Journal: bioRxiv

Article Title: Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

doi: 10.64898/2026.05.05.722861

Figure Lengend Snippet: A. Schematic of intestinal developmental timepoints analysed by single cell multiomics and in situ spatial transcriptomics (ST). Created with BioRender. B. Overview of single cell multiomics and ST cohorts; each point represents an individual donor. C. UMAP of integrated single cell multiomics data showing all cells, coloured by broad cell type and age group. Colour scheme consistent with D. D. Pseudobulk principal component analysis (PCA) of epithelial, stromal and immune compartments identified by single cell multiomics. Data were downsampled to equal cell numbers and coloured by age group. E. Heatmaps of promoter accessibility and gene expression for development-associated genes in ileal epithelium; values are mean-scaled per sample. Age group colour scheme consistent with D. F. Representative terminal ileum section (3 months) profiled by MERSCOPE ST, coloured by broad cell type. G. Scaled Wasserstein distance from a location-matched fetal anchor plotted against log2-transformed post-conceptual weeks (PCW). Each point represents an individual donor. LOESS fits are shown, highlighting the inflection and peak of the trajectories. H. Integrated transcriptomic atlas combining scRNA-seq data with datasets from Elmentaite et al. 2021 and Fawkner-Corbett et al. 2021; each point represents a donor. I. Performance of a multilayer perceptron (MLP) regressor on the validation dataset. Predicted age corresponds to the median across cells per donor. Points are coloured by location and sized by cell number. J. Volcano plots showing top genes (left) and regulons (right) from the colonic ElasticNet model. Axes indicate signed feature importance and change in mean absolute error (MAE) following in silico perturbation.

Article Snippet: The MERSCOPE Vizgen Platform was used for high-resolution subcellular spatial transcriptomics, following the manufacturer’s instructions for resistant tissue with modifications (Vizgen, MERSCOPE, 91600112, Rev B).

Techniques: Single Cell, In Situ, Spatial Transcriptomics, Gene Expression, Transformation Assay, Biomarker Discovery, In Silico

Journal: bioRxiv

Article Title: Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

doi: 10.64898/2026.05.05.722861

Figure Lengend Snippet: A. Flow cytometry gating strategy for isolation of stromal (CD45⁻) and immune (CD45⁺) compartments. B. UMAP of integrated MERSCOPE ST data, coloured by broad cell type, age group and location. C. Pseudobulk principal component analysis (PCA) of RNA and peak assays across epithelial, stromal and immune compartments identified by single cell multiomics, coloured by age group. D. UMAP of integrated single cell multiomics data, coloured by location. Paired UMAP overlay with cell cluster and age is shown in . E. UMAP of metacells derived from integrated single cell multiomics data, coloured by cell type. F. UMAP overlays showing gene expression (top), promoter accessibility (middle) and enhancer accessibility (bottom) for marker genes (MKI67, ADAMDEC1, F3). G. UMAP overlay showing cell type annotations and regulon activity in scDOGMA-seq data. Transcription factor regulons mark distinct lineages, including PHOX2B and SOX10 (enteric nervous system), HAND1 (mesenchymal), IRF8 (immune), PAX4 (endocrine) and CDX2 (epithelial), whereas KLF6 and ETS2 show broad activity across cell types.

Article Snippet: The MERSCOPE Vizgen Platform was used for high-resolution subcellular spatial transcriptomics, following the manufacturer’s instructions for resistant tissue with modifications (Vizgen, MERSCOPE, 91600112, Rev B).

Techniques: Flow Cytometry, Isolation, Single Cell, Derivative Assay, Gene Expression, Marker, Activity Assay

Journal: bioRxiv

Article Title: Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

doi: 10.64898/2026.05.05.722861

Figure Lengend Snippet: A. Circular heatmap of predicted cell-type proportions from single cell multiomics. Age effects were modelled using quasibinomial generalized linear models with a natural cubic spline for age; predicted proportions are z-score normalized across age groups and annotated by broad compartments. B. Circular heatmap of predicted cell-type proportions from MERSCOPE ST data, z-score normalized across age groups and annotated by broad compartments. C. Representative MERSCOPE ST sections from 9 PCW (top) and 3 months (bottom) terminal ileum, coloured by broad cell type (left) and epithelial and T cell clusters (right). D. Spatial expression of CTNNB1 in MERSCOPE ST sections at 9 PCW and 4 weeks.

Article Snippet: The MERSCOPE Vizgen Platform was used for high-resolution subcellular spatial transcriptomics, following the manufacturer’s instructions for resistant tissue with modifications (Vizgen, MERSCOPE, 91600112, Rev B).

