We present several algorithms for cell image analysis including microscopy image restoration, cell event detection and cell tracking in a large population. The algorithms are integrated into an automated system capable of quantifying cell proliferation metrics in vitro in real-time. This offers unique opportunities for biological applications such as efficient cell behavior discovery in response to different cell culturing conditions and adaptive experiment control. We quantitatively evaluated our system's performance on 16 microscopy image sequences with satisfactory accuracy for biologists' need. We have also developed a public website compatible to the system's local user interface, thereby allowing biologists to conveniently check their experiment progress online. The website will serve as a community resource that allows other research groups to upload their cell images for analysis and comparison.

Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and objectively determine the appropriate time for cell passaging. Optimal stem cell culturing that maintains cell pluripotency while maximizing cell yields will be especially important for efficient, cost-effective stem cell-based therapies. Toward this goal we developed a real-time computer vision-based system that monitors the degree of cell confluency with a precision of 0.791±0.031 and recall of 0.559±0.043. The system consists of an automated phase-contrast time-lapse microscope and a server. Multiple dishes are sequentially imaged and the data is uploaded to the server that performs computer vision processing, predicts when cells will exceed a pre-defined threshold for optimal cell confluency, and provides a Web-based interface for remote cell culture monitoring. Human operators are also notified via text messaging and e-mail 4 hours prior to reaching this threshold and immediately upon reaching this threshold. This system was successfully used to direct the expansion of a paradigm stem cell population, C2C12 cells. Computer-directed and human-directed control subcultures required 3 serial cultures to achieve the theoretical target cell yield of 50 million C2C12 cells and showed no difference for myogenic and osteogenic differentiation. This automated vision-based system has potential as a tool toward adaptive real-time control of subculturing, cell culture optimization and quality assurance/quality control, and it could be integrated with current and developing robotic cell cultures systems to achieve complete automation.

Phase contrast time-lapse microscopy is a non-destructive technique that generates large volumes of image-based information to quantify the behaviour of individual cells or cell populations. To guide the development of algorithms for computer-aided cell tracking and analysis, 48 time-lapse image sequences, each spanning approximately 3.5 days, were generated with accompanying ground truths for C2C12 myoblast cells cultured under 4 different media conditions, including with fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2), FGF2 + BMP2, and control (no growth factor). The ground truths generated contain information for tracking at least 3 parent cells and their descendants within these datasets and were validated using a two-tier system of manual curation. This comprehensive, validated dataset will be useful in advancing the development of computer-aided cell tracking algorithms and function as a benchmark, providing an invaluable opportunity to deepen our understanding of individual and population-based cell dynamics for biomedical research.

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Automated visual-tracking systems of stem cell populations in vitro allow for high-throughput analysis of time-lapse phase-contrast microscopy. In these systems, detection of mitosis, or cell division, is critical to tracking performance as mitosis causes branching of the trajectory of a mother cell into the two trajectories of its daughter cells. Recently, one mitosis detection algorithm showed its success in detecting the time and location that two daughter cells first clearly appear as a result of mitosis. This detection result can therefore helps trajectories to correctly bifurcate and the relations between mother and daughter cells to be revealed. In this paper, we demonstrate that the functionality of this recent mitosis detection algorithm significantly improves state-of-the-art cell tracking systems through extensive experiments on 48 C2C12 myoblastic stem cell populations under four different conditions.