基于各向同性暗斑的三维纳米分辨显微方法的研究

Stimulated emission depletion (STED) is a method that can be used for fluorescence nanoscopy in the far-field. Currently, STED systems still have many disadvantages, including the limited axial resolution which stems from the fact that the axial length of the STED spot is much larger than its transverse one, the high average power which will easily bleach the fluorescent samples and the poor stability of the system. To solve the problems above, we proposed a new scheme for the CW-STED system. (1) Using vectorial modulation methods to modulate the polarization, phase and amplitude of the STED beam and making the beam interfere at the sample plane, which creates an isotropic dark spot, both the axial length and transverse length of the spot are reduced ,thus improving the resolution of the system. Based on this isotropic dark spot, we will be able to reduce the photobleaching effect of the sample and attain a spatial resolution of 10-30 nm with lower average power.(2) By automatic detection and compensation for the beam drift, we improve the stability of the system. (3) Combining TCSPC module together with STED microscopy, we realize the simultaneous superreslution imaging for both the intensity and the lifetime of the fluorescent sample. The implementation and accomplishment of our project not only provide methods for researchers to investigate and measure in the realm of biomedical science, but also solve some important generic problems in the high-end microscopes. In a word, our work will have great importance both on theoretical research and practical use.

受激发射损耗显微（STED）是一种远场纳米分辨荧光显微方法。针对目前三维STED系统中， STED光所成三维暗斑的轴向尺寸远大于其横向尺寸，从而限制了其轴向分辨率，STED光功率过高造成样品漂白，系统稳定性差等问题，本项目提出了一种新的连续激光STED显微方法。（1）拟采用偏振、位相、振幅等矢量调控技术使STED光束在样品面处发生矢量干涉，形成各向同性的三维STED暗斑，大大压缩了其轴向尺寸，提高系统分辨率。基于该各向同性暗斑，可以在较低的STED光功率下降低样品漂白、实现三维10-30nm的系统分辨率。（2）拟采用多光束漂移自动监测与补偿方法来提高系统的稳定性。（3）将TCSPC模块与STED技术有机结合，实现超分辨下荧光强度与荧光寿命同时成像。本课题的实施，既为生物医学领域提供纳米分辨下的多功能观测手段，又能解决高端显微镜中的一些共性关键技术，具有十分重要的理论价值和应用前景。

受激发射损耗显微（ STED）是一种远场纳米分辨荧光显微方法。本项目针对目前三维STED 系统中， STED 光所成三维暗斑的轴向尺寸远大于其横向尺寸， 从而限制了其轴向分辨率， STED 光功率过高造成样品漂白，系统稳定性差等问题，提出了一种新的连续激光STED 显微方法：（ 1）采用了偏振、位相、振幅等矢量调控技术使 STED 光束在样品面处发生矢量干涉， 形成各向同性的三维 STED 暗斑， 大大压缩了其轴向尺寸， 提高了系统分辨率。基于该各向同性暗斑，可以在较低的 STED 光功率下降低样品漂白、实现了横向方向30nm的系统分辨率。（ 2）采用了多光束漂移自动监测与补偿方法来提高系统的稳定性。（ 3） 将 TCSPC 模块与 STED 技术有机结合，实现了超分辨下荧光强度与荧光寿命同时成像。本课题为生物医学领域提供了纳米分辨下的多功能观测手段，又解决了高端显微镜中的一些共性关键技术，具有十分重要的理论价值和应用前景。