Sample Submission Guidelines
What Is 10x Visium HD
Visium HD is 10x Genomics' high-definition upgrade to the Visium Spatial Gene Expression platform, commercially launched in March 2024. While the original Visium FF method provides ~5,000 barcoded spots of 55 µm diameter with gaps between them, Visium HD replaces the entire slide architecture with over 11 million continuous 2 µm features arranged in an uninterrupted array — covering every square micron of the 6.5 × 6.5 mm capture area without gaps. Every cellular and subcellular position in the tissue section is barcoded and sequenced.
Rather than poly(A) capture, Visium HD uses a probe hybridization and ligation chemistry: whole-transcriptome probe sets (targeting approximately 18,000 human or 20,000 mouse genes, with approximately 1–3 probes per gene) are hybridized directly to the tissue section and then ligated to the 2 µm spatial barcodes on the Visium HD slide via the CytAssist instrument. CytAssist facilitates the precise transfer of probe–analyte complexes from standard glass tissue slides to the Visium HD capture slide, enabling the use of tissues stained and processed on conventional histology slides — including FFPE, fresh frozen, and fixed frozen samples.
After sequencing, Space Ranger HD processes the data into count matrices at three analysis resolutions: 2 µm (maximum resolution), 8 µm (near-single-cell), and 16 µm (cluster-level). Analysis is performed on 8 µm bins as the default for cell-type-level spatial mapping — roughly equivalent to the average mammalian cell diameter. Nuclei segmentation using DAPI staining can further assign expression to individual segmented nuclei, approaching true single-cell spatial resolution. Compared to standard Visium, Visium HD detects a substantially higher number of transcriptionally distinct spatial clusters in the same tissue, resolving cellular neighborhoods and cell-type boundaries invisible to multicellular spot-based approaches.
Visium HD vs. Visium FF vs. Standard Visium
Visium HD builds on the Visium platform with a fundamentally different slide architecture and chemistry, delivering single-cell-scale resolution and FFPE compatibility — at the cost of a species-specific validated probe panel requirement.
| Feature | Standard Visium (v1) | Visium FF (Direct Placement) | Visium HD (This Service) |
|---|---|---|---|
| Feature size | 55 µm spots | 55 µm spots | 2 µm continuous features |
| Feature count per 6.5 mm area | ~5,000 spots (gaps between) | ~5,000 spots (gaps between) | 11,000,000+ (no gaps) |
| Cell coverage | 1–10 cells/spot | 1–10 cells/spot | Single-cell scale (8 µm bins) |
| Capture chemistry | Poly(A) (FF) or probe-based (FFPE) | Poly(A) — whole-transcriptome | Probe-based — whole-transcriptome panel |
| CytAssist required | Optional (CytAssist version) / No (direct placement) | No (direct placement on FF slide) | Yes — required for probe transfer |
| Sample compatibility | FF (direct) or FFPE (CytAssist probe) | Fresh frozen only | FFPE, fresh frozen, fixed frozen |
| Species compatibility | Any (FF) or human/mouse (FFPE probe) | Any with reference genome | Human, mouse (validated panels) |
| Spatial clusters detected (colon tissue) | ~3 clusters | ~3 clusters | ~18 clusters (6× more) |
| Analysis output resolutions | Spot-level only | Spot-level only | 2 µm / 8 µm / 16 µm bins |
| Best use case | Broad spatial discovery | Non-model organisms, discovery | Single-cell-scale atlases, FFPE cohorts, high-resolution TME |
Service Workflow
Visium HD projects follow a CytAssist-enabled workflow applicable to FFPE, fresh frozen, and fixed frozen tissue — all processing steps are performed in-house under validated conditions.
Step 1 — Sample QC & Tissue Sectioning: FFPE blocks are assessed for RNA quality using DV200 (≥30% required) extracted from a representative section. Fresh frozen blocks undergo RIN scoring (≥7 recommended). Tissue is sectioned to 5 µm (FFPE standard) or 10 µm (fresh frozen / fixed frozen) on conventional glass slides. Section morphology is inspected before proceeding. A pre-library QC report is issued with go/no-go recommendation per sample.
