Article | Published:

Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data

Nature Methods volume 11, pages 951958 (2014) | Download Citation

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Abstract

The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min−1 on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

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Change history

  • 20 August 2014

    In the version of this article initially published online, equation (2) in the Online Methods, which describes the expression used to calculate the distance between the location of a nucleus () and the plane defined by the vertices of a triangle on the convex hull (, and ) was incorrect. The operations between the positions of the nucleus and triangle vertices (, , and ) were incorrectly shown as a scalar product. The correct operation is a subtraction. The operations between the pairwise differences of the positions of the triangle vertices (, and ) were also incorrectly shown as a scalar product. The correct operation is a vector product. These errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank A. Cardona and the participants of the Janelia CATMAID hackathon for help with modifying the open source code of CATMAID; R. Chhetri and A. Pavlopoulos for invaluable contributions to ground truth annotations of the microscopy data sets; the Ilastik development team for help using Ilastik; M. Schroeder, H. Lacin, J. Truman and T. Lee for helpful discussion about the Drosophila nervous system; S. Srinivas and T. Watanabe (University of Oxford) for their generous help in exploring imaging assays for mouse embryonic development, helpful discussions about mouse embryo culturing and providing the CAG-TAG1 transgenic mouse strain; A. Denisin for her outstanding help developing SiMView live imaging assays; C. Akitake (Carl Zeiss) for her generous help executing the Lightsheet Z.1 experiments; S. Olenych and O. Selchow (Carl Zeiss) for supporting the Lightsheet Z.1 experiments; and C.-P. Heisenberg (Institute of Science and Technology Austria) for kindly providing the Tg(β-actin:H2B-mCherry) and Tg(β-actin:H2B-eGFP) zebrafish lines. 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.

    • Fernando Amat
    • , William Lemon
    • , Daniel P Mossing
    • , Katie McDole
    • , Yinan Wan
    • , Kristin Branson
    •  & Philipp J Keller
  2. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

    • Eugene W Myers

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Contributions

P.J.K. and F.A. conceived of the research with input from E.W.M. F.A. developed the cell lineaging framework and performed the cell lineage reconstructions with input from P.J.K. and K.B. W.L. performed the Drosophila imaging experiments. D.P.M. curated and analyzed the reconstruction of early Drosophila nervous system development. K.M. performed the mouse imaging experiments. Y.W. and W.L. performed the zebrafish imaging experiments. F.A. and P.J.K. analyzed the data with input from K.B. F.A. and P.J.K. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Fernando Amat or Philipp J Keller.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Table 1–6, and Supplementary Notes 1–5

Zip files

  1. 1.

    Supplementary Software 1

    Automated modules of the cell lineaging framework

  2. 2.

    Supplementary Software 2

    Modified CATMAID module for visualizing image and cell lineage data, manually curating cell lineage data and annotating cell lineage reconstructions

  3. 3.

    Supplementary Data 1

    Cell lineage reconstruction of early Drosophila embryonic nervous system development

Videos

  1. 1.

    SiMView imaging of Drosophila embryogenesis

    Simultaneous multi-view imaging of Drosophila embryonic development, using an embryo homozygous for the nuclear label Histone 2A-mRFP (w-; P{w[+mC]=His2Av-mRFP1}; +, stock number 23560 from the Bloomington Drosophila Stock Center). The embryo was recorded in 30-second intervals over a period of 24 hours, starting at 3 h AEL. The first 551 time points, which cover the full time period analyzed in the automated cell lineage reconstruction, are included in this video. The complete data set consists of 515 gigabytes of image data. The video shows separate maximum-intensity projections of the dorsal and ventral halves of the embryo, based on the fused and background-corrected three-dimensional image stacks. The image frame size was down-sampled to reduce video size. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  2. 2.

    Automated segmentation and tracking in SiMView data set of Drosophila embryogenesis (gradient color code)

    Automated computational cell lineage reconstruction of the image data set shown in Supplementary Video 1. Each circle represents one cell nucleus. The tails of the circles (solid lines) indicate the history of object positions for the past ten time points. The color scheme was initialized in the first frame using a color gradient from anterior to posterior, using different colors on the dorsal and ventral sides and ensuring continuity in color space at the anterior and posterior ends of the embryo (see color bar in Figure 2a). After this initial color assignment, the color information was propagated in time using the tracking information, thus providing a color-coded single-cell resolution fate map. The accuracy of this automated procedure is quantified in Figure 2e. Some cell nucleus detections correspond to background objects, arising from autofluorescence and limitations in image quality. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  3. 3.

