Tracking individual cells has allowed a new understanding of cellular behavior in human health and disease by adding a dynamic component to the already complex heterogeneity of single cells. Technically, despite countless advances, numerous experimental variables can affect data collection and interpretation and need to be considered. In this review, we discuss the main technical aspects and biological findings in the analysis of the behavior of individual cells. We discuss the most relevant contributions provided by these approaches in clinically relevant human conditions like embryo development, stem cells biology, inflammation, cancer and microbiology, along with the cellular mechanisms and molecular pathways underlying these conditions. We also discuss the key technical aspects to be considered when planning and performing experiments involving the analysis of individual cells over long periods. Despite the challenges in automatic detection, features extraction and long-term tracking that need to be tackled, the potential impact of single-cell bioimaging is enormous in understanding the pathogenesis and development of new therapies in human pathophysiology.

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