Step 2 — H&E Staining & High-Resolution Imaging: Mounted sections are stained with Hematoxylin and Eosin (H&E) or optionally with immunofluorescence (IF) if DAPI-based nuclei segmentation is required for sub-bin cell-level analysis. Sections are imaged at high resolution on a brightfield or fluorescence microscope. The resulting images are used by Space Ranger HD for precise spatial barcode registration and — if nuclei segmentation is applied — for cell boundary definition.
Step 3 — Probe Hybridization & CytAssist Transfer: Whole-transcriptome probe sets (human or mouse) are hybridized directly to the tissue section on the glass slide. Unhybridized probes are washed away, and a ligation step covalently joins hybridized probes to form capture-ready molecules. The CytAssist instrument then transfers probe–analyte complexes from the standard glass slide to the Visium HD capture slide with high spatial precision — a step that also images the tissue for co-registration.
Step 4 — Library Preparation & Illumina Sequencing: Transferred probe–analyte complexes are amplified and converted to Illumina-compatible sequencing libraries. Library quality is assessed by Bioanalyzer and Qubit before sequencing on NovaSeq. Recommended sequencing depth is approximately 25,000–50,000 reads per 8 µm bin, corresponding to approximately 1–2 billion reads per 6.5 mm capture area depending on tissue coverage.
Step 5 — Space Ranger HD Processing & Bioinformatics: Raw FASTQ files are processed through the Space Ranger HD pipeline, generating count matrices at 2 µm, 8 µm, and 16 µm bin resolutions, tissue-registered position tables, and H&E-aligned cloupe files for Loupe Browser. Downstream analysis uses Seurat or Squidpy at the 8 µm bin level as standard. Nuclei segmentation (Cellpose or StarDist) can assign expression to individual cells, enabling cell-type-resolved spatial mapping without deconvolution.
Key Applications
Visium HD is optimized for projects where single-cell-scale spatial resolution is required and FFPE clinical archives are the primary sample source — contexts where Visium FF's multicellular spot resolution is insufficient.
High-Resolution Tumor Microenvironment Profiling
Visium HD's single-cell-scale bins resolve spatially distinct immune cell subpopulations — macrophage subtypes, T cell states, natural killer cells — within the tumor microenvironment at their precise anatomical positions. Differential gene expression between spatially adjacent cells (tumor vs. stromal vs. immune) is captured at a resolution that reveals cellular interactions invisible to multicellular spot-based approaches. Our bioinformatics team applies spatial niche analysis to map functional immune cell gradients across the TME.
FFPE Clinical Biobank Mining
Visium HD's probe-based chemistry and CytAssist instrument enable high-quality spatial transcriptomics from FFPE tissue — the standard format for decades of archived clinical specimens. Researchers with existing biobanks can apply Visium HD to retrospective cohort studies, linking spatial gene expression maps to clinical outcomes, treatment responses, and pathological diagnoses without requiring fresh tissue collection. DV200 ≥ 30% is the minimum quality threshold for FFPE samples.
Single-Cell-Scale Spatial Atlases
The 2 µm feature size of Visium HD makes it possible to generate spatial atlases of cell-type distribution, gene expression variability, and spatial trajectory at near-single-cell resolution across complete tissue sections. When combined with nuclei segmentation, cell-level gene expression can be extracted directly — enabling spatially resolved analyses equivalent to dissociated single-cell RNA sequencing but with native tissue context preserved.
Brain Laminar Architecture & Neural Cell Mapping
The brain's densely packed, functionally heterogeneous cytoarchitecture has always challenged multicellular spot-based spatial methods. Visium HD resolves cortical layer-specific transcriptional identities at the single-cell level — identifying spatially constrained neuronal subtypes, glial cell positions, and cell-to-cell communication patterns in mouse and human brain tissue from both fresh frozen and FFPE preservation formats.
Spatial Validation & Xenium Integration
Visium HD is the natural companion for 10x Xenium In Situ validation studies. Discovery findings from whole-transcriptome Visium HD are validated at subcellular resolution by Xenium targeted in situ panels on adjacent sections — a workflow that combines unbiased discovery with single-molecule confirmation. For broader context on our spatial multi-omics portfolio, see our spatial multi-omics services overview.
