Whole transcriptome spatial gene expression analysis is a transformative method that provides researchers with unparalleled insights into the intricate molecular landscapes of various cell types arranged within complex tissue structures. By offering unbiased mapping of whole transcriptome gene expression, this spatial biology technique holds immense potential for uncovering novel discoveries. In addition to constructing spatially resolved cell atlases, it has facilitated the identification of rare cellular phenotypes and elucidated cell-cell interactions critical for both physiological functions and pathological conditions. However, despite the remarkable achievements made possible by this approach, numerous mysteries of biology remain unsolved. The introduction of Visium HD Spatial Gene Expression marks a significant advancement in spatial biology, offering comprehensive insights into whole transcriptome dynamics at the scale of individual cells. Leveraging innovative slide architecture and a Visium CytAssist–enabled workflow, Visium HD enables researchers to obtain high-resolution spatial transcriptomics data from human or mouse formalin-fixed paraffin-embedded (FFPE) tissue samples, facilitating groundbreaking discoveries akin to those demonstrated in previous iterations of the Visium assay.

Bringing high definition to spatial biology: How does Visium HD work?

Operationalizing Visium HD involves integrating standard histological procedures with a straightforward molecular biology protocol, enabling simultaneous acquisition of hematoxylin and eosin (H&E) or immunofluorescence (IF) images of tissue sections alongside whole transcriptome spatial gene expression profiles resolved at the single-cell level. Starting from FFPE tissue blocks or pre-sectioned slides, researchers can seamlessly transition through tissue staining, probe hybridization, and ligation steps before transferring gene expression probes onto Visium HD slides using the CytAssist instrument.

Figure 1. Diagram of probe transfer from FFPE tissue sections on glass slides onto a Visium HD slide, facilitated within the CytAssist instrument. 

The unique architecture of the Visium HD slide Capture Area is instrumental in providing high-resolution spatial insights, featuring millions of 2 x 2 µm barcoded squares that capture gene expression probes. These spatial barcodes enable precise mapping of gene expression readouts back to tissue section images, facilitating spatially resolved analysis. 

Figure 2. Diagram of Visium HD slide architecture. Visium HD slides contain two 6.5 x 6.5 mm Capture Areas with a continuous lawn of oligonucleotides arrayed in millions of 2 x 2 µm barcoded squares without gaps, achieving single cell–scale spatial resolution. 

Following probe capture and spatial barcoding, the Visium HD Slide undergoes downstream library preparation and sequencing to generate spatially resolved whole transcriptome gene expression data. This data, provided at 2 µm resolution and various bin sizes, can be overlaid onto matched tissue images for visualization and exploration. Bin sizes can be scaled small enough to capture single cells, meaning this approach is able to characterize the main cell type(s) that are present in each bin. Given this binning strategy and the average size of eukaryotic cells (between 8 and 100 µm), each binned area may cover components of more than one cell; as such, Visium HD delivers what we term single cell–scale spatial resolution.

What tissue types have been tested with the Visium HD assay?

The versatility of Visium HD extends to its compatibility with a wide range of FFPE tissue samples, including archived clinical specimens and tissue microarrays. Utilizing a universal FFPE-compatible protocol, Visium HD eliminates the need for tissue-specific optimization, ensuring robust performance across different tissue types and storage conditions.

Figure 3. A collage of the human and mouse FFPE tissues that have been tested with the Visium HD assay. 

Applications

Visium and Visium HD solutions with CytAssist are suitable for profiling of human and mouse tissues; either technology is applicable to spatial discovery applications in both healthy and diseased tissues. Among its many applications, the technology in its current and previous versions has been used to examine:

· Tumor heterogeneity in human prostate cancer (4)

· Spatial architecture in human squamous cell carcinoma (5)

· Spatial topography of the human dorsolateral prefrontal cortex, an area implicated in a number of neuropsychiatric disorders (6)

· Anatomical organization of the fibroblast response to influenza (7)

· Spatiotemporal analysis of human intestinal development (8)

