Microfluidics for Integrated Spatial Transcriptomics and Protein Imaging on the Same Tissue Section

Spatial biology increasingly depends on technologies that can map gene expression and protein localization directly within intact tissues. However, many existing spatial omics workflows rely on separate assays performed on adjacent sections, which introduces alignment errors and limits true multimodal integration. As the authors write, “Spatially mapping the transcriptome and proteome in the same tissue section can profoundly advance our understanding of cellular heterogeneity and function.” Achieving this within a single preserved tissue slice requires precise spatial indexing, controlled reagent delivery, and preservation of tissue architecture, all of which are fundamentally microfluidic challenges.

To address this need, the authors developed DBiTplus, a microfluidic deterministic barcoding in tissue platform that integrates sequencing-based spatial transcriptomics with high-plex multiplexed immunofluorescence imaging on the same tissue section. In their words, they “developed DBiTplus, which combines unbiased transcriptome-wide spatial sequencing with mxIF (CODEX or CellScape) on the same tissue section, compatible with both OCT-frozen and FFPE samples.” This microfluidic spatial omics workflow preserves tissue morphology for imaging while enabling transcriptome-wide sequencing through spatially defined barcode delivery. By unifying microfluidic barcoding and imaging-based protein mapping, DBiTplus enables image-guided deconvolution and single-cell-resolved spatial transcriptome atlases.

At the heart of DBiTplus is a PDMS-based microfluidic chip that performs deterministic spatial barcoding directly on tissue. The microfluidic device fabrication follows established DBiT-seq protocols, using patterned PDMS channels aligned orthogonally across the tissue section. Tissue samples, including OCT-frozen and FFPE sections mounted on poly-L-lysine-coated slides, are processed within a microfluidic flow cell. For fresh frozen tissues, fixation and permeabilization are followed by in situ polyadenylation, enabling efficient capture of RNA molecules. Spatial DNA barcodes are delivered through the microfluidic channels in two sequential orthogonal flows, creating a grid of uniquely indexed microfluidic pixels across the tissue. The authors optimized RNase H-mediated enzymatic cDNA retrieval within this microfluidic framework while preserving tissue integrity for downstream multiplexed protein imaging. After removal of the microfluidic device, the same tissue section undergoes high-plex imaging. Computational co-registration aligns the microfluidic spatial transcriptomic grid with cell segmentation masks from imaging, and transcriptomic spots are subdivided into cell-type-specific sub-spots for single-cell-level reconstruction.

The performance of this integrated microfluidic spatial omics system was demonstrated in frozen mouse embryos and in FFPE human lymph node and lymphoma tissues. Importantly, the microfluidic barcoding strategy enabled transcriptome-wide profiling without predefined gene panels, while the imaging modality provided high-resolution protein-based cell typing. In lymphoma samples, DBiTplus enabled spatial mapping of disease progression and transformation, revealing transcriptional remodeling associated with aggressive states. The platform also captured small RNAs, including miRNAs, extending the molecular scope accessible through microfluidic spatial sequencing. By combining microfluidic deterministic barcoding, sequencing-based transcriptomics, and multiplexed protein imaging, the authors generated spatially resolved single-cell atlases from clinically relevant specimens.

In summary, “DBiTplus represents a spatial multi-omics approach that integrates sequencing-based and imaging-based spatial assays on the same tissue section, enabling image-guided deconvolution into single-cell-resolved spatial transcriptomes.” From a microfluidics perspective, this study highlights how precise microfluidic flow control, spatially patterned barcode delivery, and PDMS-based device integration can serve as the backbone for next-generation spatial multi-omics. It underscores the central role of microfluidic engineering in enabling comprehensive molecular mapping within intact tissues.

Figures are reproduced from Enninful, A., Zhang, Z., Klymyshyn, D. et al. Integration of imaging-based and sequencing-based spatial omics mapping on the same tissue section via DBiTplus. Nat Methods (2026). https://doi.org/10.1038/s41592-025-02948-0 under a Creative Commons Attribution 4.0 International License.

Read the original article: Integration of imaging-based and sequencing-based spatial omics mapping on the same tissue section via DBiTplus

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