Sample Requirements
Visium HD accepts three tissue preservation formats via CytAssist. Sample quality assessment is performed on every submission before library preparation begins. Contact us before collection for tissue-type-specific handling guidance.
| Sample Format | Section Thickness | Quality Requirement | Shipping Condition | Notes |
|---|---|---|---|---|
| FFPE tissue block | 5 µm (recommended) | DV200 ≥ 30% (minimum); ≥ 50% preferred | Room temperature (FFPE blocks stable); sections on glass slides at RT | Deparaffinization and decrosslinking performed in-house; FFPE sections must be cut fresh before submission |
| Fresh frozen OCT block | 10 µm | RIN ≥ 7 recommended; ≥ 6 minimum | Dry ice (blocks); cryo-shipment for sections | Same CytAssist workflow as FFPE after rehydration and staining; submit blocks, not pre-cut sections |
| Fixed frozen tissue | 10–20 µm | Consult our team for quality metrics specific to fixation method | Dry ice | Compatible with certain fixatives (e.g., methanol, PFA); confirm fixation protocol with our team before collection |
- Tissue size: Tissue sections must fit within the 6.5 × 6.5 mm (standard) or 11 × 11 mm (large format, requires CytAssist firmware 2.4.0) capture area. Two tissue sections can be processed per standard Visium HD slide.
- Species: Validated probe panels available for human (approximately 18,000 genes) and mouse (approximately 20,000 genes). Contact us for custom probe design or non-validated species enquiries.
- Section evaluation: We assess tissue morphology and perform DV200 or RIN measurement from an adjacent section before proceeding. Failed QC samples are reported with detailed findings before any billable work begins.
Bioinformatics Analysis & Deliverables
Our Visium HD bioinformatics pipeline leverages Space Ranger HD's multi-resolution output alongside established spatial analysis tools optimized for high-feature-density data. All deliverables are formatted for direct publication use and interactive exploration.
- Raw Data & Space Ranger HD: FASTQ files; Space Ranger HD output matrices at 2 µm, 8 µm, and 16 µm bin resolutions; tissue position files; H&E-registered cloupe file for Loupe Browser.
- QC Report: Per-section metrics — median genes per bin (at 8 µm), median molecular barcodes per bin, fraction bins under tissue, sequencing saturation, mapping rate, and sensitivity comparison to Visium v1 if applicable.
- Multi-Resolution Spatial Clustering: Unsupervised clustering of 8 µm bins using Seurat (SCTransform normalization, graph-based clustering) with spatial maps overlaid on H&E at multiple zoom levels. Comparison of cluster resolution vs. standard Visium spot-level analysis.
- Cell Type Deconvolution: Proportional cell type predictions per 8 µm bin using RCTD or cell2location, using a matched scRNA-seq reference provided by the client or derived from public atlases. Spatial cell type maps overlaid on H&E image.
- Nuclei Segmentation (optional): When DAPI-stained sections are available, Cellpose or StarDist segmentation assigns expression from 2 µm bins to individual nuclei — enabling cell-level, rather than bin-level, spatial expression analysis.
- Spatially Variable Genes & Differential Expression: SpatialDE or Seurat-based identification of genes with significant spatial patterns. Differential expression analysis between pathologist-defined tissue regions or computationally identified spatial clusters.
Multi-sample integration, ligand-receptor interaction analysis, and pathway enrichment spatial mapping are available as extended options. All visualization files are delivered in both publication-ready PDF/PNG and interactive Loupe Browser format.
References
- Oliveira MF, Romero JP, Chung M, et al. High-definition spatial transcriptomic profiling of immune cell populations in colorectal cancer. Nat Genet. 2025;57:1512–1523. https://doi.org/10.1038/s41588-025-02193-3
- Ren P, Sheng W, Peng X, et al. Systematic benchmarking of high-throughput subcellular spatial transcriptomics platforms across human tumors. Nat Commun. 2025;16:9649. https://doi.org/10.1038/s41467-025-64292-3
- Kleshchevnikov V, Shmatko A, Dann E, et al. Cell2location maps fine-grained cell types in spatial transcriptomics. Nat Biotechnol. 2022;40(5):661–671. https://doi.org/10.1038/s41587-021-01139-4
For Research Use Only. Not for use in diagnostic or clinical procedures.
