High-plex spatial RNA imaging in one round with conventional microscopes using color-intensity barcodes

Spatial RNA imaging has not been widely adopted because conventional fluorescence microscopy is limited to only a few channels, and the cyclic reactions needed to increase multiplexing in techniques such as sequential fluorescence in-situ hybridization (FISH) require sophisticated instrumentation. Here, we introduce ‘Profiling of RNA In-situ through Single-round iMaging’ (PRISM), a method that expands coding capacity through color intensity grading. Using a radius vector filtering strategy to ensure the distinguishability of codewords in color space, PRISM achieves up to 64-plex color-barcoded RNA imaging in a single imaging round with conventional microscopes. We validate PRISM’s versatility across various tissues by generating a 3D atlas of mouse embryonic development from E12.5 to E14.5, a quasi-3D tumor-normal transition landscape of human hepatocellular carcinoma (HCC), and a 3D cell atlas and subcellular RNA localization landscapes of mouse brain. Additionally, we show the critical role of cancer-associated fibroblasts (CAFs) in mediating immune infiltration and immune response heterogeneity within and between tumor microenvironments.