title: Analysis of in vivo single cell behavior by high throughput, human-in-the-loop segmentation of three-dimensional images.
abstract: BACKGROUND
  Analysis of single cells in their native environment is a powerful method to address key questions in developmental systems biology. Confocal microscopy imaging of intact tissues, followed by automatic image segmentation, provides a means to conduct cytometric studies while at the same time preserving crucial information about the spatial organization of the tissue and morphological features of the cells. This technique is rapidly evolving but is still not in widespread use among research groups that do not specialize in technique development, perhaps in part for lack of tools that automate repetitive tasks while allowing experts to make the best use of their time in injecting their domain-specific knowledge.
  
  
  RESULTS
  Here we focus on a well-established stem cell model system, the C. elegans gonad, as well as on two other model systems widely used to study cell fate specification and morphogenesis: the pre-implantation mouse embryo and the developing mouse olfactory epithelium. We report a pipeline that integrates machine-learning-based cell detection, fast human-in-the-loop curation of these detections, and running of active contours seeded from detections to segment cells. The procedure can be bootstrapped by a small number of manual detections, and outperforms alternative pieces of software we benchmarked on C. elegans gonad datasets. Using cell segmentations to quantify fluorescence contents, we report previously-uncharacterized cell behaviors in the model systems we used. We further show how cell morphological features can be used to identify cell cycle phase; this provides a basis for future tools that will streamline cell cycle experiments by minimizing the need for exogenous cell cycle phase labels.
  
  
  CONCLUSIONS
  High-throughput 3D segmentation makes it possible to extract rich information from images that are routinely acquired by biologists, and provides insights - in particular with respect to the cell cycle - that would be difficult to derive otherwise.
authors.affiliation: Department of Developmental & Cell Biology, University of California at Irvine, Irvine, USA. mchiang3@uci.edu.|Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. mchiang3@uci.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Chiang
authors.affiliation: Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. shallman@uci.edu.|Department of Computer Science, University of California at Irvine, Irvine, USA. shallman@uci.edu.
authors.forename: Sam
authors.initials: S
authors.lastname: Hallman
authors.affiliation: Department of Developmental & Cell Biology, University of California at Irvine, Irvine, USA. acinquin@uci.edu.|Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. acinquin@uci.edu.
authors.forename: Amanda
authors.initials: A
authors.lastname: Cinquin
authors.affiliation: Department of Developmental & Cell Biology, University of California at Irvine, Irvine, USA. nreyesde@uci.edu.|Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. nreyesde@uci.edu.
authors.forename: Nabora Reyes
authors.initials: NR
authors.lastname: de Mochel
authors.affiliation: Department of Developmental & Cell Biology, University of California at Irvine, Irvine, USA. apaz@uci.edu.|Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. apaz@uci.edu.
authors.forename: Adrian
authors.initials: A
authors.lastname: Paz
authors.affiliation: Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. skawauch@uci.edu.
authors.forename: Shimako
authors.initials: S
authors.lastname: Kawauchi
authors.affiliation: Department of Developmental & Cell Biology, University of California at Irvine, Irvine, USA. alcalof@uci.edu.|Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. alcalof@uci.edu.|Department of Anatomy & Neurobiology, University of California at Irvine, Irvine, USA. alcalof@uci.edu.
authors.forename: Anne L
authors.initials: AL
authors.lastname: Calof
authors.affiliation: Department of Developmental & Cell Biology, University of California at Irvine, Irvine, USA. kwcho@uci.edu.|Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. kwcho@uci.edu.
authors.forename: Ken W
authors.initials: KW
authors.lastname: Cho
authors.affiliation: Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. fowlkes@uci.edu.|Department of Computer Science, University of California at Irvine, Irvine, USA. fowlkes@uci.edu.
authors.forename: Charless C
authors.initials: CC
authors.lastname: Fowlkes
authors.affiliation: Department of Developmental & Cell Biology, University of California at Irvine, Irvine, USA. ocinquin@uci.edu.|Center for Complex Biological Systems, University of California at Irvine, Irvine, USA. ocinquin@uci.edu.
authors.forename: Olivier
authors.initials: O
authors.lastname: Cinquin
mesh_terms: D000465:Algorithms; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000254:growth & development* / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D019295:Computational Biology / Q000379:methods; D005260:Female; D006066:Gonads / Q000166:cytology / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D009831:Olfactory Mucosa / Q000166:cytology* / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D012984:Software