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DNA advanced high-plex RNA imaging for 3D deep tissue and single-cell spatial transcriptomics

Award Number: R35GM165539
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/15/2026
PERIOD OF PERFORMANCE END DATE: 04/30/2031

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DNA advanced high-plex RNA imaging for 3D deep tissue and single-cell spatial transcriptomics - High-plex fluorescence imaging is critical to reveal spatial bimolecular complexity to understand tissue functions and disease mechanisms. Unambiguous fluorescent signal switching between different target molecules is essential to achieve high-plex fluorescent imaging. Traditional multiplex fluorescence imaging is typically limited to 3–5 colors due to the spectral overlap of fluorescent dyes. Fluidic exchange-based sequential DNA imaging has overcome this limitation and become the most widely used multiplexed imaging approach. It has achieved hundreds to thousands of RNA imaging in cells and tissues. Despite the success, they face significant drawbacks: (1) slow signal switching, taking tens of minutes to hours, and (2) complicated fluidic setups prone to issues like air bubbles and tissue damage. As a result, these methods are not widely adopted as a routine method for multiplexed imaging. Furthermore, tissue samples are intrinsically 3D organization of cells, and the capability of imaging 3D deep tissue gives context-rich 3D transcriptomic information to significantly enhance our understanding of tissue functions and diseases mechanisms. However, fluidic-exchange with DNA imagers for high-plex imaging in 3D tissue is extremely time-consuming, where days of incubation times further impede their use. To address these challenges, we propose rapid and wash-free fluorescent signal-switching methods based on rapid and orthogonal DNA displacement reaction-based signal exchange (DIRSE) for high-plex RNA imaging in cells and tissues. The method allows theoretically unlimited rapid and wash-free multiplexed RNA imaging with no additional accessories, and each round of signal exchange can be taken within 30 seconds. In objective 1, we will establish the DIRSE multiplexed imaging platform and develop a set of 60 different toehold probes with rapid kinetics through computational design and experimental screening. By using three fluorophore channels for the 60 probes, we’ll achieve sequential, rapid, and wash-free multiplexed imaging of 60 distinct RNA species in cells and tissues. In objective 2, we will adapt DIRSE with a high-plex in situ amplification to develop a rapid high-plex (up to 60-plex) RNA imaging in deep 3D tissues (> 500 µm thickness) and benchmark the platform with deep 3D tissues in mouse brain CA3 regions. The method will pave the way for future high- plex RNA imaging at the whole-brain scale. Finally, in objective 3, we will scale up RNA imaging multiplexity to the transcriptome scale by combinatorially encoding the 60 developed DNA probes. The new transcriptome imaging platform will be designed to be simple and rapid, with wide applicability in a standard fluorescence microscope, requiring no additional accessories. Our proposed method will provide researchers with a new suite of spatial transcriptomics technologies that address the unmet challenges in 3D deep tissue and throughput. This innovation has the potential to significantly advance imaging based transcriptomic profiling, offering broad applicability across biological research.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $422,125 )
2026	2026	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	000	1	8/5/2026	NEW	$422,125
														Subtotal = $422,125

Grand Total All Awards = $422,125

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DNA advanced high-plex RNA imaging for 3D deep tissue and single-cell spatial transcriptomics

Award Number: R35GM165539

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 08/15/2026

PERIOD OF PERFORMANCE END DATE: 04/30/2031

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