Biological tissues are three dimensional structures. Cells are arranged in complex layers and neighborhoods, and their position often influences how they behave. However, many spatial transcriptomics methods rely on very thin tissue sections. While these techniques provide valuable information, they do not fully capture the three dimensional organization of intact tissues.

Researchers at the University of Chicago have developed a new method called volumetric DNA microscopy aims to overcome this limitation. This approach allows scientists to map gene expression directly within intact tissue volumes, without relying on traditional microscopy optics.

Instead of using lenses to visualize spatial information, volumetric DNA microscopy encodes location into DNA molecules themselves. Inside the tissue, complementary DNA, or cDNA, is first synthesized from RNA transcripts. These DNA molecules are then processed in a way that creates DNA nanoballs, forming a dense molecular network within the specimen. Nearby molecules are linked through proximity bridging, which captures information about which transcripts are physically close to one another.

Overview of the volumetric DNA microscopy workflow

This four-stage protocol enables 3D spatial transcriptomic profiling in intact specimens. a, In situ cDNA synthesis is performed within fixed, permeabilized specimens to preserve the native spatial distribution of RNA. b, Spatial encoding is achieved by annealing precircularized UMIs to adaptors ligated to the 3′ end of cDNA, followed by RCA to form DNA nanoballs. Each DNA nanoball containing tandem copies of a UMI encodes the spatial origin of its corresponding cDNA molecule. c, UEIs are formed through both direct oligonucleotide-mediated bridging between neighboring nanoballs and subsequent in situ IVT, collectively capturing molecular proximities across short and long length scales. d, Transcript identities and spatial proximities are decoded through high-throughput sequencing of genic cDNA amplicons and UEI amplicons, enabling de novo reconstruction of 3D specimen morphology. 

After this encoding step, the tissue is processed for short read sequencing. Computational methods then reconstruct the three dimensional spatial relationships between transcripts using geodesic spectral embedding. In simple terms, the sequencing data contains clues about how close different molecules were in space, and algorithms convert those clues into a 3D map of gene activity.

Importantly, the workflow is designed to be accessible. A trained graduate level molecular biologist can generate sequencing libraries within about a week, using standard laboratory reagents and a benchtop sequencer. This makes the technique scalable and adaptable for many biological systems.

By combining RNA sequencing with in situ spatial encoding, volumetric DNA microscopy enables researchers to study genetic and morphological features within intact tissues. This opens new possibilities for understanding how cells organize into functional structures and how microenvironments influence gene expression in health and disease.