Techniques: Single Cell, Expressing

Journal: bioRxiv

Article Title: Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

doi: 10.64898/2026.05.05.722861

Figure Lengend Snippet: A. Top, overview of diet conditions in single cell multiomics cohorts; each point represents a donor, coloured by diet and shaped by location. Bottom, scatterplot with LOESS-fitted line showing metabolic pathway scores in ileal samples, coloured by pathway. B. UMAP of ileal (top) and colonic (bottom) epithelial cells in a joint RNA–ATAC embedding, coloured by cell type, age group (as in A) and crypt–villus differentiation pseudotime. C. Relationship between mean chronological age (by age group) and crypt–villus differentiation score across epithelial cell clusters, coloured by location. Distributions show stem, differentiated, fetal and adult cNMF module usage across chronology (age group) and differentiation. D. Predicted proportions of epithelial cell types in ileum (top) and colon (bottom), estimated using sccomp and visualized as ribbon plots; colours correspond to cell types in B. E. Generalized additive model (GAM)-fitted trends of promoter and enhancer accessibility per sample and gene expression for selected genes across age in ileal epithelium. Shaded areas indicate +/- 1 standard error of the GAM fitted mean. F. Bubble plot showing age-associated regulon activity across ileal epithelial cells. G. Violin plots showing fetal gene module scores (left) and chromatin accessibility module scores (right) in colonic epithelium. H. Spatial expression of the fetal marker TTR in MERSCOPE ST sections at 9 PCW (left), 5 days (middle) and 13 months (right). I. Volcano plot showing differential expression between fetal and paediatric colonic stem cells in single cell multiomics data. Differential expression was assessed using DESeq2 (Wald test with Benjamini–Hochberg correction). Points are coloured by significance and effect size (red, |log2FC| > 1 and −log10P > 1; blue, |log2FC| < 1 and −log10P > 1; yellow, |log2FC| > 1 and −log10P < 1; grey, not significant). J. Density plots of LGR5 gene expression (left) and promoter accessibility (right) in colonic epithelial cells. K. Coverage and violin plots showing promoter accessibility and gene expression of stem cell marker genes in ileal epithelium, coloured by mean crypt–villus differentiation score per cell type.

Article Snippet: The MERSCOPE Vizgen Platform was used for high-resolution subcellular spatial transcriptomics, following the manufacturer’s instructions for resistant tissue with modifications (Vizgen, MERSCOPE, 91600112, Rev B).

Techniques: Single Cell, Gene Expression, Activity Assay, Expressing, Marker, Quantitative Proteomics

Journal: bioRxiv

Article Title: Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

doi: 10.64898/2026.05.05.722861

Figure Lengend Snippet: A. Predicted proportions of immune cell types across age groups in MERSCOPE ST data, estimated using sccomp and visualized as ribbon plots (myeloid, ILC, T cells, NK cells, B cells and plasma cells). Colours correspond to cell types in B. B. Representative MERSCOPE ST sections at 9 PCW, 2 weeks, 4 weeks and 2 months, coloured by cell type. C. Representative immunohistochemistry (IHC) images across developmental stages (fetal, newborn, 1 month, 5 months and 1 year) stained for HLA-DR. Scale bars, 100 μm. D. Heatmaps of epithelial–neighbourhood correlations across developmental stages. Pearson correlations between epithelial gene expression programmes and neighbouring cytokine-related signals were computed in terminal ileum samples, averaged within age groups (fetal, <1 month), and visualized by hierarchical clustering using a consistent colour scale. E. Proportion of S1/S2 CXCL8⁺ fibroblasts across age groups in single cell multiomics data. F. Bubble plot showing marker gene expression and regulon activity in S1/S2 CXCL8⁺ fibroblasts in single cell multiomics data. G. Cell–cell communication networks inferred using CellChat from single cell multiomics data. Left, circle plot showing interaction strength between epithelial, immune and fibroblast populations in fetal and neonatal (<2 months) samples. Right, chord diagram highlighting ligand–receptor interactions in CXCL, TGFβ and BMP signalling pathways. H. Cluster–neighbour co-localisation network from MERSCOPE ST data highlighting interactions among S1/S2 CXCL8⁺ fibroblasts, IL1B⁺CXCL8⁺ myeloid cells, REG1B/DUOX2⁺ enterocytes, S100A8/9⁺ myeloid cells and epithelial macrophages. Edges represent co-localisation strength (threshold > 0.02). Node size reflects cell abundance and colour denotes cell type. Data are from terminal ileum samples aged <1 month. I. Proportion of intraepithelial immune cells in chelated epithelial crypt single cell multiomics data across age groups. J. Bubble plot showing cytokine and immune state marker gene expression in intraepithelial immune cells; point size indicates the proportion of each cell type within age groups.

Article Snippet: The MERSCOPE Vizgen Platform was used for high-resolution subcellular spatial transcriptomics, following the manufacturer’s instructions for resistant tissue with modifications (Vizgen, MERSCOPE, 91600112, Rev B).

Techniques: Clinical Proteomics, Immunohistochemistry, Staining, Gene Expression, Single Cell, Marker, Activity Assay

Journal: bioRxiv

Article Title: Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

doi: 10.64898/2026.05.05.722861

Figure Lengend Snippet: A. Representative MERSCOPE ST sections coloured by inferred spatial niches. TI-terminal ileum. B. Heatmap of cell type–niche enrichment for a representative terminal ileum sample (ABB, 2 weeks). Values are shown as log2-transformed estimates, with statistical significance indicated by asterisks (FDR < 0.05, ** < 0.01, *** < 0.001; n.s., not significant). C. Cluster co-localisation networks within the crypt niche across developmental stages. Networks include clusters with total interaction proportion ≥ 0.2 and edges with weight ≥ 0.1. Node size reflects cell abundance, edge thickness indicates co-localisation strength, and node colour denotes cell compartment.