    Automated segmentation and tracking in SiMView data set of Drosophila embryogenesis (random color code)

    Visualization as in Supplementary Video 2, but using a random color code to initialize the first video frame. As in Supplementary Video 2, the color information was then propagated in time using the tracking information. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  4. 4.

    Automated segmentation and tracking in SiMView data set of Drosophila embryogenesis (rotating embryo, color gradient)

    Visualization as in Supplementary Video 2, but using a rotating view of the embryo instead of split static views of the dorsal and ventral halves. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  5. 5.

    Image volume slicing sequence for time point 30 of the reconstructed SiMView data set of Drosophila embryogenesis

    Three-dimensional image stack from the SiMView recording of the nuclei-labeled Drosophila embryo shown in Supplementary Video 1, superimposed with the corresponding supervoxels of the low-level over-segmentation (magenta) and the ellipsoids for all automatically reconstructed objects (green). The video shows the three-dimensional image data plane per plane for time point 30 of the image data set, shortly after the onset of gastrulation (3.2 h AEL). The image frame size was down-sampled to reduce video size. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  6. 6.

    Image volume slicing sequence for time point 180 of the reconstructed SiMView data set of Drosophila embryogenesis

    As in Supplementary Video 5, but for a time point later in development (time point 180, corresponding to 4.4 h AEL). The identification and annotation of neuroblasts for the reconstruction of early Drosophila embryonic nervous system development (Supplementary Videos 24-28) was performed at this time point. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  7. 7.

    Lightsheet Z.1 imaging of Drosophila embryogenesis

    Light-sheet-based imaging of Drosophila embryonic development with a Carl Zeiss Lightsheet Z.1 microscope, using an embryo homozygous for the nuclear label Histone 2A-mRFP (w-; P{w[+mC]=His2Av-mRFP1}; +, stock number 23560 from the Bloomington Drosophila Stock Center). The embryo was recorded in 30-second intervals over a period of 11 hours, starting at 2 h AEL. The first 387 time points, which cover the full time period analyzed in the automated cell lineage reconstruction, are included in this video. The complete data set consists of 595 gigabytes of image data. The video shows maximum-intensity projections of the ventral half of the embryo, based on the background-corrected three-dimensional image stacks. Note that, in contrast to the SiMView recording, which allows quantitatively accurate fusion of all views to produce a near-complete data set of the developing embryo, the Lightsheet Z.1 microscope only allows sequential multi-view imaging (and, thus, physical rotation of the embryo is needed) to capture the dorsal and ventral halves of the embryo. To avoid image fusion artifacts, the data sets representing the dorsal and ventral halves of the embryo were therefore not fused and only cell lineages in the ventral half of the embryo were reconstructed. Image frame size was down-sampled to reduce video size. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  8. 8.

    Automated segmentation and tracking in Lightsheet Z.1 data set of Drosophila embryogenesis (gradient color code)

    Automated computational cell lineage reconstruction of the image data set shown in Supplementary Video 7. Each circle represents one cell nucleus. The tails of the circles (solid lines) indicate the history of object positions for the past ten time points. The color scheme was initialized in the first frame using a color gradient from anterior to posterior. After this initial color assignment, the color information was propagated in time using the tracking information, thus providing a color-coded single-cell resolution fate map. Some cell nucleus detections correspond to background objects, arising from autofluorescence and limitations in image quality. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  9. 9.

    Automated segmentation and tracking in Lightsheet Z.1 data set of Drosophila embryogenesis (random color code)

    Visualization as in Supplementary Video 8, but using a random color code to initialize the first video frame. As in Supplementary Video 8, the color information was then propagated in time using the tracking information. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  10. 10.

