Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350-700 nm) and near-infrared (700-1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000-2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.