What Is 10x Xenium In Situ?

10x Xenium In Situ is an advanced imaging-based spatial transcriptomics platform developed by 10x Genomics that enables high-resolution visualization and quantification of RNA molecules directly within intact tissue sections. Instead of relying on sequencing-based capture methods, Xenium detects individual RNA transcripts at their original spatial locations, allowing researchers to study gene expression while preserving tissue architecture and cellular context.

Using highly specific probe chemistry and high-resolution fluorescence imaging, Xenium maps each detected transcript to its precise spatial coordinates and accurately assigns it to individual cells. This approach delivers true single-cell and subcellular spatial information, making it possible to investigate complex tissue organization, cellular heterogeneity, and molecular interactions with exceptional precision.

How Xenium Technology Works

The Xenium workflow begins with the hybridization of target-specific probes directly to RNA molecules within the tissue section. Once bound, the probes undergo a signal amplification process that generates stable fluorescent signals while preserving the native position of each transcript.

The Xenium Analyzer then performs multiple rounds of automated fluorescence imaging, capturing unique optical signatures that identify each targeted transcript. Advanced onboard image analysis reconstructs the spatial distribution of every detected RNA molecule, generating comprehensive datasets that include:

• • Precise spatial coordinates for individual transcripts.

• • High-resolution cell segmentation.
• • Cell-by-gene expression matrices.
• • Tissue morphology and DAPI images.
• • Quality control metrics for downstream analysis.

• Because transcript detection occurs directly within intact tissue, no conventional RNA sequencing library preparation is required.

High-Plex Spatial Gene Expression Profiling

Xenium supports highly multiplexed targeted transcriptomic analysis, enabling researchers to investigate thousands of genes simultaneously within a single experiment. Flexible panel options include validated catalog panels for major research areas as well as customized probe sets tailored to specific biological questions.

This targeted strategy combines high sensitivity with exceptional spatial resolution, making Xenium an ideal platform for studies requiring accurate localization of predefined biomarkers while maintaining cellular and tissue architecture.

Large Imaging Capacity

The Xenium imaging surface measures 10.45 × 22.45 mm, providing sufficient area to analyze multiple tissue sections within a single run. This design allows efficient comparison of different biological conditions, patient samples, treatment groups, or experimental time points while maximizing instrument throughput and reducing overall project costs.

Preserves Tissue for Downstream Analysis

A significant advantage of Xenium is its non-destructive imaging workflow. Following transcript detection, the tissue section remains physically intact and can be used for additional experimental applications, including:

• • Hematoxylin and Eosin (H&E) staining.
• • Immunofluorescence (IF) or antibody-based protein detection.
• • Long-term tissue archiving.
• • Integrated spatial biology workflows alongside complementary technologies, where applicable.

This capability enables researchers to combine RNA expression, protein localization, and tissue morphology from the same specimen, providing a comprehensive view of tissue biology through multi-modal analysis.

Xenium vs. Sequencing-Based Spatial Methods

Xenium's imaging-based single-molecule detection delivers capabilities that sequencing-based methods cannot match — particularly for subcellular localization, true single-cell resolution without deconvolution, and tissue morphology correlation.

Xenium Panel Options

Xenium provides a diverse portfolio of validated gene panels designed for a wide range of research applications. Researchers can choose from comprehensive high-plex panels, organ-specific assays, or customized panels tailored to their biological questions. In addition, custom probes can be incorporated to target genes of particular interest, offering exceptional flexibility for specialized studies.

• Xenium Prime 5K Panels – High-Plex Spatial Transcriptomics

Xenium Prime enables large-scale targeted spatial transcriptomic analysis with the ability to profile up to 5,000 genes in a single experiment.

Key Features

• - Supports high-plex analysis of up to 5,000 target genes.
• - Flexible modular design allows multiple subpanels to be combined into a single customized assay.
• - Available research domains include:
  • - Cancer
  • - Immunology & Inflammation
  • - Neuroscience
  • - Development & Cell Fate
  • - Metabolism
• - Validated primarily for human tissues, with selected panels also available for mouse studies.

Ideal Applications

• - Comprehensive cell atlas generation.
• - Tumor microenvironment (TME) characterization.
• - Immune profiling.
• - Multi-system and complex tissue biology studies.
• - Large-scale targeted spatial transcriptomics projects.

