Detection and tracking of overlapping cell nuclei for large scale mitosis analyses.
BACKGROUND
Cell culture on printed micropatterns slides combined with automated fluorescent microscopy allows for extraction of tens of thousands of videos of small isolated growing cell clusters. The analysis of such large dataset in space and time is of great interest to the community in order to identify factors involved in cell growth, cell division or tissue formation by testing multiples conditions. However, cells growing on a micropattern tend to be tightly packed and to overlap with each other. Consequently, image analysis of those large dynamic datasets with no possible human intervention has proven impossible using state of the art automated cell detection methods.


RESULTS
Here, we propose a fully automated image analysis approach to estimate the number, the location and the shape of each cell nucleus, in clusters at high throughput. The method is based on a robust fit of Gaussian mixture models with two and three components on each frame followed by an analysis over time of the fitting residual and two other relevant features. We use it to identify with high precision the very first frame containing three cells. This allows in our case to measure a cell division angle on each video and to construct division angle distributions for each tested condition. We demonstrate the accuracy of our method by validating it against manual annotation on about 4000 videos of cell clusters.


CONCLUSIONS
The proposed approach enables the high throughput analysis of video sequences of isolated cell clusters obtained using micropatterns. It relies only on two parameters that can be set robustly as they reduce to the average cell size and intensity.
Scientific Center for Computational Biology, Institut de Biologie de l'Ecole Normale Superieure, CNRS-INSERM-ENS, PSL Research University, 46, rue d'Ulm, Paris, 75005, France.|Division cellulaire et neurogenèse, Institut de Biologie de l'Ecole Normale Superieure, PSL Research University, 46, rue d'Ulm, Paris, 75005, France.
Yingbo
Y
Li
Scientific Center for Computational Biology, Institut de Biologie de l'Ecole Normale Superieure, CNRS-INSERM-ENS, PSL Research University, 46, rue d'Ulm, Paris, 75005, France.
France
F
Rose
Division cellulaire et neurogenèse, Institut de Biologie de l'Ecole Normale Superieure, PSL Research University, 46, rue d'Ulm, Paris, 75005, France.
Florencia
F
di Pietro
Division cellulaire et neurogenèse, Institut de Biologie de l'Ecole Normale Superieure, PSL Research University, 46, rue d'Ulm, Paris, 75005, France.
Xavier
X
Morin
Scientific Center for Computational Biology, Institut de Biologie de l'Ecole Normale Superieure, CNRS-INSERM-ENS, PSL Research University, 46, rue d'Ulm, Paris, 75005, France. auguste.genovesio@ens.fr.
Auguste
A
Genovesio
D002467:Cell Nucleus / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008938:Mitosis; D015233:Models, Statistical; D016011:Normal Distribution; D059008:Time-Lapse Imaging
Cell detection; Gaussian mixture; High throughput; Image analysis; Mitosis; Time-lapse microscopy