A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time- Lapse Movies Frederic D. Sigoillot1., Jeremy F. Huckins1.¤, Fuhai Li2, Xiaobo Zhou3, Stephen T. C. Wong2, Randall W. 1 King * 1 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Systems Medicine and Bioengineering, The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, Texas, United States of America, 3 Department of Translational Imaging, The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, Texas, United States of America Abstract Background: Automated time-lapse microscopy can visualize proliferation of large numbers of individual cells, enabling accurate measurement of the frequency of cell division and the duration of interphase and mitosis. However, extraction of quantitative information by manual inspection of time-lapse movies is too time-consuming to be useful for analysis of large experiments. Methodology/Principal Findings: Here we present an automated time-series approach that can measure changes in the duration of mitosis and interphase in individual cells expressing fluorescent histone 2B. The approach requires analysis of only 2 features, nuclear area and average intensity. Compared to supervised learning approaches, this method reduces processing time and does not require generation of training data sets. We demonstrate that this method is as sensitive as manual analysis in identifying small changes in interphase or mitotic duration induced by drug or siRNA treatment. Conclusions/Significance: This approach should facilitate automated analysis of high-throughput time-lapse data sets to identify small molecules or gene products that influence timing of cell division. Citation: Sigoillot FD, Huckins JF, Li F, Zhou X, Wong STC, et al. (2011) A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies. PLoS ONE 6(9): e25511. doi:10.1371/journal.pone.0025511 Editor: Kurt I. Anderson, The Beatson Institute for Cancer Research, United Kingdom Received April 16, 2010; Accepted September 7, 2011; Published September 26, 2011 Copyright: 2011 Sigoillot 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. Funding: This work was supported by grants GM66492 from the NIH http://www.nih.gov/ and the Lynch Foundation http://www.thelynchfoundation.org/ to R.W.K. and a grant from National Library of Medicine http://www.nlm.nih.gov/ to S.W. (R01LM008696). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: randy_king@hms.harvard.edu . These authors contributed equally to this work. ¤ Current address: Department of Psychology and Brain Sciences, Dartmouth College, Hanover, New Hampshire, United States of America Introduction Cell division is one of the most complex of all cellular behaviors. As a consequence, perturbations in many different cellular pathways can affect the duration of cell division, typically by delaying a specific cell cycle transition. For example, agents that damage DNA will inhibit the transition from interphase into mitosis, thereby prolonging interphase. Agents that damage the mitotic spindle prevent the transition from mitosis to interphase, thereby prolonging mitosis. Because the checkpoint mechanisms that detect these defects are highly sensitive, changes in cell cycle duration can often be observed at concentrations of perturbing agents that are below those that cause any other observable alterations in the molecular target or cellular behavior. Therefore, changes in cell cycle timing can be used as a highly sensitive detector of molecular perturbations that would be difficult to observe by other methods. Live cell time-lapse imaging enables direct measurement of the duration of interphase and mitosis in individual cells. By using various fluorescent reporter proteins, such as H2B-GFP [1] or an engineered mitotic biosensor [2], these experiments can reveal how a perturbation affects the frequency of cell division, the fidelity of cell division [3], the duration of a specific cell cycle phase such as mitosis [4], or the timing and frequency of cell death [5]. The approach is especially powerful because each cell can be followed over time, revealing how phenotypes evolve in a time- dependent manner, and enabling different behaviors to be correlated with one another. However, a limitation of the approach is that manual analysis of time-lapse movies is tedious and time-consuming, limiting the utility of the approach for high- throughput experiments. To address this problem, automated image analysis methods have been developed that can track cells over time and classify cells as to cell cycle stage (interphase versus mitosis or sub-phases of mitosis). However, a fully automated system for determining interphase and mitotic duration from wide-field fluorescence time- lapse movies has not been developed. Current approaches often require specialized imaging [2] or high magnification confocal PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25511