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Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

Nature Methods volume 9, pages 755763 (2012) | Download Citation

Abstract

Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.

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Acknowledgements

We thank M. Coleman (Coleman Technologies) for custom microscope operating software; the Janelia Farm Research Campus and European Molecular Biology Laboratory workshops for custom mechanical parts; K. Branson for helpful discussions and valuable comments on the manuscript; G. Myers for helpful discussions and for supporting F.A.; B. Coop and B. Biddle for helpful advice on instrumentation; A. Denisin for testing SiMView live-imaging assays of the Drosophila central nervous system; J. Truman, D. Mellert, J. Simpson, M. Zlatic, T. Lee, B. Lemon, E. Betzig, N. Ji, T. Planchon and L. Gao for helpful discussions; and J. Simpson, B. Pfeiffer and G. Rubin (Janelia Farm Research Campus) for transgenic Drosophila stocks. This work was supported by the Howard Hughes Medical Institute.

Author information

Affiliations

  1. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.

    • Raju Tomer
    • , Khaled Khairy
    • , Fernando Amat
    •  & Philipp J Keller

Authors

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  2. Search for Khaled Khairy in:

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Contributions

P.J.K. conceived the research and designed the microscopes. R.T. and P.J.K. built and characterized the microscopes. R.T. developed and performed the imaging experiments. K.K., F.A. and P.J.K. developed the image processing and data management framework. All authors analyzed the data. P.J.K. wrote the paper, with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Philipp J Keller.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Tables 1–3

Zip files

  1. 1.

    Supplementary Software

    SiMView module for high-throughput multiview image registration.

Videos

  1. 1.

    Simultaneous multiview imaging of the Drosophila syncytial blastoderm

    Simultaneous multiview imaging of mitotic cycles 10–13 in the Drosophila syncytial blastoderm (His2Av-GFPS65T transgenic stock). The entire embryo was recorded at 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× Andor Neo sCMOS.

  2. 2.

    Simultaneous multiview imaging of Drosophila embryogenesis (embryo 1)

    Simultaneous multiview imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock). The embryo was recorded at 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× 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.

  3. 3.

    Simultaneous multiview imaging of Drosophila embryogenesis (embryo 2)

    Simultaneous multiview imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock). The embryo was recorded at 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× 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. Supplementary Videos 1 and 2 for one-photon SiMView recordings of embryos without such patches).

  4. 4.

    Two-photon SiMView slicing series of a live Drosophila stage 16 embryo

    Simultaneous multiview 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 multiview 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× Andor Neo sCMOS.

  5. 5.

    Two-photon SiMView imaging of germ band retraction

    Simultaneous multiview imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock), using the two-photon SiMView framework. The embryo was recorded at 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× Andor Neo sCMOS.

  6. 6.

    Two-photon SiMView imaging of dorsal closure and VNC formation

    Simultaneous multiview imaging of Drosophila embryonic development (His2Av-GFPS65T transgenic stock), using the two-photon SiMView framework. The embryo was recorded at 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× Andor Neo sCMOS.

  7. 7.

    Control for post-acquisition larval hatching in SiMView imaging

    Recording of the hatched larva exiting the soft agarose cylinder after image acquisition of the C155-GAL4,UAS-mCD8::GFP embryo from Supplementary Video 12. The 0.4% agarose cylinder containing the specimen was slightly protruded from the glass capillary for simultaneous multiview 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.

  8. 8.

    Global nuclei tracking in the Drosophila syncytial blastoderm

    Superposition of image data of the histone-eGFP labeled embryo in Supplementary Video 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 Figure 5d,e and Supplementary Figure 6 for the statistical analysis of the spatio-temporal lineaging information obtained from this reconstruction.

  9. 9.

    Detection of nuclear divisions in the Drosophila syncytial blastoderm

    This movie shows the same reconstruction results as Supplementary Video 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 five 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.

  10. 10.

