Journal: bioRxiv

Article Title: MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

doi: 10.64898/2026.03.06.710199

Figure Lengend Snippet: A ) Schematic of MERFISH 1.0 chemistry in samples with high RNA integrity. Intact RNA transcripts allow multiple encoding probes to bind along the transcript length, producing strong readout signals during imaging. B ) Schematic of MERFISH 1.0 chemistry in samples with low RNA integrity. RNA fragmentation reduces the number of available probe-binding sites, leading to weaker fluorescence signals and decreased detection efficiency. C ) Schematic of MERFISH 2.0 chemistry. MERFISH 2.0 workflow is developed to enhance RNA transcript detection efficiency in samples with low quality RNA. Improvements include optimized RNA anchoring to retain fragmented RNA transcripts during sample preparation, an improved encoding probe design for efficient hybridization, and augmented readout probe structure with an enhancer to increase imaging signal-to-noise ratio. D ) Comparison of MERFISH workflows. Top, MERFISH 1.0 workflow for frozen tissue. Middle, MERFISH 1.0 workflow adapted for FFPE tissue. Bottom, MERFISH 2.0 workflow applicable to both frozen and FFPE samples.

Article Snippet: MERFISH 1.0 sample preparation followed published protocols and manufacturer’s instructions: MERSCOPE Fresh and Fixed Frozen Tissue Sample Preparation User Guide (Vizgen PN 91600002) and MERSCOPE FFPE Tissue Sample Preparation User Guide (Vizgen PN 91600112).

Techniques: Imaging, Binding Assay, Fluorescence, Sample Prep, Hybridization, Comparison

Journal: bioRxiv

Article Title: MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

doi: 10.64898/2026.03.06.710199

Figure Lengend Snippet: Fresh frozen mouse brain sections were profiled with an 815-plex MERSCOPE Pan Neuro Panel and imaged on MERSCOPE platform to compare MERFISH 1.0 and MERFISH 2.0 chemistries. A) Sensitivity comparison between MERFISH (blue) and MERFISH 2.0 (red) measured as RNA transcript counts per 100 µm .Bars represent mean values across biological replicates (N=3). MERFISH 2.0 shows increased transcript detection and reduced variability relative to MERFISH 1.0. B ) Distribution of detected transcripts per cell for MERFISH 1.0 and MERFISH 2.0, shown as histograms of log10-transformed transcript counts per cell. MERFISH 2.0 exhibits higher transcript counts per cell. C ) UMAP visualization of major neuronal and non-neuronal cell populations identified using MERFISH 1.0 and MERFISH 2.0 data. The MERFISH 1.0 and MERFISH 2.0 data was combined for analysis and co-embedding in the UMAP. D) Relative abundance of annotated cell types detected with MERFISH 1.0 and MERFISH 2.0, shown as the percentage of total cells assigned to each cell type.

Article Snippet: MERFISH 1.0 sample preparation followed published protocols and manufacturer’s instructions: MERSCOPE Fresh and Fixed Frozen Tissue Sample Preparation User Guide (Vizgen PN 91600002) and MERSCOPE FFPE Tissue Sample Preparation User Guide (Vizgen PN 91600112).

Techniques: Comparison, Transformation Assay

Journal: bioRxiv

Article Title: MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

doi: 10.64898/2026.03.06.710199

Figure Lengend Snippet: Fresh-frozen human brain sections were profiled using the 815-plex MERSCOPE Human Brain Panel and imaged on the MERSCOPE platform to compare MERFISH 1.0 and MERFISH 2.0 chemistries. A) Comparison of transcript detection between MERFISH 1.0 and MERFISH 2.0 on adjacent tissue slices. Scatter plot shows transcript counts detected by the two chemistries across the panel. B) Distribution of detected transcripts per cell for MERFISH 1.0 and MERFISH 2.0 shown as histograms of log10-transformed transcript counts per cell. MERFISH 2.0 shows increased transcript detection per cell. C-D) Correlation between bulk RNA-seq and MERFISH data with MERFISH 1.0 (C) or MERFISH 2.0 (D), demonstrating strong concordance between spatial and bulk measurements. E-F) Spatial heat map of transcript/cell across the tissue section for MERFISH 1.0 (E) and MERFISH 2.0 (F) chemistry illustrating increased transcript detection with MERFISH 2.0. G) Violin plot showing transcripts counts per cell across representative cell types identified in the human brain, including glutamatergic neurons, GABAegeric neurons, oligodendrocytes, astrocytes, vascular cells, and microglia cells. MERFISH 2.0 exhibits a broader dynamic range of transcript detection across cell types. H) Spatial distribution of representative marker genes with MERFISH 1.0 (top) and 2.0 (bottom) chemistry, including OLIG2, MOG and SOX10 with DAPI staining marking nuclei. MERFISH 2.0 shows substantially increased transcript counts for each marker gene.

