新型上转换纳米探针光控发光损耗机理及其类STED超分辨成像

With the rapid development of science and technology, the spatial resolution of the traditional optical microscope cannot satisfy the requirement of biomedical and life science research any more. As an important modality of super-resolution imaging techniques, Stimulated Emission Depletion (STED) microscopy can optically break the diffraction limit and has been widely applied in different areas, with the advantages of rapid imaging (without reconstruction process) as well as the ultrahigh lateral resolution. However, the following problems need to be addressed for the future development of STED, including small penetration depth (induced by short wavelength and large scattering), annoying photobleaching of STED probes, limited sensitivity caused by overfiltering the fluorescence signals due to the spectral overlap of the wavelengths of two employed lasers and the fluorescence spectrum...This project/proposal will be based on lanthanide-ions luminescent theory and clarify the effective optical depletion mechanisms of upconverting luminescence. By establishing high efficient optical depletion schemes, we will design and synthesize controllable novel upconverting probes for STED-like multiphoton microscopy, which would be demonstrated using two relatively long wavelength lasers. We will also construct multi-photon super-resolution microscopy system with ultrahigh resolution and large imaging depth. As lasers and luminescence will be separated, full luminescence spectrum will be realized. By using lasers centered at NIR-II region, sub-100-nm resolution would be obtained in the large depth of brain-like tissue. Meanwhile, long standing continuously microscopic imaging could be realized by taking the advantage of non-photobleaching property. Aiming for solving the above three issues, this project/proposal will be highly of significance for both the advance of STED technique and further development of UCNPs’ optical biomedical applications.

科学发展迅速，传统光学显微镜分辨率已无法满足生物医学等领域的需求。作为超分辨技术之一，受激发射损耗显微术STED从光学上打破衍射极限，有成像速度快、横向分辨率高等优势，适用范围广。但STED技术的发展遇到一些瓶颈：①短波长激光散射大，成像深度低；②常见STED荧光探针有光漂白；③双激光都与荧光光谱重叠，滤光导致荧光光谱成分牺牲，限制灵敏度。这些问题都有待解决。..本项目拟将基于稀土离子发光等理论，研究阐明光控上转换发光损耗机理，建立高效光控损耗方案，构筑新型上转换探针UCNPs实现双长波长激光的类STED成像，可控制备相应UCNPs；搭建多光子、高分辨率、大深度的超分辨成像系统；光谱不重叠，实现全荧光光谱检测；用光学窗口II区激光对脑样组织实现大深度、超分辨成像；零光漂白实现长时间持续成像。本项目将攻克三大瓶颈，对STED技术的发展意义重大，也必将推动UCNPs在生物医学应用中的长足发展。

科学发展迅速，传统光学显微镜分辨率已无法满足生物医学等领域的需求。作为超分辨技术之一，受激发射损耗显微术STED从打破光学衍射极限，成像速度快，横向分辨率高，适用范围广。但STED技术的发展遇到一些瓶颈，比如激光光强过大，荧光探针光漂白，灵敏度受限等。.本项目以理论为基础以实验为手段，围绕上述问题开展了系列研究并取得诸多进展。主要包括：⑴建立全新的基于离子间强交叉驰豫的高效受激发射荧光损耗机理（Nat. Commun, 2017, 8, 1058）；⑵搭建了上转换超分辨系统，较传统STED将功率降低了2个数量级，并标记细胞骨架微丝结构，首次实现实时无光漂白光闪烁现象的上转换超分辨生物成像（Nat. Commun, 2017, 8, 1058）；⑶面向高速度上转换超分辨成像，对上转换荧光非稳态过程建立理论模型，阐明了敏化过程、能量传递效率等影响寿命长短的作用机理，理论指导如何缩短寿命（Nanoscale 2019, 11, 4959-4969）。基于此，通过引入敏化离子高浓度团簇，将寿命缩短至原来1/5，实现了10微秒/像素的快速超分辨成像（Nanoscale 2019, 11, 1563-1569）；⑷利用上转换超饱和特性对空心光半高宽的收缩作用，实现了单激光器的上超分辨成像(Opt. Express 2017, 30885)。构建了激发正交的上转换探针，通过双光束激发检测双通道，实现单次扫描超分辨成像 (Nanoscale 2018, 10, 21025)；⑸为提高超分辨成像信噪比，研究发现分子振动多阶谐振耦合导致的能量损耗，为增强探针亮度提供新的理论基础；发展了选择性加热原子的激光退火技术，使单颗粒发光增强高达484倍（ACS Nano 2018, 12, 10572）；⑹基于对上转换发光的偏振性能的研究，探索稀土掺杂微晶阵列的各向异性（Small 2019, 15, 1904298）；设计与制备UCNPs和AuNP组成的超晶格结构，实现发光调制（Small 2020, 16, 2066）；⑺开发一种STED超分辨图像背景噪声差分抑制方法（发明专利：CN111474150A）。综上，本项目所取得的进展成果，对上转换荧光探针应用具有重要意义，推动了STED技术在生物医学应用中的长足发展。