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

Dataset DOI: [10.5061/dryad.k98sf7mkx](https://doi.org/10.5061/dryad.k98sf7mkx)

## Description of the data and file structure

Gene expression data measured using MERFISH in the developing human heart. Each distinct experiment is contained in a separate AnnData file, a standard format for storing single-cell gene expression data. Each AnnData file contains a \".X\" matrix with the normalized expression values as well as a \".layers['counts']\" matrix with the raw un-normalized expression values. Additionally, spatial coordinates of each cell are in the \".obsm['X_spatial']\" matrix. Additional file-specific contents are described below.

### Files and variables

#### File: Fig1_FirstImaging.h5ad

**Description:** Imaging of 238 genes using MERFISH in a single 2D heart section. AnnData object containing the following information for each cell: The number of genes (...,