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Vertebrate neural stem cell segmentation, tracking and lineaging with validation and editing

Nature Protocols volume 6, pages 1942–1952 (2011)Cite this article

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Abstract

This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5–15 d as the cells proliferate and differentiate. LEVER automatically segments, tracks and generates lineage trees of the stem cells from the image sequence. In addition to generating lineage trees capturing the population dynamics of clonal development, LEVER extracts quantitative phenotypic measurements of cell location, shape, movement and size. When available, the system can include biomolecular markers imaged using fluorescence. It then displays the results to the user for highly efficient inspection and editing to correct any errors in the segmentation, tracking or lineaging. To enable high-throughput inspection, LEVER incorporates features for rapid identification of errors and for learning from user-supplied corrections to automatically identify and correct related errors.

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Figure 1: Schematic illustrating the steps of the protocol.
Figure 2: The LEVER user interface.
Figure 3: Segmentation errors are easily corrected.
Figure 4: Lineage tree and automatically generated cell statistics.

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Acknowledgements

This project was supported by the University of Wisconsin-Milwaukee, and by grant number NS033529 from the US National Institutes of Health (NIH) and by New York State Department of Health (NYSDOH) Contract C024352 from the Empire State Stem Cell Fund and the Regenerative Research Foundation. This project was also supported by NIH grant number R01EB005157 and by grant no. R01NS076709 from the National Institute of Neurological Disorders and Stroke. The authors would like to thank S. Shahmohammadi, R. Ference, A. DiRisio, S. Hardwick, S.M. Goderie, E. McAuley, R. Futia and C. Davenport for their help with cell culture and lineage validation.

Author information

Author notes

  1. Mark Winter and Eric Wait: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Electrical Engineering and Computer Science, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA

    Mark Winter, Eric Wait & Andrew R Cohen

  2. Department of Electrical and Computer Engineering, University of Houston, Houston, Texas, USA

    Badrinath Roysam

  3. Neural Stem Cell Institute, Rensselaer, New York, USA

    Susan K Goderie, Rania Ahmed Naguib Ali, Erzsebet Kokovay & Sally Temple

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  1. Mark Winter
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  7. Sally Temple
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Contributions

M.W., E.W., B.R. and A.R.C. designed and implemented the computational tools and algorithms, prepared the figures and wrote the manuscript. R.A.N.A. did cell preparation and maintenance, helped with staining and verified lineage trees. S.K.G., E.K. and S.T. cultured and imaged the NSCs, provided manual lineage reconstructions for comparison, edited the automated lineages, made suggestions on the design of the software tools from a biological perspective and helped prepare figures and write the manuscript.

Corresponding authors

Correspondence to Sally Temple or Andrew R Cohen.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Comparison of the MAT tracking algorithm used by LEVER with the bipartite matching tracker originally developed in Al Kofahi, et al.5, and refined in Cohen, et al.4 Although automatic segmentation, tracking and lineaging algorithms continue to improve, even very low error rates translate to a significant number of editing operations in order to generate a fully corrected lineage tree. (DOC 34 kb)

Supplementary Video 1

This video shows the corrected lineage corresponding to Supplementary Data Set 1. This video was generated directly from LEVER (protocol, Step 26). (WMV 21380 kb)

Supplementary Video 2

This video shows a second corrected lineage. The image sequence data for this lineage was generated as described in Step 1 of this protocol, but was not provided due to space restrictions on the web server. This video was also generated directly from the LEVER program. This lineage was used to create Figure 2. (WMV 36802 kb)

Supplementary Video 3

This video shows an adult NSC lineage in the top frame and an embryonic NSC lineage below. The embryonic lineage is the same shown in Supplementary Video 2. Both lineages are zoomed to show cells on the lineage more closely. These movies were generated individually from the LEVER program. A separate MATLAB script then combined the two movies and added the white 25 µm scale bars. (WMV 29649 kb)

Supplementary Results

This Excel spreadsheet contains the results of exporting cell metrics from LEVER for Data Set 1. The data was sorted by field "origin cell". All cells whose origin cell was 243 (the lineage that we wish to analyze) were copied to the second worksheet labeled "clone 243". These cells were then sorted by the "first frame" field. The cell cycle time, average speed and average area were then converted to appropriate units and plotted to produce panel b of Figure 4. (XLSX 34 kb)

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Winter, M., Wait, E., Roysam, B. et al. Vertebrate neural stem cell segmentation, tracking and lineaging with validation and editing. Nat Protoc 6, 1942–1952 (2011). https://doi.org/10.1038/nprot.2011.422

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