Seq-Scope-eXpanded (Seq-Scope-X): Spatial Omics Beyond Optical Resolution

Sequencing-based spatial transcriptomics (sST) enables transcriptome-wide gene expression mapping but falls short of reaching the optical resolution (200–300 nm) of imaging-based methods. Here, we present Seq-Scope-X (Seq-Scope-eXpanded), which empowers submicrometer-resolution Seq-Scope with tissue expansion to surpass this limitation. By physically enlarging tissues, Seq-Scope-X minimizes transcript diffusion effects and increases spatial feature density by an additional order of magnitude. In liver tissue, this approach resolves nuclear and cytoplasmic compartments in nearly every single cell, uncovering widespread differences between nuclear and cytoplasmic transcriptome patterns. Independently confirmed by imaging-based methods, these results suggest that individual hepatocytes can dynamically switch their metabolic roles. Seq-Scope-X also works in brain and colon, and can be adapted for spatial proteomics, profiling hundreds of barcode-tagged antibody stains at microscopic resolutions in mouse spleens and human tonsils. Together, these findings establish Seq-Scope-X as a transformative platform for ultra-high-resolution whole-transcriptome and proteome profiling, providing unparalleled precision and biological insights. Overall design: Tissue Samples Mouse liver, colon, and spleen tissues were collected from 8-week-old male C57BL/6 wild-type mice, and brain tissue was obtained from a 6-month-old male mouse. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Michigan (protocol PRO00011369). Human tonsil tissue was obtained from the UCLA Pathology Biobank as a discarded surgical specimen from an adult patient with chronic tonsillitis. The sample was fully de-identified prior to acquisition and classified as IRB-exempt under institutional and federal guidelines, with no access to identifiable patient information. Seq-Scope-X Transcriptome Workflow Fresh frozen tissues were cryosectioned, mounted on charged slides, fixed with formaldehyde, and permeabilized with methanol. Anchor probes were hybridized in a formamide-containing buffer under humidified conditions. Tissues were then embedded in a polyacrylate-based monomer solution, polymerized, and cleared using digestion buffer with Proteinase K. Tissue expansion was performed by incubation in diluted SSC, and DAPI staining was used to assess tissue integrity. Expanded gels were trimmed for imaging and alignment, then placed onto Seq-Scope Chips. Probe release and transcript transfer were achieved by heat incubation, followed by standard Seq-Scope library preparation, omitting tissue digestion as tissues were not directly attached to the chip. Seq-Scope-X Proteome Workflow For spatial proteomic analysis, tissues were permeabilized with methanol, blocked with a BSA-containing solution, and incubated overnight with TotalSeq™-A oligonucleotide-tagged antibody cocktails. After washing and post-fixation, tissues were processed using the same embedding, clearing, and expansion steps as in the transcriptome workflow. Library preparation was modified to capture antibody-bound DNA oligonucleotides. Following reverse transcription, antibody tag libraries were eluted, purified, amplified by AT-PCR and indexing PCR, size-selected to isolate ~250 bp fragments, and sequenced on an Illumina NovaSeq-X platform. Data Processing and Analysis Seq-Scope-X transcriptome data were processed using the Seq-Scope pipeline with adjustments for experimentally determined expansion factors, following the NovaScope and NEDA workflows. Processing included generation of spatial barcode maps, alignment to reference genomes, construction of spatial gene expression matrices, and clustering using Seurat v5. Spatial factor discovery was performed using Latent Dirichlet Allocation, and FICTURE was used to project cell-type factors into raw pixel space. Microscopy images, which lacked fiducial markers, were aligned to transcriptomic data using QGIS and further processed with CellProfiler and Photoshop. Differential expression, clustering, and integration analyses were conducted on hexagonally binned data to enable spatial and functional interpretation.