UCLA

Fluorescence imaging in the shortwave infrared (SWIR) region is a powerful approach for the noninvasive visualization of biology due to favorable light-tissue interactions. Compared to other spectral regions, SWIR light improves depth penetration and reduces tissue scattering, and there are very few endogenous emitters contributing to autofluorescence. Collectively these properties lead to real-time deep tissue imaging with exemplary contrast. The irony is that the best wavelengths for imaging through skin and tissue usually correspond to some of the worst dyes. By nature, organic SWIR-emissive fluorophores have restricted brightness, and immense hydrophobicity, thus creating liabilities in the aqueous biological environment. While the field has rapidly evolved to address these shortcomings over the past few decades, major challenges remain to merge the optical advantages of the SWIR region with fluorophores that are bright, water-soluble, and functional all at once. This dissertation explores diverse approaches to optimize SWIR fluorophore development and advance performance for in vivo imaging studies in small mammals, illuminating design strategies that allow SWIR light to shine the brightest.Chapter One provides a perspective of the history and current landscape of clinical fluorescence imaging using the relatively small set of FDA-approved fluorophores and fluorescence imaging agents. This chapter also examines modern clinical motivations, shedding light on where the field is heading next.Chapter Two is a review of the development of SWIR-emissive chromenylium and flavylium polymethine fluorophores and excitation-based multiplexed imaging technologies, setting the stage for the advancements detailed throughout this dissertation.Chapter Three explores an array of hydrophobic SWIR-emissive fluorophores and in vivo delivery strategies, highlighting how each has enabled fundamental imaging applications in mice, as well as new SWIR imaging technologies, and an increased understanding of the optimal parameters to achieve deep tissue visualization. Ultimately, across many avenues of probe design, each of these hydrophobic dyes faced a similar in vivo fate–nonspecific uptake to the liver and other organs associated with the mononuclear phagocyte system.Chapters Four and Five detail synthetic design strategies to achieve a series of bright, water-soluble, and functional small molecule chromenylium polymethine dyes, thus enabling carrier-free in vivo delivery and cutting-edge preclinical imaging studies. Despite meeting all aforementioned requirements of optimal SWIR emitters, nonspecific in vivo interactions still dominated biodistribution patterns.Chapter Six takes a unique approach to merge SWIR brightness, water solubility, and in vivo “stealth” properties via a bright, SWIR-emissive star polymer dye scaffold (named Chromenylium Star, or CStar), leading to the first demonstration of renal clearance with chromenylium dyes. Building on this framework, Chapter Seven leverages polymer size to generate a series of size-differentiated CStars that provide precision control of in vivo biodistribution pathways.Chapter Eight sees the establishment of a versatile targeted CStar platform. Linking an affinity ligand outside the polymer shielding zone allows it to guide in vivo interactions to specifically visualize solid tumors over healthy tissues using noninvasive SWIR imaging.Finally, Chapter Nine explores a few additional developments of the CStar scaffold from a chemistry perspective. These avenues could be expanded upon for future in vivo use.Taken together, these studies integrate advances in chemistry and biological discovery to ring in a new era of SWIR fluorophore design strategies. With bright, water-soluble, and functional contrast agents that minimize nonspecific localizations in the body, the field moves one step closer to the clinical translation of SWIR imaging.