Article Snippet: The MERSCOPE Vizgen Platform was used for high-resolution subcellular spatial transcriptomics, following the manufacturer’s instructions for resistant tissue with modifications (Vizgen, MERSCOPE, 91600112, Rev B).

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

doi: 10.64898/2026.05.05.722861

Figure Lengend Snippet: A. Proportion of selected clusters in MERSCOPE ST data across age groups; points represent individual donors and bars indicate mean proportions. B. Heatmaps of epithelial–neighbourhood correlations across developmental stages. Pearson correlations between epithelial gene expression programmes and neighbouring cytokine-related signals were computed in terminal ileum samples, averaged within age groups (>1 month), and visualized by hierarchical clustering using the same colour scale as . C. Bubble plot showing expression of cytokines and receptors across broad cell types in each age group. D. Age-associated changes in the myeloid compartment. (i) UMAP of myeloid cells coloured by cluster identity. (ii) Proportion of IL1B⁺ M1 macrophages within the myeloid compartment. (iii) Differential cluster abundance comparing <1 month versus fetal (top) and >1 month versus <1 month (bottom), assessed using MiloR. E. Box plots showing the distribution of T cell states, inferred using StarCAT, in intraepithelial lymphocytes from crypt-chelated epithelium and in CD45⁺ T cells. CD8 Trm, CD8⁺ tissue-resident memory T cells; TEMRA, terminally differentiated effector memory T cells; gdT, γδ T cells.

Article Snippet: The MERSCOPE Vizgen Platform was used for high-resolution subcellular spatial transcriptomics, following the manufacturer’s instructions for resistant tissue with modifications (Vizgen, MERSCOPE, 91600112, Rev B).

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: Multimodal alignments of in vivo imaging and spatial biology datasets at cellular resolution

doi: 10.64898/2026.04.28.719500

Figure Lengend Snippet: A) Top: Panel of two-photon in vivo Ca 2+ images (averages of 300 frames taken at 30 fps) from mouse motor cortex, showing viral expression of GCaMP8s in pyramidal cells after local injection of AAV2/PHP.eB-CaMKII-jGCaMP8s. The depth below the pia is indicated in the lower left corner of each image. Bottom, Corresponding two-photon images acquired postmortem from successive 10-μm-thick tissue sections, using native GCaMP8s fluorescence. Colored squares enclose example areas in which cells can be readily matched by eye across the two sets of images. Scale bar: 200 μm. B) Top : Example two-photon in vivo Ca 2+ image taken ∼280 μm beneath the surface of motor cortex, showing the viral expression of GCaMP8s in pyramidal cells. Lower : Corresponding MERFISH image, taken with a commercial MERFISH instrument (MERSCOPE, Vizgen) and the PanNeuro 500-gene panel. The Vizualizer software (Vizgen) assigned each detected RNA molecule a different color in the image, according to which of the 500 genes it encoded. DAPI labeling of cell nuclei is shown in blue. White ellipses enclose regions in which neurons can be readily matched by eye across the two images. Areas within colored rectangles are shown at greater magnification in C . Scale bar: 100 μm. C) Magnified views of areas enclosed in the red and yellow rectangles in B , showing images taken in vivo ( left column ), of RNA molecules and DAPI acquired with MERFISH ( middle column ), and of DAPI plus a green fluorescently labeled poly-thymine (PolyT) probe used to reveal DNA ( right column ). Arrows point to cells that are matched across images. Scale bar: 20 μm. D) Dot plots showing mean normalized levels of gene expression ( Methods ) for individual transcriptomic clusters of glutamatergic cells determined using a set of 15 cortical marker genes by mapping each cell’s gene expression profile onto the Allen Institute database with MapMyCells (RRID:SCR_024672). Cell clusters are labeled according to the terminology from the Allen Institute. E) Ca 2+ activity traces for 6 example neurons, one from each cluster in D . F) Color raster plots of Ca 2+ activity traces from neurons assigned through postmortem MERFISH to each of the glutamatergic neuron-types in D , shown beneath a trace of the mouse’s locomotor speed. The total numbers of cells of each type are indicated in the parentheses.

Article Snippet: We mounted 10-μm-thick slices onto MERSCOPE slides (MERSCOPE Slide Box, Vizgen, 10500001), dried them in a cryostat for 20–30 min, washed the slides 3 times with RNase-free PBS at room temperature, and dried them for 60–90 min according to Vizgen protocols (91600002, 91700118).

