Tissue transcriptomics enabled by spectral binary barcodes

The goal of the proposed work is to develop ultrahigh multiplexing (>1000 plex) fluorescent probes and methods for using them in high-resolution spatial transcriptomics of both thin (~5-10 m) and thick (> ~200 m) tissue samples in a single round of staining and fluorescence imaging. Spatial transcriptomics allows cell phenotyping in the context of tissue structure, and has become essential in biomedical research. However, current spatial transcriptomic technologies rely on either time-consuming cyclic imaging approaches (multiple rounds of staining, imaging, and de-staining) or complex, expensive, and lower- resolution sequencing approaches, and there is no commercial technology for spatial transcriptomics in thick tissue samples. A new technology is needed to streamline spatial transcriptomics for both thick and thin tissue samples for high volume clinical and research use. To address this need, we propose to create a set of over 1000 spectrally barcoded fluorescent probes, and to develop methods for using these barcodes as spatial transcriptomics probes in both thin and thick tissue samples. To create a set of 1024 probes for diverse spatial transcriptomics applications, we will conjugate the spectral barcode fluorescent probes with DNA oligonucleotide (oligo) barcodes that will allow labeling of any mRNA target via an intermediary target- and barcode-specific oligo adapter. We will design, synthesize, and characterize a set of 1024 spectrally and DNA barcoded probes that can be used in single molecule fluorescence in situ hybridization (smFISH) to image any set of up to 1024 target RNAs, and will make this probe set available to the research community for use in spatial transcriptomics applications. We will develop these ultrahigh multiplexing fluorescent probes and methods for using them in spatial transcriptomics of thin and thick tissue samples by (1) synthesizing and characterize a set of 1024 unconjugated spectral barcodes; (2) demonstrating 128-plex single-round mRNA imaging in thin (~5-10 m) tissues; and (3) demonstrating 128-plex mRNA FISH in thick (> ~200 m) tissues. To ground our technology development with an impactful use case, Aims 2 and 3 will apply the probes to constructing a spatial cell atlas of the glomerulus in adult and aged mouse kidney tissues, which will complement our ongoing glomerulus atlas effort based on protein and carbohydrate imaging. 128-plex RNA imaging of thin tissue sections in a single round of staining and imaging would be a breakthrough technology (1-2 orders of magnitude high multiplexing than current single-round imaging methods), and ultrahigh multiplex RNA imaging is currently not possible in thick tissues; future work will increase the level of multiplexing. Project Number: 1R01GM163162-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Daniel Chiu (+1 co-PI) | Institution: UNIVERSITY OF WASHINGTON, SEATTLE, WA | Award Amount: $2,102,360 | Activity Code: R01 | Study Section: Enabling Bioanalytical and Imaging Technologies Study Section[EBIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11272021