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.

高分辨率、多路复用实验是细胞成像的主要内容。然而，由于在可见（350-700 nm）和近红外（700-1,000 nm）波长下组织中存在大量散射和自发荧光，在动物身上进行的类似实验具有挑战性。在这里，我们通过设计用于短波红外（SWIR，1,000-2,000 nm）区域的光学造影剂和成像技术的互补进步，在动物身上实现了实时、非侵入性的多色成像实验。我们开发了可调谐的、发射 SWIR 的 flavylium polymethine 染料，并为这类明亮的 SWIR 造影剂建立了结构和光物理特性之间的关系。同时，我们设计了一个具有可变近红外/SWIR激发和单通道检测的成像系统，促进用于光学引导手术的视频速率多色 SWIR 成像以及具有多路检测的清醒和移动小鼠的成像。与 980 nm 和 1,064 nm 激光相匹配的优化染料与临床批准的吲哚菁绿相结合，实现了具有高时间和空间分辨率的实时三色成像。