Techniques: In Vivo, Expressing, Injection, Fluorescence, Software, Labeling, Gene Expression, Marker, Activity Assay

Journal: bioRxiv

Article Title: Multimodal alignments of in vivo imaging and spatial biology datasets at cellular resolution

doi: 10.64898/2026.04.28.719500

Figure Lengend Snippet: A) Example data from a validation study in which we used the MERSCOPE (Vizgen) to check RNA abundance in tissue processed using our TRU-FACT procedures. Left : Ex vivo two-photon image of native GCaMP8m fluorescence from a 10-μm-thick tissue section from mouse motor cortex. Right : The corresponding single molecule FISH (smFISH) image from the MERSCOPE showing the detection of mRNA for the abundant, EEF2 housekeeping gene in the same field-of-view as that in the left panel. The two images show good maintenance of detectable RNA and that cells’ morphologies were well preserved across our MERFISH tissue processing. Colored ellipses in A and B enclose subregions with cells that are readily matched by eye across the two images. Scale bar: 100 μm. B) Overlay (white) of matched in vivo (green) and ex vivo (magenta) cells (from ). White ellipses enclose the same regions as in , in which neurons can be readily matched by eye across the two images. Areas within colored rectangles are shown at greater magnification in . Scale bar: 100 μm. C) Distributions of Soma-print scores and corresponding parametric fits for pairs of best-matched and 2 nd -best-matched cells, plotted in the same format as for the data of B . Using the likelihood ratio cutoff of 0.05 (vertical dashed line), 196 out of the 272 cells observed in vivo were successfully matched to corresponding cells found postmortem (sensitivity: 72%). Inset : The same distributions and fits shown with a logarithmic scale on the y- axis. D) Linear regression plots of the mouse’s running speed versus the z-scored Ca 2+ activity of 6 different example cells from each cluster in Fig. 6D –F (red: L2/3 Glut 2, blue: L4/5 Glut 2, yellow: L4/5 Glut 5, grey: others).

Article Snippet: We mounted 10-μm-thick slices onto MERSCOPE slides (MERSCOPE Slide Box, Vizgen, 10500001), dried them in a cryostat for 20–30 min, washed the slides 3 times with RNase-free PBS at room temperature, and dried them for 60–90 min according to Vizgen protocols (91600002, 91700118).

Techniques: Biomarker Discovery, Ex Vivo, Fluorescence, In Vivo, Activity Assay

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . Schematic overview of the three spatial transcriptomics (ST) platforms used in this study, comprising sequencing-based (sST) and imaging-based (iST) approaches. Visium HD (10x Genomics) is a sST platform that captures spatially barcoded polyadenylated transcripts, followed by cDNA library preparation and sequencing. MERSCOPE (Vizgen) and Xenium (10x Genomics) are iST platforms for in situ transcript detection using multiplexed fluorescence imaging, with MERSCOPE relying on direct fluorescent probe hybridization and Xenium on padlock probes with rolling circle amplification, followed by iterative imaging, signal removal, and rehybridization cycles for transcript decoding. B . Experimental design of the SpatialBench study using spleen sections from malaria infected and cured mice across wild-type (WT), control (CTRL) ( Tbx21 +/+ Cd23 Cre ), and B cell-specific conditional Tbx21 knockout (KO) ( Tbx21 fl/fl Cd23 Cre ) genotypes. Fresh-frozen spleen sections were profiled using Visium HD, MERSCOPE, and Xenium. Matched single-nuclei RNA seq (snRNA-seq) datasets were generated from dissociated formalin-fixed paraffin-embedded (FFPE) WT spleens. C . Sample composition of the SpatialBench dataset. Colors denote platform identity, with MERSCOPE (magenta), Xenium (blue), Visium HD (green), and snRNA-seq (yellow). Asterisk denote Xenium technical replicates with multimodal staining. D . Summary of sample number, spatial resolution, and Venn diagram displaying shared genes between panels of the ST platforms. E . Dataset-level statistics for binned ST data, with 8 µm and 16 µm bins shown in the top and bottom rows, respectively. Columns show total bins, total transcript counts, transcript counts for the subset of 90 shared genes, sparsity, and sparsity for the shared genes across platforms. Points indicate individual samples, with bars and error bars denoting the mean and standard error of the mean (SEM). F . Global Pearson correlation of gene expression between snRNA-seq and ST platforms. Density contour plots show gene-level correlations for Visium HD (green, left), MERSCOPE (magenta, middle), and Xenium (blue, right) relative to snRNA-seq. Gene counts were averaged across WT samples and log 10 -transformed counts per million (CPM). Density contours represent the distribution of genes, with increased color intensity indicating higher density. The diagonal line (slope = 1) denotes equality, and Pearson correlation coefficients (R) and the number of shared genes (n) between ST platforms and snRNA-seq are reported for each comparison. G . Computational workflow for SpatialBench , showing preprocessing, quality control, normalization, dimensionality reduction, and downstream analyses, with platforms color-coded across steps.

Article Snippet: In addition, MERSCOPE data were generated prior to Vizgen’s updated gene imaging chemistry (v2 chemistry), which was reported to substantially enhance transcript detection sensitivity ( ) .

