Time-Gated Ratiometric Detection with the Same Working Wavelength To Minimize the Interferences from Photon Attenuation for Accurate in Vivo Detection

[Image: see text] Luminescence imaging, exhibiting noninvasive, sensitive, rapid, and versatile properties, plays an important role in biomedical applications. It is usually unsuitable for direct biodetection, because the detected luminescence intensity can be influenced by various factors such as the luminescent substance concentration, the depth of the luminescent substance in the organism, etc. Ratiometric imaging may eliminate the interference due to the luminescent substance concentration on the working signal. However, the conventional ratiometric imaging mode has a limited capacity for in vivo signal acquisition and fidelity due to the highly variable and wavelength-dependent scattering and absorption process in biotissue. In this work, we demonstrate a general imaging mode in which two signals with the same working wavelength are used to perform ratiometric sensing ignoring the depth of the luminescent substance in the organism. Dual-channel decoding is achieved by time-gated imaging technology, in which the signals from lanthanide ions and fluorescent dyes are distinguished by their different luminescent lifetimes. The ratiometric signal is proven to be nonsensitive to the detection depth and excitation power densities; thus, we could utilize the working curve measured in vitro to determine the amount of target substance (hypochlorous acid) in vivo.