In systems biology, live single-cell imaging is a live cell imaging technique that combines traditional live cell imaging and time-lapse microscopy techniques with automated cell tracking and feature extraction, drawing many techniques from high-content screening. It is used to study signalling dynamics and behaviour in populations of individual living cells. Live single cell studies can reveal key behaviours that would otherwise be masked in population averaging experiments such as western blots. In a live single cell imaging experiment a fluorescent reporter is introduced into a cell line to measure the levels, localisation or activity of a signalling molecule. Subsequently, a population of cells is imaged over time with careful atmospheric control to maintain viability, and reduce stress upon the cells. Automated cell tracking is then performed upon these time series images, following which filtering and quality control may be performed. Analysis of features describing the fluorescent reporter over time, can then lead to modelling and generation of biological conclusions from which further experimentation can be guided. History The field of live single-cell imaging began with work demonstrating that green fluorescent protein (GFP), found in the jellyfish Aequorea victoria, could be expressed in living organisms. This discovery allowed researches to study the localisation and levels of proteins in living single cells, for example the activity of kinases, and calcium levels, through the use of FRET reporters, as well as numerous other phenotypes. Generally, these early studies focused on the localisation and behaviour of these fluorescently labelled proteins at the subcellular level over short periods of time. However, this changed with pioneering studies looking at the tumour suppressor p53 and the stress and inflammation related protein NF-κB, revealing there levels and localisation respectively to oscillate over periods of several hours. Live single cell approaches were also applied around this time to understand signalling in single cell organisms including bacteria, where live studies allowed the dynamics of competence to be modelled, and yeast revealing the mechanism underpinning coherent cell cycle entry. Experimental work flow Fluorescent reporters In any live single cell study, the first step is to introduce a reporter for our protein/molecule of interest into a suitable cell line. Much of the growth in the field has come from improved gene editing tools such as CRISPR, this leading to development of a wide variety of fluorescent reporters. Fluorescent tagging uses a gene encoding a fluorescent protein that is inserted into the coding