Engineered Multicolor NIR‐I/II Fluorescent Proteins for Precise In Vivo Visualization and Imaging‐Guided Surgery

ABSTRACT Even though existing near‐infrared probes have successfully achieved deep tissue imaging with high contrast, it remains challenging to image many species simultaneously under complicated biological conditions. We rationally design and synthesize a series of nonamethine dyes featuring a unique double meso‐Cl structure, which can covalently bind to proteins, resulting in significant fluorescence enhancement, thereby further filling a critical gap of multicolor fluorescent proteins (FPs) in 915 nm channel bioimaging. NIR‐940 exhibits distinct binding sites and modes with HSA compared to heptamethine dyes, which likely contributes to its high albumin binding efficiency. Given that FPs enhance fluorescence emission of the chromophore and are entirely dependent on the metabolic behavior of the host protein, this approach allows for the development of customized FPs tailored to specific imaging needs—an advantage unmatched by other NIR‐I/II contrast agents. By selecting various NIR‐I and NIR‐II chromophores with spectrally distinguishable emissions, we construct a multi‐channel imaging platform based entirely on FPs and validated its robustness and versatility in monitoring various physiological states across multiple local and systemic imaging applications, including the mesentery, retroperitoneal region, and stroke‐affected mouse brain.