The dynamics of mRNA-the synthesis, transport, and degradation-plays significant roles in a variety of neuronal processes, such as axonal guidance, synaptogenesis, and long-term plasticity.Abnormal mRNA processing and transport are implicated in neurological disorders such as autism and Alzheimers disease.However, understanding the mechanistic roles of RNA dynamics has been hampered by the lack of techniques to observe the endogenous molecules in the native tissue environment.Here I will present real-time investigation of gene expression for the first time at the level of single RNA molecules in living animals.It is enabled by a novel systems approach combining single-particle tracking, genetic engineering, and intravital microscopy.We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs.In cultured neurons, multiple β-actin mRNAs assemble together, travel by active transport, and disassemble upon KCI depolarization.Two-photon imaging of live brain tissues revealed immediate-early induction of β-actin transcription after neuronal stimulation.The technology as demonstrated in our work, including the transgenic strategy and high-resolution microscopy of living tissue, could be applied to other genes to gain insights into the dynamic regulation of gene expression within the cellular and tissue microenvironment.