Quantitative RNA spatial profiling using single-molecule RNA FISH on plant tissue cryosections

Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful tool for studying gene expression dynamics with unparalleled precision and spatial resolution in a variety of biological systems. Recent advancements have expanded its application to encompass plant studies, yet there remains a need for a simple and robust smFISH method adapted to plant tissue sections. Here, we present an optimized smFISH protocol, termed cryo-smFISH, for visualizing and quantifying single mRNA molecules in plant tissue cryosections. This method exhibits remarkable sensitivity, enabling the detection of low-expression transcripts, including long non-coding RNAs. By integrating a deep learning-based algorithm into our image analysis pipeline, our method enables precise assignment of RNA abundance in nuclear and cytoplasmic compartments. The method also enables robust integration with immunofluorescence, as cryosectioning enhances antibody penetration. This allows for the sequential visualization and quantification of both RNAs and endogenous proteins within the same cells. Finally, this study demonstrates the use of smFISH to validate single-cell RNA sequencing (scRNA-seq) expression patterns in plant tissues. By extending smFISH to plant cryosections, plant scientists will be able to exploit the full potential of quantitative transcript analysis at cellular and subcellular resolution.