远场光学超分辨技术打破了衍射极限，革新了生物成像技术。其中，受激发射损耗STED技术可三维成像且无需图像处理，应用广泛。新型光子上转换UCNPs超分辨解决了传统荧光探针预算光子数低问题，并降低了损耗光强，但STED发展仍面临四个瓶颈：①发光损耗方案特异性强，通用性差；②多色STED超分辨系统过于复杂；③饱和损耗光强仍然较大；④成像深度低。如何克服？迫在眉睫。. . 本项目将基于能级理论和上转换机理，对发光离子采取“釜底抽薪”多能级级联损耗，实现全光谱损耗/检测；对敏化离子提出“拔本塞源”策略，通过非线性效应级联放大受激发射损耗，降低激光光强，并同时损耗多种探针；实现单对CW激光照射的多色超分辨成像，简化系统大大降低成本；拓展亚细胞成像，并用长波长STED实现深度成像。本项目将独辟蹊径突破这些瓶颈，推动STED领域分支发展，拓展UCNPs的生物光子学应用。

The technology of far-field optical super-resolution fluorescence microscopy broke the long-standing diffraction limit, significantly improving the microscopic spatial resolution and revolutionized the optical bio-imaging technology. As one of them, Stimulated Emission Depletion (STED) microscopy has been widely applied in different areas, which not only can enable 3D cell sectioning but also produce digital image without any reconstruction postprocessing. This novel type technology, UCNPs-based super-resolution, solved the problem of very limited photon budget of STED probe and decreased the depletion intensity. However, the following problems urgently need to be addressed for the future development of STED, including: un-universal optical depletion method for various fluorescent probes due to the specification of excitation and depletion, the sophisticated complexity of implementing multi-color STED super-resolution microscopy system, severe photon-induced damage caused by high light intensity and the limited penetration depth (induced by short wavelength and large scattering). . .This project/proposal will be carried out based on the energy level theory and the upconversion luminescence mechanism. To achieve full luminescence spectrum depletion and detection, we will realize the multi-level cascading depletion to the activator of interest via the strategy “taking away the firewood under the cooking pot”. Furthermore, in order to greatly reduce the required laser intensity and realize a facile depletion mechanism applicable for various probes, we proposed to treat the sensitizers and the activators separately, and selectively deplete the sensitizers, which can often sensitize a class of activators with nonlinear power dependence. Simultaneously, the activator will be more easily depleted, with the help of the nonlinear cascading amplification effect. Multi-color STED super-resolution microscopy based on one single pair of CW lasers irradiation would be realized, which will greatly simplify the system and reduce the cost, facilitating the popularity of this advanced technology. By using lasers centered at NIR-II region, large depth penetration would be expected, especially in brain-like tissue imaging and subcellular imaging. With the aim to address all of the aforementioned issues, this project/proposal will be highly of significance for both the advance of STED technique and further development of UCNPs-assisted optical biomedical applications.