The original Seq-Scope technology, developed in 2021 by Jun Hee Lee and colleagues, heralded a new era in spatial omics by enabling comprehensive mapping of all expressed messenger RNA (mRNA) molecules across intact tissues at a microscopic scale. Leveraging Illumina sequencing’s spatial barcoding approach, Seq-Scope cleverly delineated gene activity in situ without the need for single-cell dissociation. However, despite its revolutionary capacity, the system faced intrinsic spatial resolution constraints dictated by the physics of molecule diffusion from tissue to capture arrays during sample preparation.

“It became clear that simply improving resolution on the sequencer side was insufficient due to a hard limit imposed by molecular diffusion, which restricts spatial accuracy to roughly one micron,” explained Lee, a Professor of Molecular & Integrative Physiology. This diffusion barrier essentially blurs the transcriptomic signals as molecules spread from their origin points before being immobilized on sequencing substrates, capping the achievable spatial precision of any sequencing-based spatial method operating under conventional protocols.

To overcome this fundamental limitation, the research team conceived a clever solution: physically enlarging the tissue samples through hydrogel embedding followed by isotropic expansion using water infusion — a strategy adapted from tissue clearing and expansion microscopy techniques. This approach effectively magnifies the spatial dimensions of the tissue, thereby increasing the physical distances between biomolecules relative to the capture surface and circumventing the diffusion-imposed resolution ceiling.

The development of this expansion approach was spearheaded by Lee’s graduate student Angelo Anacleto in collaboration with Hee-Sun Han, a Professor of Chemistry at the University of Illinois Urbana-Champaign. Their interdisciplinary collaboration integrated precise chemical protocols for controlled hydrogel embedding and swellable polymer formulation with the sophisticated gene-capturing Seq-Scope workflow developed at Michigan.

By applying the expansion process, called Seq-Scope-eXpanded, the researchers not only retained the comprehensive transcriptome profiling capacity of the original technique but also achieved an unprecedented enhancement in spatial granularity. The expanded tissues, now magnified in size, allowed the team to differentiate individual cells with far greater accuracy and to trace gene expression patterns within subcellular microenvironments such as distinct nuclear and cytoplasmic regions.

Critical to the interpretation of this flood of high-resolution spatial transcriptomic data were novel computational methods developed by Hyun Min Kang, a Professor of Biostatistics. These algorithms enabled the team to dissect mRNA localization patterns within liver cells, distinguishing transcripts synthesized in the nucleus from those populating the cytoplasm. Such insights into spatially resolved gene regulation dynamics deepen understanding of cellular function and pathology at a molecular level previously impossible to achieve.

Lee emphasized that Seq-Scope-X pushes spatial transcriptomics into an entirely new regime, surpassing prior limitations by nearly an order of magnitude. This leap forward aligns with a broader trend of rapid improvements in spatial omics technologies, which have been advancing roughly fourfold in resolution each year over the past decade. “Our work places the University of Michigan at a critical inflection point where biological discovery potential accelerates sharply thanks to unprecedented spatial detail,” Lee remarked.

Beyond liver tissue, the technology’s versatility suggests broad applicability across diverse tissues and research domains, including cancer biology, developmental biology, and neuroscience. Its capacity to resolve molecular conversations at cellular and subcellular scales could identify novel disease biomarkers, map complex tissue microenvironments, and elucidate intricate cellular interactions underlying health and disease.

While the techniques rely on sophisticated chemistry and advanced sequencing platforms, the underlying concept of physically expanding tissues before molecular profiling introduces a powerful avenue to break through inherent biophysical constraints that have long hampered spatial omics methods. This hybridization of chemical manipulation and sequencing biology represents a paradigm shift in spatial molecular profiling.

The research team also acknowledges the importance of technological transfer and further development to bring Seq-Scope-X from the laboratory to widespread usage. Jun Hee Lee holds key intellectual property rights covering this innovation, emphasizing a strategic approach to manage patenting and collaboration to maximize impact while safeguarding scientific integrity.