• Organ-Specific and Disease-Focused Panels

Xenium also offers carefully curated panels developed for specific tissues and disease areas, enabling focused analysis of biologically relevant markers.

Available panel categories include:

• - Breast
• - Brain
• - Lung
• - Colon
• - Additional organ- and disease-specific panels

These assays are designed to capture the major cellular populations and clinically relevant biomarkers associated with each tissue type. Selected applications also support complementary spatial protein detection using Xenium-compatible protein assays.

Ideal Applications

• - Organ-specific biology.
• - Disease mechanism studies.
• - Biomarker validation.
• - Translational and clinical research.
• - Precision pathology investigations.

• Custom Gene Panel Design

Researchers can expand existing catalog panels by incorporating custom-designed probes targeting genes relevant to their specific projects.

Our custom panel service includes:

• - Addition of one or multiple user-defined gene targets.
• - Flexible customization based on available panel capacity.
• - Probe design, synthesis, and quality validation.
• - Optimization to ensure high specificity and reliable spatial detection.

- Custom probe development generally requires 4–6 weeks, depending on project complexity and panel requirements.

Ideal Applications

• - Novel biomarker discovery.
• - Proprietary or unpublished gene targets.
• - Species-specific transcript detection.
• - Customized research panels for specialized studies.

• Integrated RNA and Protein Spatial Profiling

Xenium supports integrated multi-modal workflows that combine spatial RNA analysis with protein detection on the same tissue specimen.

Following RNA imaging, compatible tissue sections can be processed using immunofluorescence (IF)-based antibody staining to investigate protein localization alongside gene expression. Since DAPI nuclear staining is incorporated into the standard Xenium workflow, no additional nuclear preparation is required.

This combined approach enables researchers to directly compare RNA expression with protein abundance while preserving tissue architecture and spatial context.

Ideal Applications

• - Correlation of transcript and protein expression.
• - Validation of protein biomarkers.
• - Immune cell phenotyping.
• - Cell signaling studies.
• - Comprehensive spatial multi-omics characterization.

Service Workflow

Step 1: Panel Selection & Sample Quality Assessment

Every project begins with selecting the most suitable Xenium gene panel based on your research objectives. Depending on your study design, you may choose from standard catalog panels, tissue- or organ-specific panels, or customized panels incorporating additional target genes.

Once the panel is finalized, submitted FFPE or fresh frozen OCT-embedded tissue samples undergo comprehensive quality assessment. For optimal performance, FFPE samples should have a DV200 ≥30% (≥50% recommended), while fresh frozen samples are recommended to have an RNA Integrity Number (RIN) of 7 or higher. The Xenium imaging area (10.45 × 22.45 mm) supports multiple tissue sections within a single run, and our team optimizes sample placement to maximize throughput and cost efficiency.

Step 2: Tissue Preparation & Probe Hybridization

Tissue samples are sectioned according to recommended specifications—5 µm for FFPE and 10 µm for fresh frozen tissue—and mounted onto Xenium-compatible imaging slides.

Following DAPI nuclear staining, highly specific probe sets are hybridized directly to their corresponding RNA targets while preserving the native tissue architecture. The hybridized probes are subsequently circularized and amplified through Rolling Circle Amplification (RCA), producing stable fluorescent amplification products at the original location of each transcript.

Because transcript detection occurs directly within the tissue, this workflow eliminates the need for conventional library preparation or sequencing.

Step 3: Automated Imaging & Transcript Detection

The prepared slides are processed using the 10x Xenium Analyzer, which performs fully automated cyclic fluorescence imaging across the tissue section.

During successive imaging cycles, unique combinations of fluorescent signals generate optical barcodes for each target transcript. The instrument automatically decodes these barcodes, identifies individual transcripts, and records their precise three-dimensional spatial coordinates within the tissue.

Processing time varies depending on panel size, with large high-plex panels typically completed within several days, while smaller targeted panels can be processed more rapidly.

Step 4: Cell Segmentation & Spatial Data Generation

Following image acquisition, DAPI-stained nuclei are computationally segmented using the Xenium analysis pipeline to identify individual cells throughout the tissue.

Cell boundaries are estimated from nuclear segmentation and can be further refined using optional membrane or whole-cell staining when required. Each detected transcript is then assigned directly to its corresponding cell based on its spatial position, generating an accurate cell-by-gene expression matrix without relying on computational deconvolution or cell-type estimation.

