significantly higher contrast than conventional phase imaging techniques, such as phase-contrast microscopy. The higher contrast facilitates more robust cell segmentation and tracking than achievable with conventional phase imaging. New techniques that use a combination of traditional image segmentation techniques and deep learning to segment cells are also becoming more widely used as well. Data analysis In the final stage of a live single-cell imaging study modelling and analysis of time series data extracted from tracked cells is performed. Pedigree tree profiles can be constructed to reveal heterogeneity in individual cell response and downstream signalling. A large overlap between analysis of single cell live data, and modelling of biological systems using ordinary differential equations exists. Results from this key data analysis step will drive further experimentation, for example by perturbing aspects of the system being studied and then comparing signalling dynamics with those of the control population. Applications By analysing the signalling dynamics of single cells across entire populations, live single cell studies are now letting us understand how these dynamics affect key cellular decision making processes. For example, live single cell studies of the growth factor ERK revealed it to possess digital all-or-nothing activation. Moreover, this all-or-nothing activation was pulsatile, and the frequency of pulses in turn determined whether mammalian cells would commit to cell cycle entry or not. In another key example, live single-cell studies of CDK2 activity in mammalian cells demonstrated that bifurcation in CDK2 activity following mitosis, determined whether cells would continue to proliferate or enter a state of quiescence; now shown, using live single-cell methods, to be caused by stochastic DNA damage inducing upregulation of p21, which inhibits CDK2 activity. Moving forward, live single cell studies will now likely incooperate multiple reporters into single cell lines to allow complex decision making processes to be understood, however challenges still remain in scaling up live single cell studies. References Cell imaging