High-Resolution Multicolor Shortwave Infrared Dynamic In Vivo Imaging with Chromenylium Nonamethine Dyes

Imaging in the shortwave infrared (SWIR) region offers fast, high-resolution visualization of in vivo targets in a multiplexed manner. These methods require bright, bathochromically shifted fluorescent dyes with sufficient emission at SWIR wavelengths-ideally above 1500 nm for high-resolution deep tissue imaging. Polymethine dyes are a privileged class of contrast agents due to their excellent absorption and high degree of modularity. In this work, we push flavylium and chromenylium dyes further into the SWIR region through polymethine chain extension. This panel of nonamethine dyes boasts absorbances as red as 1149 nm and tail emission beyond 1500 nm. These dyes are the brightest organic fluorophores at their respective bandgaps to date, with ε_max ∼ 10⁵ M^-1 cm^-1 and Φ_F up to 0.5%. Using two nonamethine dyes, Chrom9 and JuloFlav9, we performed two-color all-SWIR multiplexed imaging (Excitation at 1060 and 1150 nm; Emission collection at >1500 nm), enhancing the depths and resolutions able to be obtained in multicolor SWIR imaging with small molecule contrast agents. Finally, we combine the nonamenthine dyes with other SWIR-emissive fluorophores and demonstrate five-color awake imaging in an unrestrained mouse, simultaneously pushing the multiplexing, resolution, and speed limits of in vivo optical imaging.