基于荧光受激差分的纳米分辨显微方法研究

Because of the diffraction barrier, conventional optical microscopes cannot be used to detect those structures whose dimensions are below 100nm, thus limiting their application in biomedical and material researches.Although several super-resolution microscopy methods have been proposed in recent years, each method still has its own limitation.In this project, we will conduct researches on the applications of differential techniques in nano-resolved optical microscopy.(1) Based on differential excitation concepts, we propose a novel super-resolution imaging method which has the capability to realize spatial resolution beyond the diffraction barrier with low power cost and high imaging speed. When fluorescence saturation is applied, the resloving ability of this microscopy can be further enhanced.(2)We combine present super-resolution microscopy methods with differential excitation techniques and improve their imaging performance through excitation intensity subtraction and fluorescence lifetime subtraction.(3)We design and build a super-resolution imaging system.Meanwhile,we also integrate the hardware and software of the system.The implementation and accomplishment of our project will not only enhance the performance of present super-resolution methods, but also provide a novel cocept for the development of optical microscopy. In the meantime,some important generic problems in the production of high-end microscopes can also be solved.

常规光学显微术由于受到光学衍射极限的限制，无法满足对于亚百纳米尺度结构的探测需求，从而影响了其在生物医学、材料学等领域中的广泛应用。虽然近年来多种纳米分辨光学显微技术被提出，但是它们都还存在各自的特点与不足。本项目拟研究荧光受激差分在纳米分辨光学显微中的应用。（1）提出一种新型的荧光受激差分的纳米分辨光学显微术，可以在低功耗、高成像速度下实现超衍射极限的分辨率。当引入荧光饱和非线性效应时，此种显微术的分辨率可以进一步提高。（2）将荧光受激差分技术与现有超分辨显微技术相结合，通过受激荧光强度差分和荧光寿命差分的途径来改善现有超分辨方法的成像效果。（3）搭建一套多工作模式的超分辨显微系统，同时完成系统的软硬件集成。本项目的实施不仅将使现有光学超分辨显微方法变得更为完善，而且还将为光学超分辨的发展提供一种全新的思路，同时也解决了高端显微镜制造中的一些共性问题。

常规光学显微术由于受到光学衍射极限的限制，无法满足对于亚百纳米尺度结构的探测需求，从而影响了其在生物医学、材料学等领域中的广泛应用。虽然近年来多种纳米分辨光学显微技术被提出，但是它们都还存在各自的特点与不足。本项目研究荧光受激差分在纳米分辨光学显微中的应用。（1）提出一种新型的荧光受激差分的纳米分辨光学显微术，可以在低功耗、高成像速度下实现超衍射极限的分辨率。当引入荧光饱和非线性效应时，此种显微术的分辨率可以进一步提高。 （2）将荧光受激差分技术与现有超分辨显微技术相结合，通过受激荧光强度差分和荧光寿命差分的途径来改善现有超分辨方法，比如表面等离子基原激发的成像效果。（3）本项目实现三维FED 成像，得到生物细胞大视场三维超分辨效果。