Demo Results
Resolution comparison on normal colon mucosa (sample P3 NAT): Visium v1 detects 3 spatial clusters; Visium HD resolves 18 distinct clusters in the same tissue section — each corresponding to a histologically meaningful cell population. Data from Oliveira MF et al., Nat Genet, 2025.
Visium HD spatial cluster map of FFPE human CRC (8 µm bins) — distinct tumor epithelium, macrophage-enriched stroma, T cell-infiltrated tumor boundary, and normal mucosa domains are resolved at single-cell scale. Integration with matched scRNA-seq reference enables cell-type label transfer per bin. (Oliveira MF et al., Nat Genet, 2025)
References
- Oliveira MF et al. High-definition spatial transcriptomic profiling of immune cell populations in colorectal cancer. Nat Genet. 2025;57:1512–1523. https://doi.org/10.1038/s41588-025-02193-3
10x Visium HD FAQs
1. What is the key difference between Visium HD and Visium FF — when should I use each?
Visium HD and Visium FF differ in three fundamental ways. First, resolution: Visium HD provides 2 µm continuous features approaching single-cell scale, while Visium FF provides 55 µm multicellular spots. Second, chemistry: Visium HD uses probe-based capture (requiring validated human or mouse probe panels), while Visium FF uses unbiased poly(A) capture compatible with any species. Third, sample compatibility: Visium HD works with FFPE, fresh frozen, and fixed frozen tissue via CytAssist; Visium FF is restricted to fresh frozen direct placement. Choose Visium HD when you need single-cell-scale resolution, when your primary sample type is FFPE, or when working in human or mouse. Choose Visium FF when working with non-model organisms, when unbiased whole-transcriptome discovery is the priority, or when FFPE is not a constraint.
2. Does Visium HD work with FFPE tissue — and what quality threshold is required?
Yes — Visium HD was specifically designed to deliver high-quality spatial transcriptomics from FFPE tissue, which is the primary enabling advantage over Visium FF direct placement. The probe hybridization and ligation chemistry is inherently more tolerant of RNA fragmentation than poly(A) capture, because probes target specific short sequences rather than relying on intact poly(A) tails. The minimum FFPE quality threshold is DV200 ≥ 30%, meaning at least 30% of RNA fragments are longer than 200 nucleotides. Higher DV200 (≥50%) is preferred and delivers better sensitivity and gene detection rates. We assess DV200 from a representative section before proceeding and issue a pre-library QC report.
3. How does the CytAssist instrument work in the Visium HD workflow?
CytAssist is a benchtop instrument that transfers probe–analyte complexes from standard glass tissue slides to Visium HD capture slides. After probe hybridization and ligation steps are completed on the glass slide, the tissue slide is placed into the CytAssist instrument alongside the Visium HD capture slide. CytAssist uses fluidic transfer to move the spatially indexed molecules from the glass slide surface to the Visium HD slide, where they hybridize to complementary sequences adjacent to spatial barcodes. CytAssist also images the tissue during transfer — providing a second image that Space Ranger HD uses for co-registration with the original H&E image. This approach enables use of standard histology slides, greatly simplifying the tissue preparation workflow compared to the direct-placement Visium FF method.
4. What is the difference between 2 µm, 8 µm, and 16 µm bins in Space Ranger HD output?
Space Ranger HD outputs count matrices at three bin resolutions from the same underlying 2 µm data. The 2 µm output is the highest-resolution matrix — one count matrix entry per 2 µm feature — but most features contain very few reads per bin at standard sequencing depths, making this resolution most useful for visualization rather than statistical analysis. The 8 µm bin (approximately the diameter of an average mammalian cell) aggregates 4×4 adjacent 2 µm features, yielding enough transcriptomic depth per bin for unsupervised clustering, differential expression, and cell-type deconvolution. The 16 µm bin further aggregates for higher expression depth at lower spatial resolution, useful for lower-complexity analyses or tissues with low capture efficiency. Standard analysis workflows use 8 µm bins.
