A Growing Toolbox to Image Gene Expression in Single Cells: Sensitive Approaches for Demanding Challenges

The spatiotemporal regulation of gene expression is key to many biological processes. Recent imaging approaches opened exciting perspectives for understanding the intricate mechanisms regulating RNA metabolism, from synthesis to decay. Imaging techniques allow their observation at high spatial and temporal resolution, while keeping cellular morphology and micro-environment intact. Here, we focus on approaches for imaging single RNA molecules in cells, tissues, and embryos. In fixed cells, the rapid development of smFISH multiplexing opens the way to large-scale single-molecule studies, while in live cells, gene expression can be observed in real time in its native context. We highlight the strengths and limitations of these methods, as well as future challenges. We present how they advanced our understanding of gene expression heterogeneity and bursting, as well as the spatiotemporal aspects of splicing, translation, and RNA decay. These insights yield a dynamic and stochastic view of gene expression in single cells.