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 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 three-dimensional (3D) atlas of mouse embryonic development from E12.5 to E14.5, a quasi-3D tumor-normal transition landscape of human hepatocellular carcinoma and a 3D cell atlas and subcellular RNA localization landscapes of mouse brain. Additionally, we show the critical role of cancer-associated fibroblasts in mediating immune infiltration and immune response heterogeneity within and between tumor microenvironments.