5. Is Visium HD compatible with non-human species other than mouse?
Currently, validated whole-transcriptome probe panels from 10x Genomics are available for human and mouse only. For other species — including non-human primates, rat, zebrafish, and agricultural animals — Visium HD requires custom probe design, which is feasible but involves additional lead time and validation. For most non-human species projects, we recommend Visium FF or Stereo-seq, which use poly(A) capture compatible with any organism with a reference genome. Contact our scientific team to discuss the most appropriate platform for your species and experimental design.
10x Visium HD Case Studies
Published Research Highlight
Systematic Benchmarking of High-Throughput Subcellular Spatial Transcriptomics Platforms across Human Tumors
Journal: Nature Communications
Impact Factor: 14.7
Published: October 17, 2025
DOI: 10.1038/s41467-025-64292-3
Background
As high-resolution spatial transcriptomics platforms have proliferated — including Visium HD, Stereo-seq, CosMx, and Xenium — researchers need rigorous, head-to-head benchmarking data to select the most appropriate technology for their specific biological questions. Ren et al. generated the first systematic cross-platform evaluation of four high-throughput subcellular spatial transcriptomics technologies using matched serial sections from colon adenocarcinoma, hepatocellular carcinoma, and ovarian cancer — providing quantitative performance comparisons across sensitivity, specificity, spatial accuracy, cell segmentation quality, and cell type annotation concordance.
Materials & Methods
Sample Preparation
- Serial tissue sections from 3 cancer types: colon adenocarcinoma, hepatocellular carcinoma, ovarian cancer
- All samples processed from FFPE blocks on matched serial sections
- CODEX protein profiling on adjacent sections for ground truth
- Single-cell RNA-seq on same samples for reference atlas
Platforms Benchmarked
- Visium HD FFPE (10x Genomics)
- Stereo-seq v1.3 (STOmics / BGI)
- CosMx 6K (NanoString)
- Xenium 5K In Situ (10x Genomics)
- CODEX protein profiling (ground truth)
Analysis Dimensions
- Capture sensitivity and specificity
- RNA diffusion and spatial fidelity
- Cell segmentation accuracy
- Cell annotation concordance with scRNA-seq
- Spatial clustering performance
Results
- Visium HD Demonstrates Superior Spatial Fidelity
- Across all three cancer types, Visium HD FFPE showed the highest spatial fidelity scores — meaning transcripts were most accurately localized to their true spatial positions with minimal diffusion artifacts compared to the other tested platforms.
- Visium HD achieved competitive whole-transcriptome sensitivity, capturing a broad range of transcripts per spatial bin while maintaining tight spatial boundaries between adjacent tissue compartments.
Cross-platform benchmarking of Visium HD FFPE, Stereo-seq, CosMx, and Xenium on serial tumor sections from three cancer types — Visium HD demonstrates superior spatial fidelity and competitive sensitivity. (Ren P et al., Nat Commun, 2025)
- Platform-Specific Strengths Guide Technology Selection
- Visium HD showed superior performance in whole-transcriptome spatial coverage and spatial fidelity, particularly in colon adenocarcinoma where FFPE biobank samples are clinically most relevant.
- Imaging-based platforms (CosMx, Xenium) showed advantages in cell segmentation accuracy at the subcellular level, while sequencing-based platforms (Visium HD, Stereo-seq) offered broader transcriptome coverage.
- The benchmarking data supports a multi-platform strategy: Visium HD for discovery-scale whole-transcriptome spatial profiling, Xenium for targeted subcellular validation — a workflow directly available through CD Genomics.
Conclusion
This rigorous, multi-platform benchmarking study provides the most comprehensive performance comparison of high-throughput subcellular spatial technologies to date. Its findings confirm Visium HD's position as the leading sequencing-based platform for spatial fidelity in FFPE tumor tissue — directly supporting its use as the primary discovery tool in tumor microenvironment research and clinical biobank spatial transcriptomics studies. The study's recommendation of a multi-platform strategy (Visium HD for discovery, Xenium for validation) is operationally available through CD Genomics' integrated spatial multi-omics portfolio.
Reference
- Ren P, Sheng W, Peng X, et al. Systematic benchmarking of high-throughput subcellular spatial transcriptomics platforms across human tumors. Nat Commun. 2025;16:9649. https://doi.org/10.1038/s41467-025-64292-3
