Understanding how the brain ages at the molecular level has long challenged neuroscientists. Traditional spatial transcriptomics methods depend on optical imaging — a process that is slow, expensive, and restricted in tissue coverage. IRISeq, short for Imaging Reconstruction using Indexed Sequencing, changes that entirely. This breakthrough platform maps gene expression across brain tissue without any microscope or predefined capture array. It is cost-effective, highly scalable, and delivers results at a resolution of 5 to 50 micrometers. Published in Nature Neuroscience, IRISeq is setting a new standard for spatial genomics in aging research.
What Is IRISeq?
IRISeq is an optics-free spatial transcriptomics platform developed by researchers at The Rockefeller University. It serves as a powerful alternative to existing tools such as 10x Visium and Slide-seq. Unlike those methods, IRISeq requires no optical imaging and no pre-patterned capture arrays. Instead, it uses DNA-barcoded beads to record gene expression across thousands of spatial locations simultaneously. The platform then reconstructs each bead’s position based on its interaction signals with neighboring beads.
Crucially, IRISeq costs approximately $30 per tissue section — far less than most commercial spatial genomics platforms. This affordability brings high-throughput spatial profiling within reach of individual research laboratories, not just well-funded institutions.
How IRISeq Works
Bead Preparation and Tissue Capture
The IRISeq workflow begins with two specialized types of DNA-barcoded beads. Receiver beads carry poly(T) sequences designed to capture messenger RNA (mRNA) from nearby cells. Sender beads carry a photocleavable linker and a poly(A) sequence. Both types receive unique barcodes through a split-pool ligation process. Researchers distribute the beads evenly across a glass slide. Ultraviolet light then triggers local diffusion, allowing sender oligos to reach and bind with adjacent receiver beads.
Next, frozen tissue sections are placed directly onto the bead array. Cellular mRNA hybridizes to the receiver beads through this contact. After tissue digestion, researchers collect the beads and perform reverse transcription, second-strand synthesis, tagmentation, PCR amplification, and finally sequencing.
Spatial Reconstruction Without Imaging
Sequencing produces two critical outputs: a bead-bead interaction matrix and a gene expression matrix. Researchers apply principal component analysis (PCA) and UMAP algorithms to infer spatial positions from interaction patterns alone. The reconstruction preserves local neighborhood structure accurately and reproducibly — without any camera, lens, or imaging system. This optics-free design is the platform’s defining innovation.
Building a Spatial Brain Transcriptome Atlas
Researchers applied IRISeq to more than 70 coronal sections from adult (4-month-old) and aged (23-month-old) wild-type mouse brains. Sections spanned the frontal cortex, hippocampus, thalamus, hypothalamus, and surrounding regions. Altogether, the team generated over 460,000 spatially distinct transcriptome profiles. Integration with previously published single-cell datasets enabled precise annotation of 25 distinct brain regions. Cross-replicate correlation confirmed that spatial transcriptomic profiles were highly reproducible across tissue sections and experimental replicates.
Age-Associated Gene Expression Changes
Global Aging Signatures
Differential gene expression analysis across 25 brain regions identified 538 upregulated genes and 386 downregulated genes in aged brains, relative to adult brains. Broadly downregulated genes include those tied to mitochondrial function (e.g., Cox8a, Cox17), ribosomal activity (e.g., Rpl30, Rps15a), and cilia function (e.g., Cfap74, Catsperd). Together, these changes point to declining energy production, impaired protein synthesis, and weakening ciliary performance — all hallmarks of cellular aging.
Region-Specific Inflammation
In contrast, genes related to immune response — especially complement and interferon pathways — rose sharply in aged brains. Critically, the largest increases in interferon signaling occurred in ventricular regions, which are primary sites of neuroinflammation. Glial activation markers such as Gfap and Serpina3n confirmed this regional inflammatory profile. Additionally, lymphocyte-associated gene expression, including Cd24a, Ighm, and Cd52, increased most dramatically in the ventricles, white matter, and hypothalamus.
Cell-Type Shifts During Brain Aging
Using the RCTD deconvolution method, researchers mapped over 300 brain cell subtypes across all spatial regions. Differential abundance analysis uncovered 123 region-specific population changes with aging. Neuroblasts and neural progenitor cells declined most steeply in the olfactory bulb and ventricular regions, consistent with reduced adult neurogenesis along the lateral ventricle walls. Vascular smooth-muscle cells and specific endothelial subtypes also depleted significantly, indicating deteriorating vascular integrity with age.
Meanwhile, certain cell populations expanded considerably. Disease-associated microglia (DAM), reactive oligodendrocytes, and border-associated macrophages all increased — particularly in white matter and ventricular areas. Furthermore, IRISeq detected a spatially significant colocalization among DAM, reactive oligodendrocytes, and activated astrocytes. This cluster, termed the “DAM niche,” emerged as a defining spatial feature of the aging brain. Independent validation using the 10x Visium platform confirmed this pattern.
Lymphocytes Drive Brain Inflammation
Lymphocyte-Deficient Mouse Models
To directly test lymphocyte involvement in brain aging, researchers profiled 45 coronal sections from aged wild-type mice and two immunodeficient mouse models — Rag1 and Prkdc mutants — that lack functional lymphocytes. Three biological replicates per group ensured statistical robustness. This additional dataset yielded 363,777 spatially distinct transcriptome profiles. Both mutant models consistently showed gene expression changes distinct from wild-type aged brains.
Interferon Suppression and Ependymal Cell Preservation
Strikingly, lymphocyte depletion reduced interferon-related gene expression most sharply in lateral ventricular regions and white matter. Consequently, ependymal cell populations — which typically decline during aging — were preserved in lymphocyte-deficient brains. The Prkdc mutant additionally upregulated cellular senescence genes, likely due to its role in DNA repair. Single-cell analysis of 783,264 high-quality transcriptomes confirmed that lymphocyte loss suppresses inflammatory interferon pathways while simultaneously promoting neurogenesis-associated gene expression. Moreover, a novel microglial state — marked by Dhcr7 (cholesterol biosynthesis) and Tmtc2 (calcium homeostasis) — appeared exclusively in lymphocyte-deficient brains, revealing previously unknown immune-glial dynamics
Key Findings and Future Implications
IRISeq offers four major advantages over existing spatial transcriptomics methods. First, it is highly cost-effective at approximately $30 per tissue section. Second, it scales easily to multiple large tissue sections simultaneously. Third, it provides adjustable resolution spanning 5 to 50 micrometers. Fourth, it eliminates optical imaging entirely, removing a major bottleneck in spatial genomics workflows.
Beyond the platform itself, the study produces important biological insights. Lymphocytes emerge as central drivers of age-related brain inflammation, particularly through interferon signaling in ventricular regions. Targeting this pathway may eventually offer therapeutic strategies to preserve ependymal cells, slow ventricular degeneration, and maintain cerebrospinal fluid homeostasis in aging brains. Additionally, IRISeq is compatible with protein profiling, epigenomic analysis, and formalin-fixed paraffin-embedded tissues — positioning it as a versatile tool for the broader field of spatial multi-omics.