This approach provides highly reliable single-cell spatial transcriptomic data while preserving the original tissue architecture.

Step 5: Bioinformatics Analysis & Data Delivery

The generated spatial transcriptomics dataset undergoes comprehensive downstream analysis using established bioinformatics workflows. Standard analyses include quality assessment, cell clustering, cell type annotation, spatial neighborhood characterization, ligand–receptor interaction analysis, and advanced visualization of spatial gene expression patterns.

When complementary datasets—such as single-cell RNA sequencing (scRNA-seq) or Visium spatial transcriptomics—are available, integrated multi-modal analyses are performed to enhance biological interpretation and improve cell type resolution.

Final deliverables include processed datasets, publication-quality figures, interactive visualization files, detailed quality control reports, and comprehensive bioinformatics analyses, providing researchers with a complete spatial transcriptomics solution from sample submission to biological insight.

Key Applications of 10x Xenium In Situ

1. Subcellular Transcript Localization

Xenium enables ultra-high-resolution spatial mapping of RNA molecules, allowing researchers to determine whether individual transcripts are localized within the nucleus, cytoplasm, or near the cell membrane. This level of subcellular precision cannot be achieved using sequencing-based spatial transcriptomics technologies.

By preserving the native spatial organization of transcripts, Xenium supports investigations into biologically important processes such as nuclear RNA retention, localized protein translation, intracellular RNA trafficking, and membrane-associated signaling, providing valuable insights into cellular function and regulation.

2. Tumor Microenvironment Characterization and Immune Cell Mapping

Xenium provides true single-cell spatial resolution, enabling precise identification and localization of diverse immune and stromal cell populations within the tumor microenvironment. Cell types such as cytotoxic T cells, regulatory T cells, macrophage subsets, natural killer (NK) cells, dendritic cells, and other immune populations can be accurately mapped without relying on computational deconvolution.

Advanced spatial analyses reveal cellular neighborhoods, immune infiltration patterns, and interactions between tumor and immune cells, helping researchers better understand tumor biology, immune responses, and potential biomarkers associated with prognosis and therapeutic outcomes.

3. Targeted Biomarker Profiling for Translational and Clinical Research

For studies focused on predefined biomarkers, Xenium offers a highly efficient targeted spatial transcriptomics workflow. Validated gene panels enable sensitive profiling of selected transcripts while maintaining cellular and tissue context.

The platform is fully compatible with FFPE tissue, making it particularly valuable for retrospective analysis of archived clinical specimens and biobank collections. Researchers can correlate spatial gene expression with clinical characteristics, disease progression, therapeutic response, and pathological findings across large patient cohorts.

4. Integrated Discovery and Validation with Visium and Xenium

Combining Visium and Xenium creates a comprehensive spatial transcriptomics strategy that leverages the strengths of both technologies.

Whole-transcriptome spatial profiling with Visium enables unbiased discovery of novel genes, pathways, and cellular states across tissue samples. Xenium can then be used to validate selected biomarkers with single-cell and subcellular resolution, providing highly accurate spatial confirmation of discovery findings.

Our integrated spatial multi-omics workflow streamlines experimental design, data generation, bioinformatics analysis, and biological interpretation across both platforms, delivering a unified view of tissue architecture and molecular function.

5. Neuroscience and Brain Tissue Research

The complex cellular organization of the nervous system requires exceptionally high spatial resolution to accurately distinguish closely packed neuronal and glial populations. Xenium enables detailed characterization of neuronal subtypes, interneurons, astrocytes, oligodendrocytes, microglia, and other brain cell populations within their native anatomical context.

Dedicated brain-focused gene panels support comprehensive studies of cortical layers, hippocampus, cerebellum, and other regions in both human and mouse tissues. By combining precise spatial localization with targeted transcript profiling, Xenium provides powerful insights into brain development, neural circuitry, neurodegenerative disorders, and other neurological diseases.