Article Snippet: MERFISH 1.0 sample preparation followed published protocols and manufacturer’s instructions: MERSCOPE Fresh and Fixed Frozen Tissue Sample Preparation User Guide (Vizgen PN 91600002) and MERSCOPE FFPE Tissue Sample Preparation User Guide (Vizgen PN 91600112).

Techniques: Comparison, Transformation Assay, RNA Sequencing, Marker, Staining

Journal: bioRxiv

Article Title: MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

doi: 10.64898/2026.03.06.710199

Figure Lengend Snippet: Archival fresh-frozen human brain sections with reduced RNA quality were profiled using the 815-plex MERSCOPE Human Brain Panel and imaged on the MERSCOPE platform to compare MERFISH 1.0 and MERFISH 2.0 chemistries. A) Sensitivity comparison between MERFISH 1.0 (blue) or MERFISH 2.0 (red), measured as RNA transcript counts per 100 µm². Bars represent mean values across biological replicates (n = 3). MERFISH 2.0 shows substantially increased transcript detection. B) Distribution of detected transcripts per cell for MERFISH 1.0 and MERFISH 2.0, shown as histograms of log10-transformed transcript counts per cell, demonstrating increased transcript detection with MERFISH 2.0. C) UMAP visualization of different cell types identified in human brain with MERFISH 1.0, with 10,463 cells pass QC. Left: clustering of cell types based on gene expression profiles. Right: spatial distribution of annotated cell types across the tissue section. D) UMAP visualization of cell populations identified in human brain with MERFISH 2.0, with 21,236 cells pass QC. Left: clustering of cell types. Right: spatial distribution of annotated cell types. MERFISH 2.0 recovers substantially more cells and improves detection of distinct cell populations. E) Absolute number of cells assigned to each cell-type cluster with MERFISH 1.0 and MERFISH 2.0 chemistry. F) Relative abundance of cell types detected with MERFISH 1.0 and MERFISH 2.0 chemistry, shown as the percentage of total cells assigned to each cluster. MERFISH 2.0 identifies a higher fraction of astrocytes and uniquely detects MGE interneurons that are not recovered with MERFISH 1.0.

Article Snippet: MERFISH 1.0 sample preparation followed published protocols and manufacturer’s instructions: MERSCOPE Fresh and Fixed Frozen Tissue Sample Preparation User Guide (Vizgen PN 91600002) and MERSCOPE FFPE Tissue Sample Preparation User Guide (Vizgen PN 91600112).

Techniques: Comparison, Transformation Assay, Gene Expression

Journal: bioRxiv

Article Title: MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

doi: 10.64898/2026.03.06.710199

Figure Lengend Snippet: Formalin-fixed paraffin-embedded (FFPE) human tissues, including breast cancer specimens and lung and breast cancer tissue microarrays (TMAs), were profiled using the 815-plex Human Breast Cancer Panel or the Human Immuno-Oncology Panel following the MERFISH 1.0 and MERFISH 2.0 workflows. A) Sensitivity comparison between MERFISH 1.0 and 2.0 across FFPE human samples. RNA transcript counts per 100 µm² were used as a metric of sensitivity. Each condition includes at least 3 biological replicates (N=3). MERFISH 2.0 consistently increases the sensitivity for transcripts detection across samples. B) Concordance analysis between MERFISH 1.0 and MERFISH 2.0 measurements within each tissue. Person correlation coefficients calculated across samples demonstrate strong agreement between the two chemistries (r > 0.8). C) . UMAP visualization of single-cell transcriptomic profiles from FFPE human breast cancer displaying the different major cell types identified with MERFISH 1.0 (left), and MERFISH 2.0 (right). A total of 367,662 cells passed QC for MERFISH 1.0 and 654,332 cells passed QC for MERFISH 2.0. D) Transcript detection across individual TMA cores. Top, histograms showing the distribution of transcripts per cell for MERFISH 1.0 and MERFISH 2.0 across cores (R1–R5), with mean transcript counts indicated. Bottom, spatial distribution of Leiden clusters across tissue cores detected with MERFISH 1.0 (middle) and 2.0 chemistry (bottom), demonstrating much greater transcript coverage and cell recovery with MERFISH 2.0. E) Concordance between MERFISH-derived cell type profiles and reference single cell RNA sequencing data. Heatmap and summary correlation analysis show improved agreement with scRNA-seq when using MERFISH 2.0 compared to MERFISH 1.0. F) Violin plot showing the dynamic range of RNA transcripts counts per cell across representative cell types identified in human breast cancer.