· Spatial mapping of cells in the human endometrium and myometrium (9)

· Spatial characterization of human nociceptors (10)

· B-cell responses within intratumoral tertiary lymphoid structures in renal cell carcinoma (11)

· Tumor-intrinsic biomarkers and putative drug targets in patient tumor biopsies (12)

Join the new era of spatial discovery with Visium HD 

By offering unparalleled spatial insights with continuous tissue coverage and single-cell-scale resolution, Visium HD enhances the depth and breadth of spatial discovery research. Its unbiased approach provides researchers with a comprehensive view of FFPE samples, unlocking previously overlooked biological phenomena in complex tissue microenvironments.

A notable example of Visium HD’s impact is showcased in the work of Dr. Omer Bayraktar and his team at the Wellcome Sanger Institute, where they utilized Visium HD to delineate the cellular trajectories of heterogeneous malignant cell states in glioblastoma (GBM). By integrating Visium HD data with other omics techniques, they constructed a comprehensive atlas of GBM malignant and tumor microenvironment cell states, revealing previously unappreciated spatial relationships and anatomical features within tumor samples.

Visium HD

Figure 4. Visium HD solution

In conclusion, the introduction of Visium HD represents a significant milestone in spatial biology, offering researchers unprecedented insights into whole transcriptome dynamics at the highest resolution to date. As Visium HD continues to drive innovation in sequencing-based spatial biology, it holds the potential to catalyze breakthroughs in understanding complex biological systems and ultimately improve human health.

References:

1. Madissoon E, et al. A spatially resolved atlas of the human lung characterizes a gland-associated immune niche. Nat Genet 55: 66–77 (2022). doi: 10.1038/s41588-022-01243-4
2. Watanabe R, et al. Spatial gene expression analysis reveals characteristic gene expression patterns of de novo neuroendocrine prostate cancer coexisting with androgen receptor pathway prostate cancer. Int J Mol Sci 24: 8955 (2023).
3. Zhang S, et al. Spatial transcriptomics analysis of neoadjuvant cabozantinib and nivolumab in advanced hepatocellular carcinoma identifies independent mechanisms of resistance and recurrence. bioRxiv (2023). doi: 10.1101/2023.01.10.523481
4. Berglund E, et al. Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity. Nat Commun 9: 2419 (2018). doi: 10.1038/s41467-018-04724-5
5. Ji AL, et al. Multimodal analysis of composition and spatial architecture in human squamous cell carcinoma. Cell 182: 497–514.e22 (2020). doi: 10.1016/j.cell.2020.05.039
6. Maynard KR, et al. Transcriptome-scale spatial gene expression in the human dorsolateral prefrontal cortex. Nat Neurosci 24: 425–436 (2021). doi: 10.1038/ s41593-020-00787-0
7. Boyd DF, et al. Exuberant fibroblast activity compromises lung function via ADAMTS4. Nature 587: 466–471 (2020). doi: 10.1038/s41586-020-2877-5
8. Fawkner-Corbett D, et al. Spatiotemporal analysis of human intestinal development at single-cell resolution. Cell 184: 810–826.e23 (2021). doi: 10.1016/j.cell.2020.12.016
9. Garcia-Alonso L, et al. Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro. Nat Genet 53: 1698–1711 (2021). doi: 10.1038/s41588-021-00972-2
10. Tavares-Ferreira D, et al. Spatial transcriptomics of dorsal root ganglia identifies molecular signatures of human nociceptors. Sci Transl Med 14: eabj8186 (2022). doi: 10.1126/scitranslmed.abj8186
11. Meylan M, et al. Tertiary lymphoid structures generate and propagate anti-tumor antibody-producing plasma cells in renal cell cancer. Immunity S1074-7613(22)00081-4 (2022). doi: 10.1016/j.immuni.2022.02.001
12. Lyubetskaya A, et al. Assessment of spatial transcriptomics for oncology discovery. Cell Rep Methods 2: 100340 (2022). doi: 10.1016/j.crmeth.2022.100340