Techniques: Spatial Transcriptomics, Sequencing, Imaging, cDNA Library Assay, In Situ, Fluorescence, Hybridization, Amplification, Infection, Control, Knock-Out, RNA Sequencing, Generated, Formalin-fixed Paraffin-Embedded, Staining, Gene Expression, Transformation Assay, Comparison

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . Matched spleen sections from wild-type (WT709, WT713) and conditional Tbx21 knockout (KO167, KO168) mice profiled using Visium HD (green), MERSCOPE (magenta), and Xenium (blue). Heatmaps display total transcript counts per 8 µm bin, scaled per platform. Scale bars, 1 mm. B . Bin-level (8 µm) evaluation of median transcript counts per bin (top) and median detected genes per bin (bottom) for the 90 commonly shared genes across Visium HD, MERSCOPE, and Xenium plaforms. Points indicate individual samples. C . Bin-level (8 µm) evaluation of median transcript counts per bin (top) and median detected genes per bin (bottom) for the 14,999 commonly shared genes between Visium HD and snRNA-seq assays. Points indicate individual samples. D . Rarefaction curves showing the expected numbers of detected genes (top) and transcripts (Unique Molecular Identifiers; UMI) (bottom) in downsampled data as a function of mapped reads. M, millions. E . Total gene targeting and negative control probe counts per sample in MERSCOPE and Xenium. Box plots show sample-level totals ( n = 9 per platform). Points indicate individual samples. F . Distributions of observed Moran’s I values for gene targeting probes and background probes (Blanks in MERSCOPE; Unassigned in Xenium) across all genes and samples, summarized by box plots. G . Background to signal ratio (BSR) of background probe detection across MERSCOPE and Xenium. Data are presented as mean ± SEM. H . Rank-ordered mean probe counts (log 10 (mean count + 1)) for MERSCOPE and Xenium across all samples. Shaded area marks the platform-specific 95th percentile of background probe counts. Highlighted points indicate gene targeting probes at or below this threshold. I . Multidimensional scaling (MDS) of pseudo-bulk expression profiles for the 90 common genes across platforms. Point color denotes platform and shape denotes sample group. J . Variance partitioning of pseudo-bulk gene expression profiles attributed to platform (75.8%), biological group (12.1%), and residual (10.7%) variation. K . Cross-platform gene-level correlations. Density contour plots show pairwise comparisons of averaged log 10 CPM expression across shared genes in matched Visium HD, MERSCOPE, and Xenium samples, with Pearson correlation coefficients (R). Points indicate individual genes. Source data are provided as a Source Data file.

Article Snippet: In addition, MERSCOPE data were generated prior to Vizgen’s updated gene imaging chemistry (v2 chemistry), which was reported to substantially enhance transcript detection sensitivity ( ) .

Techniques: Knock-Out, Negative Control, Expressing, Gene Expression

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A, B . Representative ( A ) MERSCOPE tissue section (KO166) and ( B ) Xenium tissue section (CTRL174 rep2) with two regions of interest (ROIs) enlarged to visualise cell segmentation masks. Composite intensity images of cell membrane (yellow) and DAPI (blue) staining are shown alongside the membrane channel alone (grayscale). Segmentation outputs from the vendor default, Proseg, and custom Cellpose methods are displayed (magenta filled mask with white outlines) on top of membrane-only signal. Scale bar, 1 mm (overview) and 10 µm (zoomed ROI). C, D . Box plots of sample-level total cell count, median cell area, fraction of transcripts assigned to cells, and median transcripts per cell for( C ) MERSCOPE ( n = 9) and ( D ) Xenium multimodal ( n = 5) samples for each segmentation method. Each point indicate individual sample. E . Box plots of sample-level mutually exclusive correlation rate (MECR) for each segmentation method in MERSCOPE and Xenium. Each point indicate individual sample. F . Relationship between the fraction of transcripts assigned to cells and negative marker purity across segmentation methods in MERSCOPE and Xenium. Each point represents a platform–segmentation combination, with platform indicated by shape and segmentation method by color. G . Heatmap of cell type specific negative marker purity across segmentation methods in MERSCOPE and Xenium. Exact values are displayed. H . Uniform Manifold Approximation and Projection (UMAP) of segmented single-cell expression profiles from MERSCOPE (top) and Xenium (bottom) for the vendor default (left), Proseg (middle), and custom Cellpose (right) segmentation methods, colored by annotated cell type. I . Bar plot of mean cells per sample by cell type and segmentation method in MERSCOPE and Xenium. Each point indicate individual sample. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: In addition, MERSCOPE data were generated prior to Vizgen’s updated gene imaging chemistry (v2 chemistry), which was reported to substantially enhance transcript detection sensitivity ( ) .

Techniques: Membrane, Staining, Cell Characterization, Marker, Single Cell, Expressing