    Image volume slicing sequence for time point 90 of the reconstructed Lightsheet Z.1 data set of Drosophila embryogenesis

    Three-dimensional image stack from the Lightsheet Z.1 recording of the nuclei-labeled Drosophila embryo shown in Supplementary Video 7, superimposed with the corresponding supervoxels of the low-level over-segmentation (magenta) and the ellipsoids for all automatically reconstructed objects (green). The video shows the three-dimensional image data plane per plane for time point 90 of the image data set, at the onset of gastrulation (3 h AEL). The image frame size was down-sampled to reduce video size. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  11. 11.

    Image volume slicing sequence for time point 240 of the reconstructed Lightsheet Z.1 data set of Drosophila embryogenesis

    As in Supplementary Video 10, but for a time point later in development (time point 240, corresponding to 4.3 h AEL). Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  12. 12.

    Confocal imaging of zebrafish embryogenesis

    Confocal microscopy recording of zebrafish embryonic development with a Carl Zeiss LSM 710 laser-scanning confocal microscope, using an embryo heterozygous for the fluorescent nuclear label H2B-mCherry. The embryo was recorded in 120-second intervals over a period of 3.4 hours. The data set consists of 9.4 gigabytes of image data. The video shows maximum-intensity projections with a view of the animal pole of the embryo. The image frame size was down-sampled to reduce video size. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  13. 13.

    Automated segmentation and tracking in confocal data set of zebrafish embryogenesis (gradient color code)

    Automated computational cell lineage reconstruction of the image data set shown in Supplementary Video 12. Each circle represents one cell nucleus. The tails of the circles (solid lines) indicate the history of object positions for the past ten time points. The color scheme was initialized in the first frame using a radially-symmetrical color gradient from the animal pole to the periphery of the blastoderm. After this initial color assignment, the color information was propagated in time using the tracking information, thus providing a color-coded single-cell resolution fate map. Some cell nucleus detections correspond to background objects, arising from autofluorescence and limitations in image quality. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  14. 14.

    Automated segmentation and tracking in confocal data set of zebrafish embryogenesis (random color code)

    Visualization as in Supplementary Video 13, but using a random color code to initialize the first video frame. As in Supplementary Video 13, the color information was then propagated in time using the tracking information. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  15. 15.

    Image volume slicing sequence for time point 60 of the reconstructed confocal data set of zebrafish embryogenesis

    Three-dimensional image stack from the LSM 710 recording of the nuclei-labeled zebrafish embryo shown in Supplementary Video 12, superimposed with the corresponding supervoxels of the low-level over-segmentation (magenta) and the ellipsoids for all automatically reconstructed objects (green). The video shows the three-dimensional image data plane per plane for time point 60 of the image data set. The video frame size is the original size of the image data. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  16. 16.

    SiMView imaging of mouse embryogenesis

    Simultaneous multi-view imaging of mouse embryonic development, using an embryo expressing Histone2B-eGFP in all nuclei. The E6.25 embryo was recorded in 5-minute intervals over a period of 2 hours. The data set consists of 8.4 gigabytes of image data. The video shows maximum-intensity projections of the embryo, based on the fused and background-corrected three-dimensional image stacks. The video frame size is the original size of the image data. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  17. 17.

    Automated segmentation and tracking in SiMView data set of mouse embryogenesis (random color code)

    Automated computational cell lineage reconstruction of the image data set shown in Supplementary Video 16. Each circle represents one cell nucleus. The tails of the circles (solid lines) indicate the history of object positions for the past ten time points. The color scheme was initialized in the first frame using a random color code. After this initial color assignment, the color information was propagated in time using the tracking information. Some cell nucleus detections correspond to background objects, arising from autofluorescence and limitations in image quality. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  18. 18.

    Image volume slicing sequence for time point 24 of the reconstructed SiMView data set of mouse embryogenesis

    Three-dimensional image stack from the SiMView recording of the nuclei-labeled mouse embryo shown in Supplementary Video 16, superimposed with the corresponding supervoxels of the low-level over-segmentation (magenta) and the ellipsoids for all automatically reconstructed objects (green). The video shows the three-dimensional image data plane per plane for time point 24 of the image data set. The video frame size is the original size of the image data. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  19. 19.

