MERFISH+, a large-scale, multi-omics spatial technology resolves the transcriptomic holograms of the 3D human developing heart

Hybridization-based spatial transcriptomics technologies have advanced our ability to map cellular and subcellular organization in complex tissues. However, existing methods remain constrained in gene coverage, multimodal compatibility, and scalability. Here, we present MERFISH+, an enhanced version of Multiplexed Error-Robust Fluorescence in Situ Hybridization (MERFISH), which integrates covalent probe anchoring in protective hydrogels with automated high-throughput microfluidics and microscopy. This optimized design supports >100 hybridization cycles and centimeter-scale 3D serial imaging. MERFISH+ quantified over 1,800 genes and resolved the 3D organization of chromatin loci and epigenomic marks in developing human hearts. To unify these molecular features, we developed a generative integration framework for spatial multimodal data (Spateo-VI), generating a comprehensive 3D spatial multi-omic atlas encompassing ~3.1 million cells across 34 cellular populations. This 3D atlas provides a holistic view of an entire human organ enabling the characterization of 3D cellular neighborhoods and transcriptional variation within substructures such as valves and major artery branches. Thus, MERFISH+ offers a robust, large-scale platform for spatial multi-omics that enables high resolution mapping of gene expression at subcellular resolution and the characterization of cellular organization within 3D organs.