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . UMAP of snRNA-seq, Visium HD (8 µm bins), and cell-level MERSCOPE and Xenium data showing major spleen cell type annotations used for downstream analyses. B . Bar plot of average silhouette width (ASW) per platform, computed from the Principal component analysis (PCA) embeddings underlying the UMAP shown in . C . Spatial maps of annotated cell types for WT713 and KO167 spleen sections across matched Visium HD (top), MERSCOPE (middle), and Xenium (bottom) samples. Each color represent a cell type. Scale bars, 1 mm. D . Bar plot of cell type proportions in WT and KO samples by platform. Visium HD includes WT and KO samples ( n = 2 each), and MERSCOPE and Xenium include WT and KO samples ( n = 3 each) (top). Data are presented as mean ± SEM. E . Box plot of extracted GC B cell proportions from 4D in WT and KO samples by platform. Each point indicate individual sample. F . Spatial maps of Tbx21 expression in WT713 and KO167 samples measured by Visium HD, MERSCOPE, and Xenium, with expression scaled within each platform. Scale bars, 1 mm. G . Bar plot of the percentage of Tbx21 -positive units in WT and KO samples across platforms, defined as 8 µm bins for Visium HD and cells for MERSCOPE and Xenium. Data are presented as mean ± SEM. H . Bar plot showing the cell type composition of Tbx21 -positive units across platforms, colored by annotated cell type as in panel 4C. I . Volcano plots of pseudo-bulk KO versus WT differential expression in näive B cells (top) and germinal center B cells (bottom) across Visium HD, MERSCOPE, and Xenium. Genes are colored by differential expression status (|log 2 FC| > 1, adjusted P < 0.05). Tbx21 is highlighted in yellow with other key expected down-regulated genes. J . Bar plot of the proportion of significantly differentially expressed genes (adjusted P < 0.05) overlapping an external Tbx21 (encodes for T-bet) knockout gene signature from Ly et al. in näive B and germinal center B cells across platforms. K . Dot plot of pseudo-bulk Tbx21 differential expression across annotated cell types for Visium HD, MERSCOPE, and Xenium. Dot color show log 2 fold change (KO versus WT), with adjusted P -values indicated by dot size and annotated significance indicated by star symbol.

Article Snippet: In addition, MERSCOPE data were generated prior to Vizgen’s updated gene imaging chemistry (v2 chemistry), which was reported to substantially enhance transcript detection sensitivity ( ) .

Techniques: Expressing, Quantitative Proteomics, Knock-Out

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . Spatial maps of annotated cell types for WT713 and KO167 spleen samples profiled by Visium HD, MERSCOPE, and Xenium. Germinal centers (GC) are delineated with dark and light zones annotated, and representative marker gene expression is shown for Bcl6 (GC), Aicda (dark zone), and Cd83 (light zone). Dotted outlines indicate GC boundaries. Color intensity indicates log-normalised expression. Scale bars, 1 mm (overview) and 200 µm (ROIs). B . Dot plots of dark zone and light zone marker gene expression across platforms. Average expression is shown by color and the percentage of expressing units is indicated by dot size. Visium HD and Xenium show expression stratified by annotated dark and light zones, while MERSCOPE shows expression within GC B cells. C . Bar plot of GC composition in WT and KO samples for Visium HD, MERSCOPE, and Xenium, showing the relative contribution of dark zone, light zone, and unassigned GC regions. Bars represent mean proportions across samples, with colors indicating GC sub-compartments. D-E . Percentage change in ( D ) GC density and ( E ) GC diameter in KO relative to WT samples across platforms. F . Venn diagram showing the overlap of differentially expressed genes (adjusted P < 0.05) between dark and light zones in WT samples and a published GC zone gene set from Victora et al. . G . Volcano plot of pseudo-bulk differential expression between dark and light zones in WT samples profiled with Visium HD. Genes are colored by differential expression status using | log 2 FC| > 1 and adjusted P < 0.05. H .Gene Ontology (GO) biological process enrichment for genes differentially expressed between dark and light zones in WT samples. Bar plots show significantly enriched GO terms for dark zone (top) and light zone (bottom) gene sets, ranked by enrichment significance (log 10 P ). Representative contributing genes are shown for each term. Source data are provided as a Source Data file.

Article Snippet: In addition, MERSCOPE data were generated prior to Vizgen’s updated gene imaging chemistry (v2 chemistry), which was reported to substantially enhance transcript detection sensitivity ( ) .

Techniques: Marker, Gene Expression, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . Schematic overview of the three spatial transcriptomics (ST) platforms used in this study, comprising sequencing-based (sST) and imaging-based (iST) approaches. Visium HD (10x Genomics) is a sST platform that captures spatially barcoded polyadenylated transcripts, followed by cDNA library preparation and sequencing. MERSCOPE (Vizgen) and Xenium (10x Genomics) are iST platforms for in situ transcript detection using multiplexed fluorescence imaging, with MERSCOPE relying on direct fluorescent probe hybridization and Xenium on padlock probes with rolling circle amplification, followed by iterative imaging, signal removal, and rehybridization cycles for transcript decoding. B . Experimental design of the SpatialBench study using spleen sections from malaria infected and cured mice across wild-type (WT), control (CTRL) ( Tbx21 +/+ Cd23 Cre ), and B cell-specific conditional Tbx21 knockout (KO) ( Tbx21 fl/fl Cd23 Cre ) genotypes. Fresh-frozen spleen sections were profiled using Visium HD, MERSCOPE, and Xenium. Matched single-nuclei RNA seq (snRNA-seq) datasets were generated from dissociated formalin-fixed paraffin-embedded (FFPE) WT spleens. C . Sample composition of the SpatialBench dataset. Colors denote platform identity, with MERSCOPE (magenta), Xenium (blue), Visium HD (green), and snRNA-seq (yellow). Asterisk denote Xenium technical replicates with multimodal staining. D . Summary of sample number, spatial resolution, and Venn diagram displaying shared genes between panels of the ST platforms. E . Dataset-level statistics for binned ST data, with 8 µm and 16 µm bins shown in the top and bottom rows, respectively. Columns show total bins, total transcript counts, transcript counts for the subset of 90 shared genes, sparsity, and sparsity for the shared genes across platforms. Points indicate individual samples, with bars and error bars denoting the mean and standard error of the mean (SEM). F . Global Pearson correlation of gene expression between snRNA-seq and ST platforms. Density contour plots show gene-level correlations for Visium HD (green, left), MERSCOPE (magenta, middle), and Xenium (blue, right) relative to snRNA-seq. Gene counts were averaged across WT samples and log 10 -transformed counts per million (CPM). Density contours represent the distribution of genes, with increased color intensity indicating higher density. The diagonal line (slope = 1) denotes equality, and Pearson correlation coefficients (R) and the number of shared genes (n) between ST platforms and snRNA-seq are reported for each comparison. G . Computational workflow for SpatialBench , showing preprocessing, quality control, normalization, dimensionality reduction, and downstream analyses, with platforms color-coded across steps.

