1924 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 59, NO. 7, JULY 2012 Online 3-D Tracking of Suspension Living Cells Imaged with Phase-Contrast Microscopy Chao-Hui Huang ∗ , Member, IEEE, Shvetha Sankaran, Daniel Racoceanu, Member, IEEE, Srivats Hariharan, and Sohail Ahmed Abstract—Neural stem cells/neural progenitors (NSCs/NPs) are cells that give rise to the main cell types of the nervous system: oligo- dendrocytes, neurons, and astrocytes. Studying NSCs/NPs with time-lapse microscopy is critical to the understanding of the biology of these cells. However, NSCs/NPs are very sensitive to phototoxic damage, and therefore, fluorescent dyes cannot be used to follow these cells. Also, since in most of NSC/NP-related experiments, a large number of cells neesd to be monitored. Consequently, the acquisition of a huge amount of images is required. An additional difficulty is related to our original suspension living, tracking ob- jective, behavior much closer to the natural, in vivo, way of de- velopment of the cells. Indeed, unlike adherent cells, suspension cells float freely in a liquid solution, thus, making their dynam- ics very different from that of adherent cells. As a result, existing visual tracking algorithms that have primarily been developed to track adherent cells are no longer adequate to tackle living cells in suspension. This paper presents a novel automated 3-D visual tracking of suspension living cells for time-lapse image acquisition using phase-contrast microscopy. This new tracking method can potentially strongly impact on current 3-D video microscopy meth- ods, paving the way for innovative analysis of NSCs/NPs and as a result, on the study of neurodegenerative diseases. Index Terms—Autofocusing, MetaMorph, micromanager, neu- ral stem cells (NSCs), neural progenitors, phase-contrast mi- croscopy, suspension living cells, 3-D visual tracking. I. INTRODUCTION T HE study of neural stem cells (NSCs) and neural pro- genitors (NPs) is crucial to the understanding of brain development and neurodegenerative disorders like Alzheimer’s Manuscript received October 14, 2011; revised February 26, 2012; accepted March 8, 2012. Date of publication April 11, 2012; date of current version June 20, 2012. This work was supported by the Joint Council Office (JCO), Agency for Science, Technology and Research (A-STAR), Singapore under Project JCOAG03_FG01_2009. Asterisk indicates corresponding author. ∗ C.-H. Huang is with the Bioinformatics Institute (BII), Agency for Science, Technology, and Research (A-STAR), Singapore 138671 (e-mail: huangch.tw@gmail.com). S. Sankaran and S. Ahmed are with the Institute of Medical Biology (IMB), Agency for Science, Technology, and Research (A-STAR), Singapore 138648 (e-mail: shvetha.sankaran@imb.a-star.edu.sg; sohail.ahmed@ imb.a-star.edu.sg). D. Racoceanu is with the University Pierre and Marie Curie, Paris 75005, France, and also with the French National Center for Scientific Research, Image and Pervasive Access Laboratory (IPAL) UMI CNRS, Paris 75794, France (e-mail: daniel.racoceanu@upmc.fr). S. Hariharan is with Olympus Co. Ltd., Singapore 248373 (e-mail: srivats.hariharan@gmail.com). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TBME.2012.2194487 Fig. 1. Most NSCs/NPs tend to show dynamic movement patterns resulting in 90% of the cells moving away from the microscopic field of view within the first few hours of imaging, unless the operator is manually able to retain the cells in the field of view. However, it is impractical for a human operator to continuously monitor more than 100 cells every 15–30 min for several days. (a) 0 h, (b) 2 h, (c) 4 h. and Parkinson’s. In this study, we investigate the behavior of NSCs/NPs using time-lapse imaging. NSCs/NPs grow as free floating 3-D structures called neuro- spheres when propagated in vitro [1]. Neurospheres are naturally occurring 3-D clusters that are composed of both stem cells and progenitors. Recent evidence suggests links between aberrant NSC growth and brain tumors [2]–[4]. Hence, a better under- standing of NSC biology will in turn play a crucial role in the study of tumor biology. The tracking of NSCs/NPs raise several challenges for long- term time-lapse microscopy [5]; one of the major ones is the motility of these cells. Some recent research results [6], [7] suggested that the motility of NSCs/NPs can provide important information for cell fate prediction. Since NSCs/NPs are grown in suspension, most of the living cells show dynamic movement patterns resulting in 90% of the cells moving away from the microscopic field of view within a few hours (see Fig. 1). The only way to follow cells is for the operator to manually track all the cell positions. Subsequently, the experiments are very labor intensive [8]. Several methods have been reported to address similar issues. The most commonly used techniques include physical entrap- ment using microwells [9] and using fluorescence to track single cells [10]–[12]. The study of NSCs/NPs has also tended to rely heavily on 2-D fluorescence imaging that involve fixing (which means killing) the cells followed by marker labeling [13]–[15]. Beside, NSCs/NPs cannot be labeled with fluorescent markers for continuous live imaging since exposure to fluorescent light is phototoxic. Time-lapse sequences of cells labeled with sev- eral markers such as Cell Tracker Green, Hoechst 33342, PMT- EYFP, and Actin-GFP (cells from transgenic mice) have been attempted. Exposure to fluorescent light results in cell death as early as after the first exposure. 0018-9294/$31.00 © 2012 IEEE