    Lineaging and morphological segmentation in the syncytial blastoderm

    Enlarged view of the reconstructed Drosophila embryo with nuclei tracking information on the left (see also Supplementary Videos 8 and 9) and morphological nuclear segmentation on the right. The video shows the developmental period 63–84 minutes post fertilization at 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 Supplementary Videos 8 and 9 for unfiltered segmentation, tracking and cell division detection results for the entire embryo.

  11. 11.

    SiMView reconstruction of neuroblast and epidermoblast lineages

    SiMView recording of the histone-labeled Drosophila embryo shown in Supplementary Video 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 Figure 10. To reduce the file size of this video, frames were down-sampled by a factor of 1.4.

  12. 12.

    One-photon SiMView imaging of Drosophila neural development (C155-GAL4, embryo 1)

    Simultaneous multiview imaging of Drosophila nervous system development (C155-GAL4,UAS-mCD8::GFP transgenic embryo). The entire embryo was recorded at 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 Figure 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× Andor Neo sCMOS.

  13. 13.

    One-photon SiMView imaging of Drosophila neural development (C155-GAL4, embryo 2)

    Simultaneous multiview imaging of Drosophila nervous system development (C155-GAL4,UAS-mCD8::GFP transgenic embryo). The entire embryo was recorded at 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 Figure 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: 2× Nikon CFI75 LWD 16×/0.80 W. Cameras: 2× Andor Neo sCMOS.

  14. 14.

    Volume rendering of neural development recording (C155-GAL4, embryo 2)

    Rotating Amira volume rendering of the SiMView time-lapse microscopy data set visualized in Supplementary Video 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 Supplementary Video 13. To reduce the file size of this video, frames were down-sampled by a factor of 3.

  15. 15.

    Axonal outgrowth in Drosophila neural development (C155-GAL4, embryo 1)

    The video shows a subregion visualization of only the dorsal posterior part of the whole-embryo recording in Supplementary Video 12. The vitelline membrane was computationally removed to highlight the fine spatio-temporal dynamics of axonal outgrowth captured with simultaneous multiview light-sheet microscopy.

  16. 16.

    High-resolution SiMView imaging of the Drosophila CNS (Ftz-ng-GAL4, embryo 1)

    High-magnification simultaneous multiview 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 at 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 multiview light-sheet microscopy. See Supplementary Video 19 for subregions of the optical slices recorded in this experiment. Imaging framework: One-photon SiMView. Detection objectives: 2× Carl Zeiss Plan-Apochromat 40×/1.0 W. Cameras: 2× Andor Neo sCMOS.

  17. 17.

    Volume rendering of CNS time-lapse recording (Ftz-ng-GAL4, embryo 1)

    Rotating Amira volume rendering of the SiMView time-lapse microscopy data set visualized in Supplementary Video 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 Supplementary Video 16. To reduce the file size of this video, frames were down-sampled by a factor of 8.

  18. 18.

    High-resolution SiMView imaging of the Drosophila CNS (Ftz-ng-GAL4, embryo 2)

    High-magnification simultaneous multiview 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 at 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 Supplementary Videos 19 and 20 for high-resolution subregions of the optical slices recorded in this series of experiments. Imaging framework: One-photon SiMView. Detection objectives: 2× Carl Zeiss Plan-Apochromat 40×/1.0 W. Cameras: 2× Andor Neo sCMOS.

  19. 19.

    High-resolution SiMView imaging of axonal morphogenesis (Ftz-ng-GAL4, embryo 1)

    The video shows full-resolution maximum-intensity projections of a subregion of the recording visualized in Supplementary Video 16. The subregion represents 2.5% of the volume covered by the complete recording (cropped by 75% laterally and 90% axially).

  20. 20.

    High-resolution SiMView imaging of the Drosophila VNC (Ftz-ng-GAL4, embryo 3)

    The video shows full-resolution maximum-intensity projections of a subregion of the ventral nerve cord (VNC), recorded with one-photon SiMView. The subregion 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 at 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: 2× Carl Zeiss Plan-Apochromat 40×/1.0 W. Cameras: 2× Andor Neo sCMOS.

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https://doi.org/10.1038/nmeth.2062

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