Article Snippet: The MERFISH imaging process was performed according to the MERSCOPE Instrument User Guide (Vizgen, number 91600001).

Techniques: Spatial Transcriptomics, Sequencing, Imaging, cDNA Library Assay, In Situ, Fluorescence, Hybridization, Amplification, Infection, Control, Knock-Out, RNA Sequencing, Generated, Formalin-fixed Paraffin-Embedded, Staining, Gene Expression, Transformation Assay, Comparison

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . Matched spleen sections from wild-type (WT709, WT713) and conditional Tbx21 knockout (KO167, KO168) mice profiled using Visium HD (green), MERSCOPE (magenta), and Xenium (blue). Heatmaps display total transcript counts per 8 µm bin, scaled per platform. Scale bars, 1 mm. B . Bin-level (8 µm) evaluation of median transcript counts per bin (top) and median detected genes per bin (bottom) for the 90 commonly shared genes across Visium HD, MERSCOPE, and Xenium plaforms. Points indicate individual samples. C . Bin-level (8 µm) evaluation of median transcript counts per bin (top) and median detected genes per bin (bottom) for the 14,999 commonly shared genes between Visium HD and snRNA-seq assays. Points indicate individual samples. D . Rarefaction curves showing the expected numbers of detected genes (top) and transcripts (Unique Molecular Identifiers; UMI) (bottom) in downsampled data as a function of mapped reads. M, millions. E . Total gene targeting and negative control probe counts per sample in MERSCOPE and Xenium. Box plots show sample-level totals ( n = 9 per platform). Points indicate individual samples. F . Distributions of observed Moran’s I values for gene targeting probes and background probes (Blanks in MERSCOPE; Unassigned in Xenium) across all genes and samples, summarized by box plots. G . Background to signal ratio (BSR) of background probe detection across MERSCOPE and Xenium. Data are presented as mean ± SEM. H . Rank-ordered mean probe counts (log 10 (mean count + 1)) for MERSCOPE and Xenium across all samples. Shaded area marks the platform-specific 95th percentile of background probe counts. Highlighted points indicate gene targeting probes at or below this threshold. I . Multidimensional scaling (MDS) of pseudo-bulk expression profiles for the 90 common genes across platforms. Point color denotes platform and shape denotes sample group. J . Variance partitioning of pseudo-bulk gene expression profiles attributed to platform (75.8%), biological group (12.1%), and residual (10.7%) variation. K . Cross-platform gene-level correlations. Density contour plots show pairwise comparisons of averaged log 10 CPM expression across shared genes in matched Visium HD, MERSCOPE, and Xenium samples, with Pearson correlation coefficients (R). Points indicate individual genes. Source data are provided as a Source Data file.

Article Snippet: The MERFISH imaging process was performed according to the MERSCOPE Instrument User Guide (Vizgen, number 91600001).

Techniques: Knock-Out, Negative Control, Expressing, Gene Expression

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A, B . Representative ( A ) MERSCOPE tissue section (KO166) and ( B ) Xenium tissue section (CTRL174 rep2) with two regions of interest (ROIs) enlarged to visualise cell segmentation masks. Composite intensity images of cell membrane (yellow) and DAPI (blue) staining are shown alongside the membrane channel alone (grayscale). Segmentation outputs from the vendor default, Proseg, and custom Cellpose methods are displayed (magenta filled mask with white outlines) on top of membrane-only signal. Scale bar, 1 mm (overview) and 10 µm (zoomed ROI). C, D . Box plots of sample-level total cell count, median cell area, fraction of transcripts assigned to cells, and median transcripts per cell for( C ) MERSCOPE ( n = 9) and ( D ) Xenium multimodal ( n = 5) samples for each segmentation method. Each point indicate individual sample. E . Box plots of sample-level mutually exclusive correlation rate (MECR) for each segmentation method in MERSCOPE and Xenium. Each point indicate individual sample. F . Relationship between the fraction of transcripts assigned to cells and negative marker purity across segmentation methods in MERSCOPE and Xenium. Each point represents a platform–segmentation combination, with platform indicated by shape and segmentation method by color. G . Heatmap of cell type specific negative marker purity across segmentation methods in MERSCOPE and Xenium. Exact values are displayed. H . Uniform Manifold Approximation and Projection (UMAP) of segmented single-cell expression profiles from MERSCOPE (top) and Xenium (bottom) for the vendor default (left), Proseg (middle), and custom Cellpose (right) segmentation methods, colored by annotated cell type. I . Bar plot of mean cells per sample by cell type and segmentation method in MERSCOPE and Xenium. Each point indicate individual sample. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: The MERFISH imaging process was performed according to the MERSCOPE Instrument User Guide (Vizgen, number 91600001).

