Spatial transcriptomics has revolutionized the study of tissue biology by enabling researchers to measure gene expression while preserving tissue architecture. With the introduction of 10x Genomics Visium HD and Visium HD 3′, researchers now have two high-resolution workflows that share the same 2 μm spatial resolution but differ in chemistry, sample compatibility, and experimental applications.

If you’re planning a spatial transcriptomics study, understanding these differences can help you select the workflow that best aligns with your research goals.

A Shared High-Resolution Platform

Both Visium HD and Visium HD 3′ are built on the same high-density capture array, providing 2 μm spatial resolution for near single-cell spatial gene expression analysis. Both workflows preserve tissue morphology and integrate histological imaging with transcriptomic data, enabling researchers to investigate tissue architecture, cellular neighborhoods, and spatial gene expression patterns.

The primary differences lie in how transcripts are captured and which sample types they support.

Technology Principles

Visium HD

Visium HD supports both Fresh Frozen (FF) and FFPE tissues, but the chemistry differs depending on the sample type.

For Fresh Frozen samples, RNA released from the tissue is captured directly by spatially barcoded oligonucleotides on the slide, enabling whole-transcriptome analysis.

For FFPE samples, where RNA is often fragmented, Visium HD uses probe hybridization chemistry to recover transcripts. This approach allows robust spatial gene expression profiling from archived clinical specimens that are not suitable for poly(A)-based capture.

Visium HD 3′

Visium HD 3′ uses a poly(A)-capture strategy, similar to the 10x Chromium Single Cell Gene Expression workflow. Messenger RNA molecules are captured through their poly(A) tails, and sequencing focuses on the 3′ end of each transcript.

Because this workflow does not rely on species-specific probe panels, it can be applied to a broad range of eukaryotic species, provided that high-quality fresh frozen tissue is available.

Sample Compatibility

Sample type is often the first factor to consider when choosing between the two workflows.

If your project involves FFPE or archived tissue samples, Visium HD is currently the recommended workflow. For studies using high-quality fresh frozen tissues, both workflows are available, with the final choice depending on your research goals and species.

Species Compatibility

Another important consideration is the species being studied.

Because Visium HD 3′ captures polyadenylated mRNA directly, it is not restricted by predefined probe panels. This makes it suitable for many eukaryotic organisms, including non-model species.

By contrast, Visium HD FFPE relies on probe hybridization and therefore requires validated species-specific probe panels. At present, this workflow is primarily available for human and mouse samples.

If your project involves zebrafish, pig, dog, non-human primates, plants, or other non-model organisms, Visium HD 3′ may provide greater flexibility, assuming fresh frozen tissue with good RNA quality is available.

RNA Quality Requirements

RNA integrity is another key factor that influences workflow selection.

Because poly(A)-based capture requires relatively intact RNA, Visium HD 3′ generally has more stringent RNA quality requirements than Visium HD.

Which Workflow Is Right for Your Study?

Choose Visium HD if you:

· Are working with FFPE or archived clinical samples

· Need compatibility with both FF and FFPE tissues

· Study human or mouse samples

· Expect partially degraded RNA

· Require a well-established workflow for clinical or translational research

Choose Visium HD 3′ if you:

· Have high-quality fresh frozen tissue

· Work with non-model organisms

· Want unbiased transcriptome profiling without probe limitations

· Need flexibility across different eukaryotic species

Key Differences at a Glance

Final Thoughts

Visium HD and Visium HD 3′ are complementary spatial transcriptomics workflows built on the same high-resolution platform. The optimal choice depends on your sample type, RNA quality, species, and research objectives rather than on spatial resolution alone.

For projects involving FFPE samples or established human and mouse studies, Visium HD remains the preferred option. For exploratory studies using high-quality fresh frozen tissues—particularly in non-model organisms—Visium HD 3′ offers greater flexibility through its probe-free, poly(A)-based transcript capture strategy.

At Novogene, our spatial transcriptomics specialists work closely with researchers to evaluate sample suitability, optimize experimental design, and recommend the workflow that best supports each project’s scientific goals.

Why choose Novogene for Spatial Transcriptomics

PhD-Level Expert Team

Novogene is supported by a highly experienced team of PhD-level scientists specializing in both experimental workflows and bioinformatics analysis. Our experts bring over 20 years of combined experience in clinical pathology and tissue-based research, ensuring optimal handling of complex samples and accurate interpretation of spatial transcriptomics data. As a certified partner of 10x Genomics and STOmics Stereo-seq, we deliver trusted, industry-leading solutions.

Advanced Technology Platforms

We provide access to cutting-edge spatial transcriptomics platforms, including 10x Genomics Visium HD, Visium HD 3’, and STOmics Stereo-seq. Our laboratory is equipped with state-of-the-art instruments such as the Leica CM3050S cryostat and Aperio Versa 8 fluorescence microscope, enabling high-quality tissue preparation, imaging, and spatial capture for reliable downstream analysis.

Proven Experience Across Diverse Projects

With over 4,000 completed spatial and transcriptomics-related projects, Novogene has extensive experience working with more than 50 species and 100+ tissue types. Our robust workflows have been validated across a wide range of research areas, including oncology, immunology, neuroscience, and translational medicine, ensuring consistent and reproducible results.

High-Impact Scientific Outcomes

Novogene has contributed to over 350 peer-reviewed publications in top-tier journals such as Nature, Cell, and other high-impact scientific publications, with a cumulative impact factor exceeding 4,800. Our data quality and analytical rigor support breakthrough discoveries and enable researchers to publish in leading journals.