Rational Design of NIR‐II Ratiometric Fluorescence Probes for Accurate Bioimaging and Biosensing In Vivo

Abstract Intravital fluorescence imaging in the second near‐infrared window (NIR‐II, 900–1700 nm) has emerged as a promising method for non‐invasive diagnostics in complex biological systems due to its advantages of less background interference, high tissue penetration depth, high imaging contrast, and sensitivity. However, traditional NIR‐II fluorescence imaging, which is characterized by the “always on” or “turn on” mode, lacks the ability of quantitative detection, leading to low reproducibility and reliability during bio‐detection. In contrast, NIR‐II ratiometric fluorescence imaging can realize quantitative and reliable analysis and detection in vivo by providing reference signals for fluorescence correction, generating new opportunities and prospects during in vivo bioimaging and biosensing. In this review, the current design strategies and sensing mechanisms of NIR‐II ratiometric fluorescence probes for bioimaging and biosensing applications are systematically summarized. Further, current challenges, future perspectives and opportunities for designing NIR‐II ratiometric fluorescence probes are also discussed. It is hoped that this review can provide effective guidance for the design of NIR‐II ratiometric fluorescence probes and promote its adoption in reliable biological imaging and sensing in vivo.