Journal of Microscopy, Vol. 234, Pt 1 2009, pp. 62–79 Received 13 May 2008; accepted 15 November 2008 Automated tracking of migrating cells in phase-contrast video microscopy sequences using image registration A.J. HAND ∗ , T. SUN ∗ , D.C. BARBER †, D.R. HOSE † & S. MACNEIL ∗ ∗ Kroto Research Institute, North Campus, University of Sheffield, Broad Lane, Sheffield, S3 7HQ, United Kingdom †Medical Physics, Royal Hallamshire Hospital, University of Sheffield, Glossop Road, Sheffield, S10 2JF, United Kingdom Key words. Cell motility, cell tracking, image registration. Summary Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell- tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal microscopy techniques. The results showed that using fluorescence microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively Correspondence to: Prof. S. MacNeil. Tel: +44 114 222 5993; fax: +44 114 222 5945; e-mail: s.macneil@sheffield.ac.uk small (<5% difference in tracking success rate), whereas much greater improvements in performance were seen when using fluorescence microscopy with Volocity and ImageJ. Introduction The analysis of in vitro cell motility and morphology is an area that is gaining increasing importance with recent advances in computing and imaging technology. Accurate knowledge about changes in cell behaviour will allow more in-depth comparisons between the effects of different drugs for the treatment of diseases, as well as enabling more thorough testing of suitable scaffolds for cell culture in the area of tissue engineering. It is also a vital tool for the validation of computational models of cell behaviour, such as the Epitheliome project (Walker et al., 2004), which aims to model the social interactions of epithelial cells during wound healing and is currently in progress at the Universities of Sheffield and York. The traditional way to track cells is via manual marking of time-lapse microscopy images. Although this is the ‘gold standard’ method of cell tracking, it is very time-consuming and can be tiring for the operator when tracking large numbers of cells over long time periods. This in itself can lead to errors in the tracking process. For this reason, a significant amount of research is currently underway to develop automated cell- tracking systems that are capable of tracking cells in time- lapse sequences with a minimum of user interaction (see, e.g. Zimmer et al., 2002; Rabut & Ellenberg, 2004; Debeir et al., 2005; Gor et al., 2005). However, many of these systems are being developed primarily for use with fluorescently labelled cells, which produce high-contrast images. Although phase-contrast microscopy produces images with bright or dark rings around the cells and may be considered to be a high-contrast imaging C  2009 The Authors Journal compilation C  2009 The Royal Microscopical Society