基于小分子荧光探针的膨胀超分辨显微成像标记方法的研究

Expansion super-resolution microscopy is an optical imaging technology that combines expansion microscopy with super-resolution imaging. Due to the limitations of current labeling methods, it is difficult to break through the resolution of 10 nm. In order to solve the problem that the labeling density and brightness of the existing small-molecule fluorescent probes are insufficient and there is no specific sample treatment scheme for small-molecule probes, a new labeling method based on small-molecule fluorescent probes for expansion super-resolution microscopy is proposed in this study. (1) Design and labeling of small-molecule fluorescent probes. We will study the labeling mechanism of fluorescent probes and carry out ingenious structural design. By means of multi-epitope labeling and the introduction of multiple fluorescent dye molecules into the probes, we will improve the labeling density, brightness, and other properties of fluorescent markers. (2) We will study the sample treatment which is suitable for small-molecule fluorescent probes, and optimize the existing expansion process to achieve the best performance of the small-molecule fluorescent probes. (3) We will optimize the imaging conditions to realize the sub-10-nm resolution of mono-color, or even multi-color, 3D expansion super-resolution microscopy. The implementation and accomplishment of this project can solve the current technical problems in the fluorescence labeling density, brightness, marker sizes, and multi-color labeling in expansion super-resolution microscopy. In a word, our work will have great importance both in theoretical research and practical use.

膨胀超分辨显微成像是将膨胀显微与超分辨成像结合起来的光学成像技术，由于目前标记方法的限制而很难突破10 nm的分辨率。针对现有的小分子荧光探针在该成像技术中标记密度和亮度不足、没有针对性的样品处理方案的问题，本研究提出一种新的基于小分子探针的膨胀超分辨显微成像标记方法。（1）小分子荧光探针的设计和标记，研究荧光探针的标记机理，进行巧妙的结构设计，通过多表位标记和在探针中引入多个荧光分子等方法，提升荧光标记的标记密度、亮度等性能；（2）研究适用于小分子荧光探针的样品膨胀处理方法，对现有的膨胀样品处理流程进行优化，以实现小分子荧光探针的最佳性能；（3）优化成像条件，实现单色3D膨胀超分辨显微成像亚10 nm的分辨率，实现具有生物学意义的双色/多色成像。本课题的实施，可以解决目前在膨胀超分辨显微成像中荧光标记的密度、亮度、标记物尺寸、多色标记等方面的技术难题，具有十分重要的理论价值和应用前景。

膨胀显微与超分辨显微成像的联用，为进一步提高超分辨显微成像的分辨率提供了可能。然而，超分辨显微成像对光子数的要求较高，但样品膨胀带来了大量的标记密度和亮度损失，因此，膨胀超分辨显微成像很难达到三维的亚10 nm分辨率。.本项目围绕基于小分子荧光探针的膨胀超分辨显微成像方法，通过设计、构建一系列膨胀样品兼容的小分子荧光探针，解决目前荧光标记方法的标记密度、亮度、尺寸和颜色限制，配合膨胀方法和流程的优化，实现亚10 nm分辨率的3D膨胀超分辨显微成像和多色成像：① 设计合成了一系列适用于膨胀超分辨显微成像的小分子荧光探针，其膨胀后的荧光亮度和信背比均为其他标记方法的2倍以上，且多色可选；② 优化了适配小分子荧光探针标记的水凝胶膨胀处理方法和整个膨胀流程，有效提高膨胀后的荧光强度和样品还原度；③ 实现单色的3D亚10 nm分辨率光学显微成像，揭示了纳米级分辨率的微丝、溶酶体、线粒体的蛋白质空间分布；④ 实现双色的3D膨胀超分辨显微成像，揭示了微丝、溶酶体、线粒体蛋白质的纳米级空间分布关系及其相互作用。.本项目所开发的基于小分子荧光探针的膨胀超分辨显微成像方法，实现了三维的亚10 nm分辨率光学成像，在细胞生物学、生物医学光子学、医疗健康等领域具有广泛的应用前景。