Article Snippet: MERFISH 1.0 sample preparation followed published protocols and manufacturer’s instructions: MERSCOPE Fresh and Fixed Frozen Tissue Sample Preparation User Guide (Vizgen PN 91600002) and MERSCOPE FFPE Tissue Sample Preparation User Guide (Vizgen PN 91600112).

Techniques: Formalin-fixed Paraffin-Embedded, Comparison, Single Cell, Cell Recovery, Derivative Assay, RNA Sequencing

Journal: bioRxiv

Article Title: MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

doi: 10.64898/2026.03.06.710199

Figure Lengend Snippet: Immune cell populations from an FFPE human breast cancer sample were analyzed to evaluate whether the increased sensitivity of MERFISH 2.0 enables improved identification of immune cell subtypes. A) Spatial mapping of major cell populations and T cells. Left: spatial distribution of major cell types in the human breast cancer tissue profiled with MERFISH 1.0 (top) and 2.0 (bottom); Middle: spatial distribution of T cells identified in each dataset. Right: spatial distribution of CD8A gene expression and corresponding quantification of transcript counts showing increased detection with MERFISH 2.0 chemistry; B ) Representative multiplexed images showing spatial localization of selected immune markers detected with MERFISH 1.0 (top) and MERFISH 2.0 (bottom) in annotated T cells (green), B cells (red), plasmablast cells (yellow) and myeloid cells (blue). MERFISH 2.0 detects substantially higher transcript counts for select marker genes across immune cell populations. C ) UMAP visualization of immune cell subtypes identified with MERFISH 1.0 (left) and MERFISH 2.0 (right). Bar charts show the absolute number and proportion of cells assigned to each immune cell cluster. MERFISH 2.0 identifies larger populations of CD4⁺ and CD8⁺ T cells and reveals additional immune subtypes, including cycling myeloid cells that are not detected with MERFISH 1.0.

Article Snippet: MERFISH 1.0 sample preparation followed published protocols and manufacturer’s instructions: MERSCOPE Fresh and Fixed Frozen Tissue Sample Preparation User Guide (Vizgen PN 91600002) and MERSCOPE FFPE Tissue Sample Preparation User Guide (Vizgen PN 91600112).

Techniques: Gene Expression, Marker

Journal: bioRxiv

Article Title: MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

doi: 10.64898/2026.03.06.710199

Figure Lengend Snippet: Spatial analyses were performed on FFPE human breast cancer samples to evaluate whether increased transcript detection with MERFISH 2.0 improves the characterization of cellular interactions and gene co-expression patterns. A) Spatial enrichment analysis of the major cell types identified with MERFISH 1.0 (top) and MERFISH 2.0 (bottom). Heatmaps show the degree of spatial association between cell types. MERFISH 2.0 reveals greater and more diverse spatial interactions among cell populations. B ) Representative images illustrating spatial relationships between selected cell types. Left: interaction between endothelial cells and perivascular-like (PVL) cells; right: interaction between T cells and B cells only visible with MERFISH 2.0 (bottom) as B-cells did not appear with MERFISH 1.0 profiling. MERFISH 2.0 reveals these spatial relationships more clearly through enhanced transcript detection and improved cell-type annotations. C ) Gene-gene correlation matrices from MERFISH 1.0 (left) and MERFISH 2.0 (right) datasets. MERFISH 2.0 reveals a larger number of correlated gene modules, indicating improved detection of coordinated gene expression programs.

Article Snippet: MERFISH 1.0 sample preparation followed published protocols and manufacturer’s instructions: MERSCOPE Fresh and Fixed Frozen Tissue Sample Preparation User Guide (Vizgen PN 91600002) and MERSCOPE FFPE Tissue Sample Preparation User Guide (Vizgen PN 91600112).

Techniques: Expressing, Gene Expression