Multiplexed RNA imaging and <i>in situ</i> profiling in living cells

Single-cell <i>in situ</i> RNA profiling and multiplexed imaging have transformed our ability to uncover cellular heterogeneity by resolving distinct gene expression signatures. While established spatial transcriptomics methods - such as fluorescence <i>in situ</i> hybridization and <i>in situ</i> sequencing-have been widely used to map RNA profiles, they are inherently limited by the need for cell fixation and permeabilization, providing only static snapshots. In contrast, recent development of advanced fluorescent probes now enables multiplexed RNA imaging in living cells, offering a powerful way to monitor RNA localization, interaction, and concentration changes in real time. These innovations open new avenues for spatiotemporal <i>in situ</i> RNA profiling, making it possible to track RNA dynamics and unravel temporal relationships among multiple RNA species - longstanding challenges that remained out of reach with conventional approaches. This perspective highlights emerging advances in live-cell multiplexed RNA imaging, while also situating them alongside fixed-cell spatial transcriptomics methods, and discusses how these technologies can reshape our understanding of RNA biology and accelerate applications in disease mechanism studies, diagnostics, and therapeutic development.