Sample Requirements

Xenium is compatible with both FFPE (Formalin-Fixed Paraffin-Embedded) and fresh frozen tissue samples. Because sample quality has a significant impact on transcript detection efficiency and overall data quality, we recommend consulting our technical team before sample collection to ensure the most appropriate tissue preparation and handling procedures for your study

• • Tissue Imaging Area

• The Xenium imaging surface measures 10.45 × 22.45 mm, allowing multiple tissue sections to be accommodated within a single imaging area. Our team carefully optimizes tissue placement to maximize usable imaging space and facilitate comparative studies, including multiple biological conditions, treatment groups, or time points within the same experiment whenever feasible.

• • Species Compatibility

• Commercial Xenium gene panels are available for human tissues, while selected catalog panels also support mouse samples. Certain human panels have demonstrated cross-species compatibility with specific mouse tissues, and dedicated mouse panels are available for several research applications.
• For studies involving other organisms—including rat, zebrafish, non-human primates, livestock, or additional model species—custom probe design can be developed based on annotated transcriptome information. Our experts can evaluate project feasibility and recommend the most suitable panel design for your species of interest.

• • Gene Panel Selection

• Standard catalog gene panels are readily available and can be scheduled without additional development time. If your project requires custom targets or non-catalog panels, probe design and synthesis typically require 4–6 weeks before experimental processing can begin.
• To minimize project delays, we recommend finalizing gene panel selection prior to sample submission.

• • Tissue Preservation After Analysis

• A key advantage of the Xenium workflow is that the tissue section remains physically intact after imaging. This enables the same specimen to be used for additional downstream applications, including:
• - Hematoxylin and Eosin (H&E) staining.
• - Immunofluorescence (IF) or antibody-based staining.
• - Long-term tissue archiving.
• - Additional spatial transcriptomics workflows, including compatible Visium CytAssist analyses when appropriate.

Bioinformatics Analysis & Deliverables

The Xenium platform generates comprehensive primary data immediately after run completion through the Xenium Ranger pipeline. To maximize biological insights, our advanced bioinformatics workflow extends beyond the standard outputs, providing in-depth cell characterization, spatial analysis, and multi-omics integration for a complete interpretation of your spatial transcriptomics data.

• Primary Xenium Data Outputs

The standard Xenium processing workflow generates a comprehensive set of primary results, including:

• - Spatial coordinates (X, Y, Z) for every detected transcript along with transcript identity.
• - High-resolution cell segmentation masks defining both nuclear and cellular boundaries.
• - Cell-by-gene expression matrices for downstream analysis.
• - DAPI and tissue morphology images.
• - Cell-level quality metrics, including transcript counts, cell area, and nuclear area.

• Quality Control Assessment

A detailed quality control report is provided for every project to evaluate data integrity and experimental performance. The report includes:

• - Gene-wise transcript detection efficiency.
• - Median transcript count per cell.
• - Cell segmentation performance.
• - Negative control (blank probe) signal assessment.
• - Background fluorescence evaluation.
• - Overall sample and section quality metrics.

• Cell Type Identification

Cells are classified using advanced single-cell analysis frameworks such as Seurat or Squidpy. Our workflow includes:

• - Unsupervised clustering of individual cells.
• - Cell type annotation based on established marker genes.
• - High-resolution spatial maps illustrating the distribution of identified cell populations across the tissue section.

• Spatial Neighborhood Analysis

To uncover tissue organization and cellular interactions, we perform comprehensive spatial analyses, including:

• - Identification of neighboring cell populations.
• - Cell–cell co-localization analysis to detect recurring spatial arrangements.
• - Spatial niche characterization based on local cellular composition.
• - Visualization of tissue architecture and cellular microenvironments.

• Ligand–Receptor Interaction Analysis

Cellular communication is investigated using established computational frameworks such as CellChat and NicheNet. By leveraging precise spatial coordinates, our analysis identifies potential signaling interactions between neighboring cell populations, enabling a more accurate interpretation of local cell-to-cell communication within the tissue microenvironment.

• Subcellular Transcript Localization

For studies requiring subcellular resolution, we provide detailed visualization of transcript distribution, including:

• - Nuclear versus cytoplasmic transcript localization.
• - Gene-specific spatial expression maps.
• - High-resolution transcript density heatmaps for selected targets.

• Multi-Modal Data Integration

When complementary datasets are available, we perform integrated analyses to generate a unified biological interpretation. Supported integrations include matched single-cell RNA sequencing (scRNA-seq) and Visium HD datasets using advanced computational approaches such as:

• - Label transfer.
• - Anchor-based integration.
• - Joint embedding and cross-platform alignment.
• - Concordance assessment across multiple spatial and single-cell technologies.