    SiMView imaging of zebrafish embryogenesis

    Simultaneous multi-view imaging of zebrafish embryonic development, using an embryo heterozygous for the nuclear label H2B-GFP, expressed under the control of the beta-actin promoter. The embryo was recorded in 60-second intervals over a period of 18 hours, starting at the sphere stage at 6 hours post fertilization. The first 541 time points are included in this video. The complete data set consists of 1.7 terabytes of image data. The video shows separate maximum-intensity projections of the animal and vegetal halves of the embryo, based on the fused and background-corrected three-dimensional image stacks. The image frame size was down-sampled to reduce video size. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  20. 20.

    Automated segmentation and tracking in SiMView data set of zebrafish embryogenesis (gradient color code)

    Automated computational cell lineage reconstruction of the image data set shown in Supplementary Video 19. Each circle represents one cell nucleus. The tails of the circles (solid lines) indicate the history of object positions for the past ten time points. The color scheme was initialized in the first frame using a radially-symmetrical color gradient from the animal pole to the periphery of the blastoderm. After this initial color assignment, the color information was propagated in time using the tracking information, thus providing a color-coded single-cell resolution fate map. Some cell nucleus detections correspond to background objects, arising from autofluorescence and limitations in image quality. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  21. 21.

    Automated segmentation and tracking in SiMView data set of zebrafish embryogenesis (random color code)

    Visualization as in Supplementary Video 20, but using a random color code to initialize the first video frame. As in Supplementary Video 20, the color information was then propagated in time using the tracking information. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  22. 22.

    Image volume slicing sequence for time point 100 of the reconstructed SiMView data set of zebrafish embryogenesis

    Three-dimensional image stack from the SiMView recording of the nuclei-labeled zebrafish embryo shown in Supplementary Video 19, superimposed with the corresponding supervoxels of the low-level over-segmentation (magenta) and the ellipsoids for all automatically reconstructed objects (green). The video shows the three-dimensional image data plane per plane for time point 100 of the image data set (7.7. hours post fertilization, imaging at 21.5°C). The image frame size was down-sampled to reduce video size. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  23. 23.

    Image volume slicing sequence for time point 400 of the reconstructed SiMView data set of zebrafish embryogenesis

    As in Supplementary Video 22, but for a time point later in development (time point 400, corresponding to 12.7 hours post fertilization; imaging at 21.5°C). Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  24. 24.

    Neuroblast identification in the SiMView data set of Drosophila embryogenesis

    Rotating maximum-intensity projection of the three-dimensional SiMView image stack of the nuclei-labeled Drosophila embryo shown in Supplementary Video 1 (at time point 180, corresponding to 4.4 h AEL), superimposed with green spheres marking the location of neuroblasts identified in the image data. Neuroblast identification was performed based on morphological appearance and cell position in the embryo. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  25. 25.

    Movements and divisions of neural precursors in the early Drosophila embryonic nervous system (with SiMView data)

    Ventral and lateral maximum-intensity projections of the SiMView time-lapse recording of the nuclei-labeled Drosophila embryo shown in Supplementary Video 1, superimposed with green spheres marking the location, movements and divisions of neural precursors from the blastoderm stage up to 5 h AEL The tails indicate the history of cell positions for the past ten time points, using a color code that gradually transitions from purple to white as a function of time. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  26. 26.

    Movements and divisions of neural precursors in the early Drosophila embryonic nervous system (static view)

    Visualization as in Supplementary Video 2, but using a cell type specific color code obtained from the cell lineage reconstruction of early Drosophila embryonic nervous system development. Neural precursors are color-coded based on neuroblast type (see Supplementary Figure 12 for color correspondence) and all other cells are shown in grey. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  27. 27.

    Movements and divisions of neural precursors in the early Drosophila embryonic nervous system (rotating view)

    Visualization as in Supplementary Video 26, but using a rotating view of the embryo instead of split static views of the dorsal and ventral halves. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

  28. 28.

    Cell lineage reconstruction of early Drosophila embryonic nervous system development

    Rotating view of neural precursor cell tracks obtained from the cell lineage reconstruction of early Drosophila embryonic nervous system development. The tracks are represented by solid lines, using a color code that indicates time (purple to white: from the blastoderm stage to the end point of the reconstruction at 5 h AEL). Cell locations at the end point of the reconstruction are marked by green spheres. Anterior is to the left, posterior to the right. Note: The DivX codec required for video playback is freely available at http://www.divx.com/downloads/divx/1

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DOI

https://doi.org/10.1038/nmeth.3036

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