Project summary/abstract Advances in in vivo biomedical imaging are critical for revolutionizing the ability of researchers and clinicians to peer inside the living body. These advances often catalyze major steps forward in our understanding of biomolecular and physiological processes. At present, the power of fluorescence imaging for deep tissue biomedical imaging is limited by the relative opacity of biological tissues and fluids at visible to short near-infrared wavelengths < 1,000 nm. The NIR-II tissue transparency window (1,000 to 1,700 nm) presents the opportunity to achieve molecular fluorescence imaging at centimeter-scale depths, for monitoring biomolecular processes at high spatial resolutions and in real time. To fully realize the transformative potential of NIR-II fluorescence deep tissue imaging, we must develop fluorophores that overcome major challenges of existing organic dyes and nanoparticles with NIR-II emission, such as low brightness, large size, low solubility, and toxicity. This proposed research program pioneers a new approach to develop small, bright, tunable, and biocompatible NIR-II emitters for targeted molecular imaging in vivo. We will exploit a novel class of NIR-emissive DNA-stabilized silver nanoclusters (AgN- DNAs) with 1-3 nm sizes, high-quantum yield emission, tunable fluorescence colors, and compatibility with nucleic acid chemistries. Recent experiments have uncovered the first AgN- DNAs with NIR-II emission and support the promise of creating a palette of these nanoclusters that emit throughout the NIR-II spectral region. Using high-throughput experimental screening and machine learning approaches, we will develop a set of bright, stable, and NIR-II emitting AgN- DNAs that are well-suited for in vivo imaging. In tandem, we will develop chemical strategies to transform these nanoclusters into biolabels for targeted molecular imaging by conjugating AgN- DNAs to aptamers, peptides, antibodies, and other biomolecules of interest. These hybrid biolabels will enable targeted staining and NIR-II fluorescence imaging of tumors, organs, and other targets. The utility of the new NIR-II biolabels for fluorescence imaging will be assessed in tissue models and then tested in vivo in mouse models for vascular imaging and for tracking novel breast cancer therapeutics. We envision that these new fluorescent probes will enable a new era of deep tissue fluorescence imaging, with a versatile range of applications from cancer research and therapeutics development to microvascular imaging.

Project narrative Fluorescence imaging is a crucial tool for monitoring biomolecular processes at high spatial resolutions and in real time but has limited utility for deep tissue imaging because the few available fluorophores in the NIR-II tissue transparency window (1,000 to 1,700 nm) suffer from challenges such as low brightness, large size, and toxicity. This proposed work pioneers a new approach to develop small, bright, tunable, and biocompatible NIR-II emitters for targeted in vivo molecular imaging, by exploiting a new class of NIR-emissive DNA-stabilized silver nanoclusters. We envision that these new NIR-II fluorescent probes will transform the field of deep tissue fluorescence imaging, with applications ranging from cancer research to vascular imaging.