• Publication-Ready Deliverables

All project outputs are provided in formats suitable for research publications and downstream exploration, including:

• - High-resolution PDF and PNG figures.
• - Interactive Xenium Explorer files.
• - Annotated expression matrices.
• - Quality control reports.
• - Cell annotation and clustering results.
• - Spatial visualization outputs.
• - Comprehensive bioinformatics summary reports
•  
• 

1. When is Xenium a better choice than Visium HD for spatial transcriptomics?

The ideal platform depends on your research objectives and the level of spatial resolution required. Xenium is the preferred option when your study requires:

• • Precise subcellular localization of RNA molecules, such as distinguishing nuclear and cytoplasmic transcripts.

• • Direct single-cell transcript assignment without computational deconvolution, as transcripts are mapped to segmented cells using DAPI-based imaging.
• • Targeted analysis with a validated gene panel rather than unbiased whole-transcriptome profiling.
• • Preservation of tissue architecture, allowing the same tissue section to be used for additional downstream analyses.

• In contrast, Visium HD is better suited for projects focused on unbiased whole-transcriptome discovery, studies involving FFPE tissues with variable RNA quality, or applications where single-cell-scale resolution is sufficient without requiring subcellular localization. For many research programs, combining both technologies provides the greatest value—using Visium HD for comprehensive discovery and Xenium for high-resolution validation of selected targets.

2. What is the difference between Xenium and Xenium Prime, and how many genes can be analyzed?

The standard Xenium platform supports targeted spatial transcriptomic analysis using gene panels that typically contain several hundred genes. Xenium Prime, introduced as an enhanced version of the platform, significantly expands profiling capacity by enabling analysis of up to 5,000 genes within a single experiment.

Xenium Prime utilizes a flexible modular panel design, allowing researchers to combine multiple predefined subpanels covering areas such as oncology, immunology, neuroscience, developmental biology, and metabolism. Custom probe sets can also be incorporated to create panels tailored to specific research objectives.

N2Jenomics Lab Pvt. Ltd. provides both Xenium and Xenium Prime services, helping researchers select the most appropriate workflow based on study design, target genes, and project goals.

3. Can Xenium be applied to species other than humans?

Yes. While commercially available Xenium gene panels are primarily optimized for human samples, and selected panels are available for mouse, the technology can also be adapted for numerous other species through custom probe development.

Researchers working with organisms such as rat, zebrafish, non-human primates, livestock, or other model species can utilize custom-designed probes targeting genes of interest, provided suitable transcriptome annotations are available. Custom panel development generally requires several weeks for probe design, validation, and manufacturing.

Our scientific team can assist with evaluating project feasibility, designing species-specific panels, and developing customized spatial transcriptomics workflows.

4. Does Xenium consume the tissue sample, or can it be used for additional analyses?

One of the major advantages of Xenium is that it is a non-destructive imaging-based workflow. Unlike sequencing-based spatial transcriptomics methods that consume tissue during library preparation, Xenium preserves the physical tissue section after data acquisition.

Following Xenium analysis, the same tissue section may be used for additional applications, including:

• • Hematoxylin and Eosin (H&E) staining and imaging.
• • Immunofluorescence (IF) staining for protein localization studies.
• • Complementary spatial transcriptomics workflows, including Visium-based analyses, where compatible with the experimental design.

This capability enables integrated multi-modal studies by combining RNA expression with histological and protein-level information from the same specimen.

5. How are cells segmented in Xenium, and are additional reagents required?

Xenium performs cell segmentation using DAPI nuclear staining, which is included as part of the standard assay workflow. During analysis, Xenium software identifies cell nuclei from DAPI images and computationally estimates cell boundaries by extending the nuclear segmentation, providing reliable cell assignment across a wide range of tissue types.

For tissues with densely packed cells or complex cellular morphology, segmentation accuracy can be further enhanced by incorporating optional whole-cell or membrane-specific markers. Depending on the tissue type and experimental objectives, markers such as pan-cytokeratin or other cell boundary stains may be included to improve cell boundary definition and transcript assignment.

Our team can recommend the most appropriate segmentation strategy based on your tissue type and research application.