Ting Han, Zi-Han Chen, Hongyue Liu, Jiang Ming, Xiaohan Wang, Zhihua Wang, Wenlin Li, Caiyun Fang, Yong Fan, Fan Zhang
Near-infrared (NIR, 750-2500 nm) imaging is crucial for disease diagnosis, yet conventional wide-field bioimaging yields only two-dimensional (2D) information without depth. Here, we report a ratiometric fluorescence strategy for tissue depth determination using α-NaTmF4@NaErF4@NaYF4 nanoprobes. Under 808 nm excitation, interfacial cascade energy transfer between Tm3+ and Er3+ ions enhances NIR-II emissions at 1532 and 1626 nm by 3.3- and 6.7-fold, respectively, compared with that of a codoped counterpart. Leveraging distinct water absorption at these wavelengths, the logarithm of the attenuated intensity ratio (ITm/IEr) varies linearly with tissue depth (0-4 mm). With this NIR ratiometric wide-field depth-mapping method, quantitative depth information about in vivo vasculature was assigned to each image pixel and rendered into 2.5D visualization by stacking 2D planes over a 40 mm × 40 mm × 4 mm volume. Dynamic tracking of nanoprobe redistribution from vessels along the projection direction was also demonstrated. This work establishes a universal depth-analysis tool and significantly advances medical optical imaging.