Multiplexed RNA imaging and in situ profiling in living cells

Single-cell in situ RNA profiling and multiplexed imaging are powerful tools for uncovering cellular heterogeneity through recognizing distinct gene expression signatures. Identifying these molecular profiles and understanding their correlations are essential in both fundamental biological research and clinical diagnostics. Established analytical techniques in spatial transcriptomics such as those based on fluorescence in situ hybridization and in situ sequencing have been widely employed for this purpose, though they all typically require cell fixation and permeabilization. Recent development of advanced fluorescent probes has improved our ability of multiplexed RNA imaging in living cells, opening new avenues for spatiotemporal in situ RNA profiling. These innovative and effective tools are poised to allow for real-time monitoring of RNA dynamics and the exploration of temporal relationships among different RNA species—longstanding challenges in the field. This perspective highlights recent advances in developing analytical methods for multiplexed RNA imaging in both fixed and live cells, and discusses how these technologies can deepen our understanding of RNA biology and their potential applications in studying disease mechanisms, diagnosis, and treatment.