In sum, Seq-Scope-eXpanded stands as a milestone in the quest to map biological information in its true spatial context with extraordinary precision. It invites a reimagining of how we explore gene expression dynamics, not as isolated snapshots but as living landscapes within tissues. This technology promises not only to enrich basic science but also to catalyze breakthroughs in diagnostics and therapy, marking an exciting horizon in biomedical research.

Subject of Research: Spatial transcriptomics, molecular resolution enhancement, tissue expansion techniques

Article Title: Seq-Scope-eXpanded: spatial omics beyond optical resolution

Web References:
https://www.nature.com/articles/s41467-026-69346-8

References:
Lee JH et al., “Seq-Scope-eXpanded: spatial omics beyond optical resolution,” Nature Communications, DOI: 10.1038/s41467-026-69346-8

Image Credits: Jun Hee Lee Laboratory

Keywords

Spatial transcriptomics, Seq-Scope, tissue expansion, hydrogel embedding, Illumina sequencing, molecular diffusion barrier, subcellular resolution, gene expression mapping, computational spatial omics, liver transcriptome, hydrogel diffusion, advanced sequencing technology

At its core, volumetric DNA microscopy capitalizes on the intrinsic properties of DNA molecules to encode spatial information directly within intact biological specimens. Unlike conventional imaging techniques that rely heavily on fluorescent labeling and microscopy, this approach harnesses the formation of dense intermolecular DNA networks — a feat accomplished through in situ biochemical processing. Through an intricate procedure involving cDNA synthesis inside tissue samples, DNA molecules self-organize into nanoball structures, effectively capturing the spatial coordinates of gene expression patterns in three dimensions.

The technical brilliance of this approach lies in the dual-scale proximity bridging mechanism between neighboring DNA nanoballs. By meticulously engineering the interplay between these nanoballs, the system constructs a complex web of spatial relationships that can be decoded solely by sequencing the resulting DNA. This innovative strategy obviates the need for optical components, making the technology both scalable and broadly accessible using standard molecular biology kits and a benchtop sequencer – tools usually available in most biomedical laboratories.

The workflow begins with fixing and permeabilizing the tissue to stabilize the RNA transcripts. Subsequently, reverse transcription occurs in situ to convert RNA into cDNA while preserving its spatial context. Following cDNA synthesis, a spatial encoding phase induces the formation of DNA nanoballs, miniaturized DNA aggregates that localize within the tissue volume. These nanoballs link through short-range and long-range DNA proximity interactions, creating a dense, three-dimensional molecular network whose topology faithfully reflects the spatial layout of the tissue.

Once this molecular web has been established, the next critical phase is sequencing. Short-read high-throughput sequencing protocols decode the dense DNA network, generating massive datasets encoding both genetic and spatial information. Computational algorithms then reconstruct the spatial transcriptomic landscape by solving these encoded proximity relationships. This is achieved via a sophisticated geodesic spectral embedding methodology, which effectively maps the DNA molecules back into their original 3D coordinates within the tissue volume with remarkable precision.

The implications of volumetric DNA microscopy for biological research are profound. By overcoming the longstanding limitation of thin-section imaging, scientists can now capture fully volumetric maps of gene expression, which means the ability to observe spatially complex cellular neighborhoods and developmental processes in their entirety. This is especially crucial for studying organs with intricate architectures such as the brain, tumors, or embryonic tissues, where context-dependent gene expression governs function and pathology in a highly spatially dependent manner.

Importantly, the technology does not require specialized optical systems or expensive imaging hardware. Its reliance on routine biochemistry and sequencing workflows democratizes access to three-dimensional spatial transcriptomics, enabling a wide array of laboratories to undertake detailed volumetric analyses. This opens up new avenues for collaborative research, leveraging standardized protocols that can be adapted across different biological specimens and experimental scales, from small biopsies to whole-organ analyses.

