10x Visium HD Spatial Transcriptomics Service
CD Genomics provides end-to-end 10x Visium HD spatial transcriptomics services for FFPE, fresh frozen, and fixed frozen tissue. We support both probe-based Visium HD Spatial Gene Expression and poly(A)-based Visium HD 3′ Spatial Gene Expression, with workflow selection based on sample preservation, RNA condition, species, and study objectives.
Both workflows use a continuous 2 μm × 2 μm spatial grid to support single-cell-scale transcriptomic analysis while preserving tissue morphology.
- Continuous 2 μm × 2 μm barcoded grid for single-cell-scale spatial profiling
- Probe-based Visium HD for FFPE, fresh frozen, and fixed frozen tissue
- Visium HD 3′ for unbiased poly(A)-based profiling of fresh frozen tissue
- Integrated tissue imaging, library construction, sequencing, Space Ranger processing, and downstream spatial analysis
Two Visium HD Workflows for Different Sample Types
Visium HD includes two spatial gene expression workflows with different transcript-capture chemistries. The probe-based assay uses paired probes that hybridize to target transcripts and are subsequently ligated, while Visium HD 3′ captures polyadenylated RNA from fresh frozen tissue without requiring a predefined gene probe panel.
Both workflows use the continuous 2 μm Visium HD spatial grid, but they differ in sample compatibility, imaging options, and molecular capture strategy.
Visium HD Probe-Based Spatial Gene Expression
A whole-transcriptome probe-based workflow suitable for FFPE, fresh frozen, and fixed frozen tissue. It is particularly useful when sample fixation, archival storage, or RNA fragmentation makes direct poly(A)-based capture less suitable.
Recommended for:
- FFPE tissue collections
- Fixed frozen tissue
- Fresh frozen tissue requiring a probe-based workflow
- Cohort studies involving archived or differently preserved specimens
Visium HD 3′ Spatial Gene Expression
An unbiased, poly(A)-based whole-transcriptome workflow designed for fresh frozen tissue. It captures 3′ gene expression without relying on a predefined transcript probe panel.
Recommended for:
- High-quality fresh frozen tissue
- Discovery-focused whole-transcriptome studies
- Projects requiring poly(A)-based transcript capture
- Studies where FFPE compatibility is not required
Workflow selection is finalized after review of tissue preservation, species, RNA quality, section dimensions, imaging requirements, and study objectives.
How to Choose Between Visium HD Workflows
| Parameter | Visium HD Probe-Based | Visium HD 3′ |
|---|---|---|
| Sample | FFPE, fresh frozen, and fixed frozen tissue | Fresh frozen tissue |
| Capture | Paired-probe hybridization and ligation | Poly(A)-based 3′ transcript capture |
| Coverage | Whole-transcriptome probe set; 6.5 mm × 6.5 mm or 11 mm × 11 mm capture area | Unbiased whole-transcriptome profiling; 6.5 mm × 6.5 mm capture area |
| Best Fit | Fixed, archived, or mixed-preservation tissue cohorts | Discovery-focused studies using high-quality fresh frozen tissue |
Single-Cell-Scale Spatial Resolution with Visium HD
Earlier Visium workflows use 55 μm capture spots, with each spot typically containing transcripts from multiple neighboring cells. Visium HD replaces this spot-based format with a continuous array of 2 μm × 2 μm barcoded squares, providing substantially finer spatial sampling across the tissue section.
Because a mammalian cell generally spans multiple 2 μm squares, Visium HD data are initially generated as spatial bins rather than as directly isolated individual cells. Raw 2 μm bins can be analyzed individually, aggregated into larger bins, or combined with tissue imaging and segmentation algorithms to derive cell-scale expression profiles.
Both probe-based Visium HD and Visium HD 3′ use this high-density spatial grid. Their primary difference lies in transcript-capture chemistry and sample compatibility rather than spatial array resolution.
Diverse Applications in Translational Research
The continuous 2 μm spatial grid supports detailed analysis of tissue architecture, cellular neighborhoods, transcriptional boundaries, and localized biological responses across diverse research areas.
Tumor Microenvironment (TME) Mapping
In oncology, evaluating the exact spatial distribution of tumor-infiltrating lymphocytes (TILs) relative to the tumor boundary is critical. Visium HD allows for the precise mapping of immune exclusion zones, identifying where cytotoxic T cells are physically halted by physical or chemical barriers in the stroma. The high-density spatial grid can improve the delineation of narrow invasive fronts, microvascular structures, and localized immune-cell distributions that may be obscured in lower-resolution spot-based data.
Developmental Biology & Lineage Tracing
Understanding embryogenesis and organogenesis requires tracking gene expression across highly stratified, developing tissues. Visium HD enables the assignment of localized transcriptomic profiles to distinct developmental layers. Researchers can map morphogen gradients without physical microdissection, preserving the delicate native architecture required to understand tissue maturation.