Techniques: Membrane, Staining, Cell Characterization, Marker, Single Cell, Expressing

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . UMAP of snRNA-seq, Visium HD (8 µm bins), and cell-level MERSCOPE and Xenium data showing major spleen cell type annotations used for downstream analyses. B . Bar plot of average silhouette width (ASW) per platform, computed from the Principal component analysis (PCA) embeddings underlying the UMAP shown in . C . Spatial maps of annotated cell types for WT713 and KO167 spleen sections across matched Visium HD (top), MERSCOPE (middle), and Xenium (bottom) samples. Each color represent a cell type. Scale bars, 1 mm. D . Bar plot of cell type proportions in WT and KO samples by platform. Visium HD includes WT and KO samples ( n = 2 each), and MERSCOPE and Xenium include WT and KO samples ( n = 3 each) (top). Data are presented as mean ± SEM. E . Box plot of extracted GC B cell proportions from 4D in WT and KO samples by platform. Each point indicate individual sample. F . Spatial maps of Tbx21 expression in WT713 and KO167 samples measured by Visium HD, MERSCOPE, and Xenium, with expression scaled within each platform. Scale bars, 1 mm. G . Bar plot of the percentage of Tbx21 -positive units in WT and KO samples across platforms, defined as 8 µm bins for Visium HD and cells for MERSCOPE and Xenium. Data are presented as mean ± SEM. H . Bar plot showing the cell type composition of Tbx21 -positive units across platforms, colored by annotated cell type as in panel 4C. I . Volcano plots of pseudo-bulk KO versus WT differential expression in näive B cells (top) and germinal center B cells (bottom) across Visium HD, MERSCOPE, and Xenium. Genes are colored by differential expression status (|log 2 FC| > 1, adjusted P < 0.05). Tbx21 is highlighted in yellow with other key expected down-regulated genes. J . Bar plot of the proportion of significantly differentially expressed genes (adjusted P < 0.05) overlapping an external Tbx21 (encodes for T-bet) knockout gene signature from Ly et al. in näive B and germinal center B cells across platforms. K . Dot plot of pseudo-bulk Tbx21 differential expression across annotated cell types for Visium HD, MERSCOPE, and Xenium. Dot color show log 2 fold change (KO versus WT), with adjusted P -values indicated by dot size and annotated significance indicated by star symbol.

Article Snippet: The MERFISH imaging process was performed according to the MERSCOPE Instrument User Guide (Vizgen, number 91600001).

Techniques: Expressing, Quantitative Proteomics, Knock-Out

Journal: bioRxiv

Article Title: SpatialBench : Comparative cross-platform benchmarking of high-resolution spatial transcriptomics using matched mouse lymphoid tissue

doi: 10.64898/2026.04.29.721531

Figure Lengend Snippet: A . Spatial maps of annotated cell types for WT713 and KO167 spleen samples profiled by Visium HD, MERSCOPE, and Xenium. Germinal centers (GC) are delineated with dark and light zones annotated, and representative marker gene expression is shown for Bcl6 (GC), Aicda (dark zone), and Cd83 (light zone). Dotted outlines indicate GC boundaries. Color intensity indicates log-normalised expression. Scale bars, 1 mm (overview) and 200 µm (ROIs). B . Dot plots of dark zone and light zone marker gene expression across platforms. Average expression is shown by color and the percentage of expressing units is indicated by dot size. Visium HD and Xenium show expression stratified by annotated dark and light zones, while MERSCOPE shows expression within GC B cells. C . Bar plot of GC composition in WT and KO samples for Visium HD, MERSCOPE, and Xenium, showing the relative contribution of dark zone, light zone, and unassigned GC regions. Bars represent mean proportions across samples, with colors indicating GC sub-compartments. D-E . Percentage change in ( D ) GC density and ( E ) GC diameter in KO relative to WT samples across platforms. F . Venn diagram showing the overlap of differentially expressed genes (adjusted P < 0.05) between dark and light zones in WT samples and a published GC zone gene set from Victora et al. . G . Volcano plot of pseudo-bulk differential expression between dark and light zones in WT samples profiled with Visium HD. Genes are colored by differential expression status using | log 2 FC| > 1 and adjusted P < 0.05. H .Gene Ontology (GO) biological process enrichment for genes differentially expressed between dark and light zones in WT samples. Bar plots show significantly enriched GO terms for dark zone (top) and light zone (bottom) gene sets, ranked by enrichment significance (log 10 P ). Representative contributing genes are shown for each term. Source data are provided as a Source Data file.

Article Snippet: The MERFISH imaging process was performed according to the MERSCOPE Instrument User Guide (Vizgen, number 91600001).

Techniques: Marker, Gene Expression, Expressing, Quantitative Proteomics