DOI: 10.5061/dryad.k98sf7mkx
title: MERFISH+, a large-scale, multi-omics spatial technology resolves the transcriptomic holograms of the 3D human developing heart

description: 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 &gt;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.
description: # 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
 ('n_genes') and transcripts ('total_counts') detected,
 and the cell type identity of each cell ('celltype3'). ####
 File: Fig1_ReImage.h5ad **Description:** Re-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
 ('n_genes') and transcripts ('total_counts') detected,
 and the cell type identity of each cell ('celltype3'). ####
 File: FigS2_Codebook1.h5ad **Description:** Imaging of 251 genes using
 MERFISH in a 2D heart section. AnnData object containing the following
 information for each cell: The number of genes
 ('n_genes_by_counts') and transcripts ('total_counts')
 detected, and cell type annotation ('celltype'). #### File:
 FigS2_Codebook2.h5ad **Description:** Imaging of 251 genes using MERFISH
 in a 2D heart section, with a re-organized codebook. AnnData object
 containing the following information for each cell: The number of genes
 ('n_genes_by_counts') and transcripts ('total_counts')
 detected, and cell type annotation ('celltype'). #### File:
 Fig2_1835genes.h5ad **Description:** Imaging of 1835 genes using MERFISH
 in a 2D heart section. AnnData object containing the following information
 for each cell: The number of genes ('n_genes_by_counts') and
 transcripts ('total_counts') detected, and cell type annotation
 ('celltype'). #### File: Fig3_multimodal_v2.h5ad
 **Description:** Imaging of 251 genes using MERFISH and 6 proteins with
 immunostaining in a 2D heart section. AnnData object containing the
 following information for each cell: The number of genes
 ('n_genes_by_counts') and transcripts ('total_counts')
 detected, cell type annotation ('celltype'), and protein
 quantification measured by immunofluorescence ('H3K4me3',
 'PolSer2', 'H3K27ac', 'Pol2Se5',
 'H3K9me3', 'H3K27me3'). Chromatin tracing data is
 stored in the .obsm['X_h_score'] matrix, which is an Nx4x50x5
 matrix where N is the number of cells, 4 is the maximum number of
 chromosomes per cell, 50 is each of the chromatin regions imaged, and 5
 are the 5 properties measured for each region: The z, x, and y coordinates
 of the region, the brightness, and the score (see methods). #### File:
 Fig4to6_3DHeart.h5ad **Description:** Imaging of 238 genes using MERFISH
 in multiple 2D sections from the same heart, which was then reconstructed
 into its 3D conformation. AnnData object containing the following
 information for each cell: The number of genes
 ('n_genes_by_counts') and transcripts ('total_counts')
 detected, the high-level cell class ('class'), cell type
 annotation ('celltype'), spatially defined sub-type
 ('celltype-extra'). The MYH11+ cells assigned to coronary artery
 vessels are annotated in the 'vessel_name' column. The
 'RCA/LCX/LAD/LCA_community' columns indicate whether a cell is
 part of the specified blood vessel or surrounding cells. The
 'celltype_extra' column contains additional sub-clusters for the
 valves and blood vessels which were defined spatially. The cell
 coordinates contained in the .obsm["X_spatial"] are 2D
 coordinates of each section, while the .obsm["X_spateo"] matrix
 are the 3D reconstructed coordinates. #### File:
 Fig5_Pulmonary_valve_vessel.h5ad **Description:** Subset of 3D heart
 including the cells consisting of the pulmonary artery, pulmonary valve,
 and outflow tract. AnnData object containing the following information for
 each cell: The number of genes ('n_genes_by_counts') and
 transcripts ('total_counts') detected, the high-level cell class
 ('class'), cell type annotation ('celltype'),
 spatially defined sub-type ('celltype-extra'), endothelial
 subcluster ('Endo_subcluster'), and VIC subcluster
 ('VIC_subcluster') #### File: Fig7_3DHeart_imputed.h5ad
 **Description:** Imputed expression of 1825 genes and 5 antibodies in a
 reconstructed 3D heart. AnnData object containing 1,825 genes and 5
 protein abundances in the .X matrix imputed computationally in the same
 cells as Fig4to6_3DHeart.h5ad. Cell names match those in
 Fig4to6_3DHeart.h5ad and can be linked to the additional information
 stored there. ## Code/software These files are in anndata format, which
 can be loaded in python using the Scanpy package: ``` !pip install scanpy
 import scanpy as sc adata = sc.read("Fig1_FirstImaging.h5ad")
 ```