Neurobiology and Pathology
The mammalian cortex and specific brain structures operate based on highly organized, layered architectures. The continuous 2 μm grid allows researchers to map pathological markers—such as localized microglial activation or astrogliosis—directly adjacent to microscopic amyloid-beta plaques or tau tangles in neurodegenerative disease models, isolating the exact transcriptomic response of the immediate cellular niche.
10x Visium HD Service Workflow
Working with archival clinical samples requires a specialized approach. Standardizing the transfer of RNA from glass slides to spatial arrays minimizes tissue handling errors. Our laboratory utilizes the 10x Genomics CytAssist instrument to facilitate a highly controlled, optimized workflow for FFPE samples.
End-to-end Visium HD workflow featuring CytAssist integration.
- Project Design and Workflow Selection
We review the sample type, tissue dimensions, RNA quality, species, and study objectives to select the appropriate Visium HD workflow. - Tissue Sectioning and Imaging
Tissue sections are prepared, stained, and imaged using conditions appropriate for the selected sample type and workflow. - Spatial Transcript Capture
Transcript signals are captured using either the probe-based Visium HD assay or the poly(A)-based Visium HD 3′ assay. - CytAssist Processing
The tissue slide is processed with the CytAssist instrument to transfer spatially indexed material to the Visium HD capture slide. - Library Preparation and Sequencing
Spatial libraries undergo quality control and high-throughput sequencing at a depth selected for the tissue and study design. - Spatial Data Analysis
Sequencing data are processed to generate tissue-aligned expression matrices, spatial clusters, cell-type maps, and project-specific downstream analyses.
Visium HD Sample Submission Guidelines
Sample requirements depend on tissue preservation and the selected Visium HD workflow. The following criteria are general project-planning guidelines. Final acceptance is determined after sample-specific feasibility review.
| Sample Type | Recommended Workflow | Capture Area | General Requirements |
|---|---|---|---|
| FFPE tissue | Visium HD Probe-Based | 6.5 mm × 6.5 mm or 11 mm × 11 mm | 5 μm sections; DV200 ≥30% recommended |
| Fresh frozen, OCT-embedded tissue | Visium HD Probe-Based | 6.5 mm × 6.5 mm or 11 mm × 11 mm | RNA quality assessment required; section thickness confirmed according to tissue type |
| Fresh frozen, OCT-embedded tissue | Visium HD 3′ | 6.5 mm × 6.5 mm | 10 μm sections recommended; RIN >7 preferred |
| Fixed frozen tissue | Visium HD Probe-Based | 6.5 mm × 6.5 mm or 11 mm × 11 mm | Fixation information and RNA quality assessment required |
Important Considerations
- Tissue sections must fit within the selected capture area.
- Please provide the tissue dimensions, species, preservation method, and available RNA quality results before sample submission.
- Final sample acceptance is based on tissue morphology, RNA quality, section integrity, and project objectives.
For additional sample handling information, see our FFPE Spatial Transcriptomics Service and Spatial Transcriptome Sequencing of Frozen Samples pages.
Advanced Bioinformatics: From 2 μm Bins to Cell Segmentation
Generating massive amounts of sequencing data is only the first phase. The continuous 2 μm grid generates an unprecedented volume of data points per sample. Processing massive 2 μm spatial matrices requires immense high-performance computing infrastructure, which our analytical team fully provides, ensuring your data is translated into actionable biological insights.
ioinformatics pipeline for processing high-density 2 μm spatial data.
Standard Space Ranger Processing
Our baseline data processing utilizes the latest 10x Genomics analytical pipelines.
- Sequence Alignment: Mapping reads to the reference genome and assigning them to specific spatial barcodes.
- Bin Aggregation: Space Ranger outputs count matrices at the raw 2 μm × 2 μm level, while also generating aggregated 8µm × 8µm bins to help users balance spatial resolution with increased transcript counts per bin for initial exploratory analysis.
- Basic Clustering: Unsupervised clustering of the aggregated spatial bins, overlaid directly onto your H&E image to define broad anatomical domains.
High-Resolution Spatial Data Mining (Segmentation)
To achieve true biological meaning, transcriptomic data must be mapped to distinct cells, not just physical squares.
- Cellular Segmentation: Using the high-resolution H&E image, advanced algorithms identify nuclear boundaries and draw polygonal boundaries around individual cells. The 2 μm bins falling within these boundaries are aggregated, allowing us to generate a highly accurate, single-cell scale gene expression matrix.
- Spatial Trajectory Inference: Reconstructing pseudotime trajectories to trace cellular development or functional state transitions across the physical tissue space.
