MERFISH+ technology, which covalently anchors fluorescent probes to polyacrylamide hydrogels enabling stable multi-month imaging, facilitates the reconstruction of the first comprehensive 3D molecular atlas of an entire developing human organ at single-cell resolution, profiling over 1,800 genes and multimodal epigenetic features across 3.1 million cells to reveal cellular neighborhoods, spatial expression gradients, and cell-cell signaling patterns that govern cardiac morphogenesis and coronary artery development.

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 chemical probe anchoring in protective hydrogels with high-throughput microfluidics and microscopy. This optimized design supports robust and repeated hybridization cycles across an entire centimeter-scale tissue sample. MERFISH+ allowed to simultaneously quantify over 1,800 genes and resolve the 3D organization of chromatin loci and their associated epigenomic marks in developing human hearts. Using a generative integration framework for spatial multimodal data (Spateo-VI), we harmonized these MERFISH+ transcriptomic and chromatin data to reconstruct a 3D spatially-resolved multi-omic atlas of the developing human heart at subcellular resolution capturing 3.1 million cells across 34 distinct populations. This 3D atlas provides a holistic view of an entire organ enabling the characterization of 3D cellular neighborhoods and transcriptional gradients of substructures such as the descending arteries. Thus, MERFISH+ offers a robust, large-format 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.

MERFISH+ is an spatial multi-omics platform that integrates hydrogel-based probe anchoring, automated high-throughput microfluidics, and large-format multimodal data production to enable comprehensive, subcellular resolution mapping of gene expression and chromatin organization across millions of cells within complex developing human organs.

Highlights

MERFISH+ expands MERFISH capabilities to measure >1,800 genes and at whole-organ 3D imaging scale

Combines chemical probe anchoring with high-throughput volumetric microscopy and microfluidics

Generates a 3D molecular atlas of a developing human heart with > 3.1 million cells at subcellular resolution

Introduces Spateo-VI, a novel generative framework integrating 3D multimodal datasets