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Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution.

Downey, MJ; Jeziorska, DM; Ott, S; Tamai, TK; Koentges, G; Vance, KW; Bretschneider, T; (2011) Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution. PLOS One , 6 (12) , Article e27886. 10.1371/journal.pone.0027886. Green open access

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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.

Type: Article
Title: Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution.
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1371/journal.pone.0027886
Publisher version: http://dx.doi.org/10.1371/journal.pone.0027886
Language: English
Additional information: © 2011 Downey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. MJD was funded by the EPSRC (www.epsrc.ac.uk) through the MOAC Doctoral Training Centre, University of Warwick. KWV was funded by a Senior Research Fellowship from the University of Warwick. The experimental work was funded by WellcomeTrust (WT 066790/E/02/Z and 066745/Z/01/Z, www.wellcome.ac.uk) and HFSP (RGP0029/2007 C, www.hfsp.org) programme grants awarded to GK. Fucci data provided by TKT was obtained through funding by BBSRC/ERASysBio+ (BB/I004823/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Keywords: Animals, Biological Markers, Cell Division, Cell Line, Cell Lineage, Cell Nucleus, Cell Tracking, Fluorescence, Genes, Reporter, Green Fluorescent Proteins, High-Throughput Screening Assays, Imaging, Three-Dimensional, Mice, Microscopy, Time Factors, Zebrafish
UCL classification: UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Cell and Developmental Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1335409
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