Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here, we introduce signal amplification by exchange reaction (SABER), which endows oligo-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplifies RNA and DNA FISH signals (5 to 450-fold) in fixed cells and tissues, apply 17 orthogonal amplifiers against chromosomal targets simultaneously, and detect mRNAs with high efficiency. We further apply 10-plexSABER-FISH to identify in vivo introduced enhancers with cell type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.

Spatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity, and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reactions (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrated 5–180-fold signal amplification in diverse samples (cultured cells, and FFPE, cryosectioned or whole mount tissues), and simultaneous signal amplification for 10 different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.

SignificanceFISH enables researchers to visualize the subcellular distribution of RNA and DNA molecules in individual cells. The recent development of FISH methods employing probes composed of synthetic DNA oligonucleotides (oligos) allows researchers to tightly control aspects of probe design such as binding energy and genomic specificity. Although oligo FISH probes are central to many recently developed massively multiplexed and superresolution imaging methods, no dedicated computational utility exists to facilitate the design of such probes on the genome-wide scale. Here, we introduce a streamlined pipeline for the rapid, genome-scale design of oligo FISH probes and validate our approach by using conventional and superresolution imaging. Our method provides a framework with which to design oligo-based hybridization experiments.