作为一种新兴的成像技术，近红外二区（NIR-II，1000-1700纳米）荧光成像具有在生物医学领域具有很大的潜力，因其高灵敏度、深组织穿透和卓越的空间和时间分辨率。然而，如何促进NIR-II荧光成像在医学科学和药学等一些急需领域的应用，一直困扰着相关研究人员。本协议详细描述了一种近红外二区荧光分子探针HLY1的构建和生物成像应用，其中D-A-D（供体-受体-供体）骨架。HLY1表现出良好的光学性质和生物相容性。此外，使用NIR-II光学成像设备在小鼠身上进行了NIR-II血管和肿瘤成像。实时高分辨率的NIR-II荧光图像获得，用于指导肿瘤和血管疾病的检测。从探针制备到数据采集，成像质量得到了极大的改善，并确保了用于活体成像的NIR-II分子探针的数据记录的真实性。

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.