Abstract
Single molecule fluorescence in situ hybridization (smFISH) is a powerful method for thevisualization and quantification of individual RNA molecules within intact cells. With its ability to probegene expression at the single cell and single-molecule level, the technique offers valuable insights intocellular processes and cell-to-cell heterogeneity. Although widely used in the animal field, its use inplants has been limited. Here, we present an experimental smFISH workflow that allows researchers toovercome hybridization and imaging challenges in plants, including sample preparation, probehybridization, and signal detection. Overall, this protocol holds great promise for unraveling theintricacies of gene expression regulation and RNA dynamics at the single-molecule level in whole plants.