The researchers behind this method highlight that the entire volumetric DNA microscopy workflow can be completed within a typical laboratory timeline of 7 to 8 days. This rapid turnaround contrasts starkly with existing spatially resolved transcriptomic techniques that often demand prolonged imaging sessions and complex sample preparations. Such efficiency does not compromise detail, ensuring that volumetric DNA microscopy provides both high throughput and high fidelity in spatial gene expression mapping.

Another remarkable feature of this new method is its capacity for scalability. The dense intermolecular DNA networks can be formed throughout large tissue volumes, enabling comprehensive spatial coverage. This scalability is a major advantage over many optical methods, which face challenges related to limited penetration depths or photobleaching effects in thick samples. The robust molecular encoding here circumvents these issues, laying the foundation for mapping ever-larger tissue samples or even entire small organisms while preserving single-cell resolution.

Moreover, the computational infrastructure integrated into the volumetric DNA microscopy workflow is as critical as the molecular biology. The geodesic spectral embedding algorithm employed for spatial reconstruction represents cutting-edge mathematical modeling, ensuring that the complex web of DNA proximity signals can be translated into accurate three-dimensional representations. This computational power facilitates not only spatial mapping but also enables subsequent biological interpretation, such as identifying cellular neighborhoods, tissue compartments, and functional gradients.

Volumetric DNA microscopy stands to redefine our capacity to explore fundamental questions in developmental biology, neuroscience, oncology, and beyond. Understanding how gene expression networks organize in three dimensions will inform on cellular differentiation pathways, tissue morphogenesis, and disease states with an unprecedented holistic view. By bridging the gap between molecular profiles and spatial context in intact tissues, this method empowers researchers to decode nature’s blueprint in all its complexity.

As the field of spatial biology races forward, technologies like volumetric DNA microscopy will likely serve as essential tools in the armamentarium of modern biologists. The ability to generate highly multiplexed, spatially resolved gene expression datasets in intact tissue volumes without elaborate imaging setups heralds a new era in spatial transcriptomics. One where biological insights are no longer limited by optical constraints but rather driven by the power of molecular and computational synergy.

This development also holds promise for translational medicine, where precise 3D spatial maps of diseased tissues could enhance diagnostics and personalized treatment strategies. In particular, complex tumor microenvironments, with their heterogeneous cell populations and spatially regulated signaling niches, stand to benefit immensely from this comprehensive spatial profiling approach. It opens the door for better understanding of tumor biology, immune cell infiltration, and treatment responses in three dimensions.

In the educational landscape, volumetric DNA microscopy exemplifies an accessible yet highly sophisticated technique that can be integrated into graduate-level curricula, training the next generation of scientists in cutting-edge spatial transcriptomic methods. The procedural transparency, using routine laboratory components and standard sequencing platforms, lowers the barrier for adoption, fostering innovation across diverse research environments.

Critically, while volumetric DNA microscopy offers many advantages, ongoing optimization and validation are imperative. Researchers must refine protocols to maximize the fidelity of spatial encoding, manage data complexity, and extend the method’s applicability to challenging specimens such as fibrous or calcified tissues. As community uptake grows, collaborative improvements and shared datasets will further enhance the robustness and versatility of this technology.

Conclusively, volumetric DNA microscopy constitutes a paradigm shift in how spatial transcriptomic data can be acquired, analyzed, and interpreted in three dimensions within intact biological specimens. Its innovative combination of molecular synthesis, proximity-driven DNA network construction, sequencing, and sophisticated computational reconstruction offers a holistic toolkit poised to transform multiple fields of biological research. The future of spatial biology is volumetric — and this method lights the path forward.

Subject of Research:
Spatial transcriptomics in three-dimensional intact biological tissues.

Article Title:
Volumetric DNA microscopy for mapping spatial transcriptomes in three dimensions.

Article References:
Qian, N., Li, J., Yasser, R. et al. Volumetric DNA microscopy for mapping spatial transcriptomes in three dimensions. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01329-3

Image Credits:
AI Generated

DOI:
https://doi.org/10.1038/s41596-025-01329-3