frame of the protein to be tagged. Texture and intensity features can be extracted from images of the tagged protein. Molecules can also be tagged in vitro and introduced into the cell with electrophoresis. This enable the use of smaller and more photostable fluorophores but requires additional washing steps. By engineering expression of FRET reporter such that donor and emitter fluorophores are only in close proximity when an upstream signalling molecule is either active or inactive, the donor to emitter fluorescence intensity ratio can be used as a measure of signalling activity. For example, in key early work using FRET reporters for live single studies FRET reporters of Rho GTPase activity were engineered. Nuclear translocation reporters use engineered nuclear import and nuclear export signals, which can be inhibited by signalling molecules, to record signalling activity via the ration of nuclear reporter to cytoplasmic reporter. Live imaging Live cell imaging of fluorescently labelled cells must then be performed. This requires simultaneous incubation of cells in stress free conditions whilst imaging is being performed. There are several factors that must be taken into account when choosing imaging conditions such as phototoxicity, photobleaching, tracking ease, rate of change of signalling activity, and Signal to noise. These all relate to imaging frequency and illumination intensity. Phototoxicity can result from being exposed to large amounts of light over long periods of time. Cells will become stressed, which can lead to apoptosis. High frequency and intensity imaging can cause the fluorophore signal to decrease through photobleaching. Higher frequency imaging generally makes automated cell tracking easier. Imaging frequencies should be able to capture necessary changes to signalling activity. Low intensity imaging or poor reporters may prevent low levels of signalling activity within the cell from being detected. Live cell tracking Following live cell imaging, automated tracking software is then employed to extract time series data from videos of cells. Live cell tracking is generally split into two steps, image segmentation of cells or their nuclei and cell/nuclei tracking based on these segments. Many challenges still exist in this stage of a live single cell imaging study. However recent progress has been highlighted in the field first objective comparison of single cell tracking techniques. Quantitative phase imaging (QPI) is particularly useful for live cell tracking. As QPI is label-free, it does not induce phototoxicity, nor does it suffer from the photobleaching associated with fluorescence imaging. QPI offers a