RT&T-AMP-MERFISH enables whole-transcriptome-scale spatial imaging of approximately 23,000 genes and 10,000 isoform-defining transcripts in single cells within intact tissues, revealing extensive region- and cell-type-specific isoform usage patterns across the mouse brain, with the choroid plexus and hippocampus serving as hotspots for isoform diversity, and demonstrating that alternative splicing generates distinct spatial and cellular expression programs including striking differences between protein-coding and non-coding isoforms of the same gene.

Cell and tissue functions arise from complex interactions among numerous genes, and a systematic understanding of these functions requires isoform-resolved, whole-transcriptome-scale analysis of single cells with high spatial resolution. Here, we introduce a spatial transcriptomics method based on a novel in situ RNA amplification strategy, enabling short RNA sequence detection and hence spatially resolved expression profiling of individual cells at the whole-transcriptome scale with splice-isoform resolution. Using this approach, we imaged ∼33,000 distinct RNAs—including ∼23,000 genes and ∼10,000 isoform-defining transcripts—in the mouse brain. Our data enabled systematic analyses of region- and cell-type-specific gene programs and ligand-receptor-based cell-cell communications. These data further revealed a rich spatial diversity and cell-type specificity in isoform usage across numerous genes and identified brain structures particularly enriched for specific isoform usage. We anticipate broad application of this method for molecular and cellular analysis of tissues, unlocking previously inaccessible discoveries in cell and organismal biology.