Downey, Mike J., Jeziorska, Danuta M., Ott, Sascha, Tamai, T. Katherine, Koentges, Georgy, Vance, Keith W. and Bretschneider, Till. (2011) Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution. PLoS ONE, Vol.6 (No.12). e27886. ISSN 1932-6203

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Abstract

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history
Divisions:	Faculty of Science > Life Sciences (2010- ) Faculty of Science > Molecular Organisation and Assembly in Cells (MOAC) Faculty of Science > Centre for Systems Biology
Library of Congress Subject Headings (LCSH):	Fluorescence microscopy -- Methodology
Journal or Publication Title:	PLoS ONE
Publisher:	PLOS
ISSN:	1932-6203
Date:	15 December 2011
Volume:	Vol.6
Number:	No.12
Page Range:	e27886
Identification Number:	10.1371/journal.pone.0027886
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Engineering and Physical Sciences Research Council (EPSRC), University of Warwick, Wellcome Trust (London, England), Human Frontiers Science Program (HFSP), Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)
Grant number:	066790/E/02/Z (WT), 066745/Z/01/Z (WT), RGP0029/2007 (HFSP), BB/I004823/1 (BBSRC)
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URI:	http://wrap.warwick.ac.uk/id/eprint/40852

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