Chromenylium and Flavylium Polymethine Fluorophores Light Up the Shortwave Infrared Region

The shortwave infrared (SWIR or NIR-II) region of the electromagnetic spectrum is exceptional for performing fluorescence imaging through skin and tissue. These long, low-energy wavelengths of light provide higher contrast, sensitivity, and imaging depth compared to visible and near infrared light. Though the advantages of SWIR imaging are well established, imaging setups are often custom-built, and there are currently no FDA-approved SWIR fluorophores. It is, however, an exciting time for fluorescence imaging in the clinic. With several new FDA-approved fluorophores in recent years, there is growing interest in advancing the landscape of fluorescence imaging for both diagnostic and therapeutic pursuits. To translate SWIR imaging from fundamental science to clinical applications, progress in both imaging technology and contrast agent design are two crucial, intimately linked factors. This Account details our journey to design biocompatible SWIR-emissive chromenylium- and flavylium-based polymethine fluorophores. Classically, the low band gaps and extended structural conjugation required to achieve SWIR emission compromise the brightness, stability, and aqueous solubility of organic dyes. The driving hypothesis of these studies is that rigorous structural derivatization can illuminate key design principles to overcome these challenges and generate bright, water-soluble, and functional SWIR dyes. Our story begins with Flav7, the first SWIR fluorophore specifically designed for in vivo imaging. We then detail lessons in heterocycle and polymethine linker design principles. From this, 7-, 2-, and C4'-position modifications provided insights for modulating the peak absorption wavelength (λ_max,abs) and fluorescence quantum yield (Φ_F). Since chromenylium and flavylium polymethine dyes maintain high absorption coefficients (ε_max), their total brightness (ε_max × Φ_F) is excellent. Overall, the chromenylium dyes (e.g., Chrom7) proved to be a privileged scaffold for SWIR imaging. To maximize both fluorescence signal and multiplexing abilities, we focused on matching the λ_max,abs of fluorophores to commercial laser lines. This approach has enabled high resolution excitation-based multiplexed imaging with up to five fluorophores in mice in real time, at video frame rates. Building on these design principles, this Account then highlights our strategies to achieve water-soluble and functional SWIR-emissive dyes. We leverage late-stage click chemistry to install hydrophilic moieties via two distinct approaches: 1) small, charged groups or 2) short poly(2-methyl-2-oxazoline) polymer chains. The first strategy resulted in small-molecule dyes SulfoChrom7, AmmonChrom7, and PhosphoChrom7 with diverse functionalities, while the second gave a unique star polymer architecture named "chromenylium star" or "CStar" (CStar30). With optimized bright, functional, and water-soluble dyes in hand, we look toward clinical applications in vascular imaging with SulfoChrom7, lymphatic imaging with CStar30, bone imaging with PhosphoChrom7, and in vivo cell tracking with AmmonChrom7. Finally, we propose that excitation-multiplexed image-guided surgery in the SWIR region can advance existing clinical technologies. Broadly, we anticipate chromenylium and flavylium fluorophores to continue to light up the SWIR region, signaling a new era in optical imaging.