- Ligand-Receptor Networks: Inferring active communication pathways specifically between physically adjacent segmented cells, providing strong evidence for localized cellular interactions.
Demo Results: Unprecedented Detail in the TME
Our standard deliverables are designed to support immediate biological interpretation and grant applications. The visual outputs clearly communicate the value of the 2 μm continuous resolution.
High-resolution spatial clustering and cell abundance mapping.
- Continuous Spatial Clustering: High-resolution UMAP clusters perfectly aligned to fine morphological structures via the 2 μm/8µm bin arrays, separating distinct histological layers.
- Inferred Cell Segmentation Maps: Polygonal boundaries identifying individual cells drawn directly over the tissue image, mapped with their corresponding transcriptomic identities.
- High-Definition Tumor Boundaries: Spatial heatmaps providing a sharp interface mapping of immune infiltration precisely at the tumor margin.
- Spatially Resolved Differential Gene Expression: Volcano plots paired with spatial distribution maps that define specific micro-niches.
- Ligand-Receptor Networks: Interaction diagrams mapped over physical tissue architecture showing exactly where communication is occurring.
Platform Selection: Visium HD vs. Visium V1 vs. Xenium
Selecting the correct spatial platform dictates the type of biological questions you can answer. Use the following guide to navigate your technology selection.
| Parameter | 10x Visium V1 | 10x Visium HD | Xenium In Situ |
|---|---|---|---|
| Resolution Grid | 55µm spots (100µm pitch) | 2 μm × 2 μm continuous | Subcellular imaging |
| Transcriptome Coverage | Whole-transcriptome probe set or unbiased poly(A)-based whole-transcriptome profiling, depending on workflow | Whole-transcriptome probe set or unbiased poly(A)-based whole-transcriptome profiling, depending on workflow | Targeted Panel (Hundreds of Genes) |
| Tissue Coverage | 6.5mm × 6.5mm (with gaps) | 6.5 mm × 6.5 mm or 11 mm × 11 mm for probe-based Visium HD; 6.5 mm × 6.5 mm for Visium HD 3′ | ~12mm × 24mm |
| Sample Processing | Direct on slide (Fresh Frozen/FFPE) | CytAssist workflow for FFPE, fresh frozen, or fixed frozen tissue | In Situ Hybridization |
Strategic Platform Selection:
- Choose probe-based Visium HD for FFPE, fresh frozen, or fixed frozen tissue. Choose Visium HD 3′ when fresh frozen tissue is available and unbiased poly(A)-based whole-transcriptome profiling is preferred.
- Choose Xenium In Situ if you require absolute subcellular imaging resolution and already have a targeted panel of known genes you wish to validate.
- Choose Visium V1 if ultra-high resolution is not biologically required and you need broad anatomical mapping of fresh frozen samples.
Case Study: Cell-Level Reconstruction in Lung Cancer TME
Source: STCS: A Platform-Agnostic Framework for Cell-Level Reconstruction in Sequencing-Based Spatial Transcriptomics (bioRxiv, 2026)
High-resolution Visium HD spatial mapping of a human lung cancer FFPE section, demonstrating cell-level segmentation.
Background
Translating the massive volume of 2 μm continuous bin data generated by Visium HD into biologically meaningful single-cell information is a major computational challenge. Researchers needed to accurately reconstruct individual cells within the complex tumor microenvironment (TME) of human lung cancer to study localized immune interactions.
Methods
The study utilized a human lung cancer FFPE dataset generated via the 10x Visium HD platform. To process the 2 μm spatial grid, the team applied an advanced computational framework designed for cell-level reconstruction. This approach integrated the high-resolution H&E morphological image with the continuous transcriptomic bin data to accurately delineate true cellular boundaries.
Results
The advanced segmentation successfully reconstructed single-cell spatial profiles from the Visium HD grid. This cell-level resolution clearly identified distinct tumor and immune cell populations, revealing intricate spatial arrangements and sharp tumor boundaries that standard bin-level aggregation might obscure.
Conclusion
Coupling Visium HD's 2 μm continuous capture technology with robust cell segmentation algorithms enables true single-cell spatial transcriptomics. This validates the platform's capability to deeply profile the TME and identify precise, localized therapeutic targets in archival FFPE tissues.
Frequently Asked Questions (FAQs)
References
- High-definition spatial transcriptomic profiling of immune cell populations in colorectal cancer (PMC, 2025)
- Visium Sample Submission Guidelines | GENEWIZ from Azenta
- Getting Started with Visium HD Spatial Transcriptomics | Omics Empower
- Bin2cell reconstructs cells from high resolution Visium HD data (Oxford Academic, 2024)
- STCS: A Platform-Agnostic Framework for Cell-Level Reconstruction in Sequencing-Based Spatial Transcriptomics (bioRxiv, 2026)