codec).
Download Movie 9 as a QuickTime file (38 MB, H.264 codec).
The movie shows 4,500 cell division events in the data set presented in Movie 7, detected by the automated processing pipeline at an efficiency of over 90 % for the time interval 100-400 mpf (minutes post fertilization at 26.5 °C). Dividing cells are marked in red and their corresponding daughter cells are shown in cyan. The movie illustrates the initial radial division waves, followed by the symmetry break and by slow peripheral waves in division cycles 10-13. Beyond that time interval, cell divisions appear in a complex pattern of asynchronous patches. The statistical analysis of the division axes of these 4,500 cell divisions is shown in Figure S6b.
Download Movie 10 as a DivX AVI file (14 MB, DivX 6.8.2 codec).
Download Movie 10 as a QuickTime file (14 MB, H.264 codec).
Visualization of the tracking of retinal progenitor cells (red nuclei) in the digital embryo (gray nuclei). The cells were identified by their spatial position in the last time point, i.e. when the optic vesicle had already formed. A reverse engineering of their migratory tracks over a time interval of six hours provides information about the morphogenetic blueprint of eye organogenesis. The automatically computed tracks are in good agreement with the manual reconstruction presented in Rembold et al. (2006). The movie illustrates the principle of organ lineaging using our digital embryos.
Download Movie 11 as a DivX AVI file (14 MB, DivX 6.8.2 codec).
Download Movie 11 as a QuickTime file (16 MB, H.264 codec).
The movie shows maximum-intensity projections of a DSLM time-lapse recoding of zebrafish embryonic development, with a view on the embryonic shield. The wild-type zebrafish embryo was injected with H2B-eGFP mRNA at the one cell stage. Imaging was started 5 hours post fertilization at approximately 35 % epiboly. The entire data set consists of 1,000 time points, recorded in 60 s intervals. At each time point, 205 images with 2048 x 2048 pixels each were recorded within 30 s with a z-spacing of 2.96 µm (i.e. a 600 µm deep volume). The images were deconvolved with the Lucy-Richardson-algorithm (10 iterations) and normalized for constant overall intensity, thus compensating for the increasing intensity over time due to GFP-production. Detection lens: Carl Zeiss C-Apochromat 10x/0.45 W.
Download Movie 12 as a DivX AVI file (24 MB, DivX 6.8.2 codec).
Download Movie 12 as a QuickTime file (38 MB, H.264 codec).
The movie shows global cell tracking for the microscopy time-lapse data set presented in Movie 12. The parameter settings are identical to those used in Movie 9. The nuclear movements are colour-encoded as follows: towards the vegetal pole (cyan), towards the animal pole (yellow), towards the body axis (green) and towards the yolk cell (pink).
Download Movie 14 as a DivX AVI file (20 MB, DivX 6.8.2 codec).
Download Movie 14 as a QuickTime file (30 MB, H.264 codec).
Global cell tracking with parameter settings identical to those used in Movie 9. The nuclear movements are colour-encoded as follows: towards the vegetal pole (cyan), towards the animal pole (yellow), towards the body axis (green) and towards the yolk cell (pink).
Download Movie 15 as a DivX AVI file (29 MB, DivX 6.8.2 codec).
Download Movie 15 as a QuickTime file (27 MB, H.264 codec).
Frontal and lateral view on embryonic slices on the dorsal and ventral hemispheres of the digital embryo (right: slice close to the shield, left: slice opposite of the shield; complete reconstructions are shown in Movies 14 and 15). The montage visualizes cell internalization in the form of a wave of mesendodermal progenitor cells folding under cell layers of ectodermal progenitors (emboly) between 40 and 50 % epiboly. In the reconstruction, four cell populations were tracked: green nuclei at the tip of the early wave, yellow nuclei in the late wave, blue nuclei at the leading edge moving towards the vegetal pole and pink nuclei close to the animal pole moving towards the vegetal pole. Internalized cells move towards the animal pole on the dorsal hemisphere. In contrast, on the ventral hemisphere, internalized cells move only briefly towards the animal pole and then change their migration direction back towards the vegetal pole. A comprehensive morphogenetic model of hypoblast formation is provided in Figure 6.
Download Movie 16 as a DivX AVI file (15 MB, DivX 6.8.2 codec).
Download Movie 16 as a QuickTime file (18 MB, H.264 codec).
Figures included in the PDF document (requires Adobe Acrobat Reader 6.0 or higher):
- Figure 1: Digital Scanned Light Sheet Microscopy
- Figure 2: Imaging and reconstruction of zebrafish embryogenesis
- Figure 3: Cell tracking and detection of cell divisions in the digital embryo
- Figure 4: Symmetry breaking of the global cell division pattern
- Figure 5: Mesendoderm internalization and migration in dorsal and ventral hemispheres
- Figure 6: A model of mesendoderm formation in zebrafish
- Figure S1: Blueprint of the Digital Scanned Laser Light Sheet Fluorescence Microscope
- Figure S2: DSLM multi-view imaging of large specimen
- Figure S3: Statistical analysis of nuclear properties in the animal hemisphere
- Figure S4: The image processing pipeline
- Figure S5: Pre-processing/visualization of DSLM recordings of zebrafish embryogenesis
- Figure S6: Global statistical analysis of cell divisions
- Figure S7: Embryo-to-embryo variability of morphogenetic key parameters
- Figure S8: Quantification of the properties of the peripheral cell division waves
- Figure S9: Analysis of the patterns of mesendoderm internalization on the dorsal and ventral hemispheres
Download high-quality Figures 1-6 and S1-S9 (6 MB, PDF).
Each digital embryo database is provided as a Matlab MAT-file, containing a multi-cell array (with each cell corresponding to one time point) and a two-dimensional parameter table in each cell characterizing the nuclei segmented at the respective time point. Each table has n rows (n being the number of nuclei at the respective time point) and 17 columns listing the following parameters:
|
Columns 1-3: |
x-y-z coordinates of the centre-of-mass of the |
|
Columns 4/5: |
Average (column 4) and centre-of-mass (column 5) |
|
Columns 6-11: |
x-y-z start and end coordinates of the bounding box of |
|
Columns 12-17: |
x-y-z FWHM coordinates of the three-dimensional intensity |
Total number of nucleus entries for all developmental blueprints: 55,707,328
Note: Raw DSLM data sets can be provided upon request. The cropped and compressed DSLM image databases are two terabytes in size and require shipping on hard disks. If you are interested in this option, please send an email to keller(at)embl.de for further instructions. The microscopy data sets are helpful e.g. for manual verification of the segmentation data. An example of such an overlay of DSLM data and the segmentation centres in the digital embryo can be downloaded here (30 MB). The overlay represents data set 2 at time point 100 (see below). The DSLM microscopy data was cropped, gamma-corrected and reduced to an 8 bit look-up-table in order to simplify the comparison with the segmentation data.
Specification of data set 2:
Number of time points and temporal sampling:
1,456 time points (100-1,555 mpf), 60 sec intervals
Total number of nucleus entries: 8,736,014
Database size: 431 MB
Related database items: Movie 7, Movie 8, Movie 9, Movie 10, Movie 11
Download the Matlab database for data set 2.
Modules included in the package:
Segmentation pipeline:
clusterNuclei, clusterCollect
clusterFilter, clusterCorrelate, clusterSaturate
clusterCombine
Data analysis pipeline:
clusterDivisions
clusterMaps, clusterVectorize
clusterIngression, clusterMovement
clusterReduce, clusterRendering
clusterStatistics
clusterTrace, clusterTracking
Download the compressed Matlab source code (65 kB, ZIP archive).