Over the past 20 years luminescent and fluorescent imaging assays have been developed to report on the dynamics of transcription and protein translocation in single cells. The combination of these measurements with mathematical analysis is having an increasingly significant impact on cell biology. There is an urgent need to translate these assays to the study of cells and tissues in vivo, which requires new tools and technologies. Emergence of these new tools and techniques will further the understanding of the role of signalling and transcriptional dynamics in the generation of cellular heterogeneity and the control of cell fate.