High-resolution imaging of exosome pulmonary spatial distribution via fluorescent mRNA labeling and fMOST

Technical limitations hinder the clinical translation of exosomes for precise visualization of their in vivo pharmacokinetics. Conventional fluorescent labelling methods suffer from low signal-to-noise ratios, which hamper three-dimensional (3D), high-resolution, and quantitative analysis of exosome distribution at the whole-organ scale. To address this challenge, this study established an integrated "fluorescent mRNA labelling-fMOST imaging" technology system. Exosomes were labelled with fluorescent mRNA; in vitro characterization confirmed that the nucleic acid tags were efficiently loaded into exosomes without compromising their structural integrity, while demonstrating significantly superior signal-to-noise ratios and stability over traditional DiO dyes. Using light-sheet microscopy and quantitative analysis, we compared the pulmonary distribution of exosomes across different administration routes. The results indicated that lung accumulation following tail-vein injection was 2.48-fold higher than that of intranasal administration. Furthermore, fluorescence Micro-Optical Sectioning Tomography (fMOST) imaging provided a high-resolution 3D map of exosomes throughout the entire lung. The technology platform established in this study achieves single-exosome spatial resolution of in vivo distribution, offering key methodological tools and data support for evaluating the targeting efficiency of exosomal drugs and optimising delivery strategies, thereby facilitating their clinical translation.