应用于三维STORM超分辨率成像的轴向纳米定位技术研究

	应用于三维STORM超分辨率成像的轴向纳米定位技术研究
其他题名	Research on Single Molecules Axial Location inThree-Dimensional Stochastic Optical Reconstruction Microscopy
	张世超
学位类型	硕士
导师	熊大曦
	2015-10
学位授予单位	中国科学院大学
学位专业	光学工程
关键词	三维超分辨率成像荧光显微镜柱透镜标定曲线
摘要	超高分辨率荧光成像技术的发展，使得突破光学衍射极限的活体实时观测成为可能，为研究细胞内特定蛋白以及基因调控提供了强有力的研究手段。其中，基于单分子定位技术的随机光学重建显微技术(STORM)空间分辨率达到了约20nm，使得STORM 成像技术在很多领域得到了应用。相对于横向分辨率的极大提高，目前仍没有合适的办法可以实现纳米级的纵向成像分辨率，研究如何在轴向提高荧光分子定位的精度仍是超分辨成像领域的热点问题。本文研究了柱透镜参数与点扩散函数椭圆率的相互关系，完成了成像仿真，设计并搭建了STORM-SIM 系统，优化了柱透镜参数的选择，利用所搭建的系统完成了对细胞微丝骨架的3D-STORM 与2D-SIM 成像。第一部分主要介绍3D-STORM 成像的算法及完成成像过程的仿真(第2 章)。讨论了3D-STORM 采用的单分子定位算法。详细描述了质心法与高斯拟合法的原理，并利用MATLAB，使用高斯拟合算法完成3D-STORM 的模拟工作。第二部分主要介绍了基于柱透镜的三维超分辨率成像系统的搭建以及柱透镜参数的优化(第3、4 章)。设计了一套基于OLYMPUS IX-83 搭建的STORM-SIM 光学系统，完成各个部分的调试工作，包括光源模块、光路切换模块和荧光成像模块等。设计柱透镜安装位置，利用三个不同的柱透镜，研究如何优化柱透镜参数的选择。该系统光路简单，调试方便，在普通实验室也能实现。结果表明，该系统3D-STORM成像轴向分辨率可以达到约90nm。第三部分主要介绍成像系统工作于SIM 模式下的情况(第5 章)。详细介绍SIM 成像过程与算法，利用DMD 生成矩形光栅，经傅里叶变换后滤波产生正负一级光束在焦平面进行干涉产生余弦条纹照明样品。着重介绍了系统中的关键技术与参数，并进行实验成像。最后，对全文做出了总结，分析了实验中的不足和可行的解决方法，并对超分辨率成像进行了展望。
其他摘要	Super-resolution microscopy greatly improves the fundamental research of biology science and provides an important tool to reveal the essence of life. Stochastic Optical Reconstruction Microscopy(STORM), based on single molecule localization technology, is a key method for super-resolution imaging. The resolution of STORM can be approximately 20~50nm in lateral and 70nm in axial direction. Despite these breakthroughs, there is no precise and easy-to-use method to localize single molecules along axial direction, which limits the application of single molecule fluorescence and super-resolution microscopy in biology. In this paper the relationship between the ellipticity of point spread function and the focal length of cylindrical lens has been investigated. We completed the simulation of imaging and built a STORM-SIM microscope. Then the parameters of cylindrical lenses was optimized. As the demonstration, we reconstructed 3D-STORM and SIM image of actin filaments. The first section mainly introduces the 3D-STORM imaging algorithm and simulation process. In this section, we discussed the difference imaging algorithm, include Centroid algorithm and Guassian fitting. With MATLAB, we finished the 3D-STORM imaging process using Guassian fitting. The second part mainly focuses on setting up the three-dimensional super-resolution microscopy, based on optical astigmatism method with cylindrical lenses and optimized cylindrical lens. The imaging system was built up based on Olympus's inverted fluorescence, including light source module, laser selection moduel, total internal reflection subsystem, and fluorescence imaging subsystem. With three different focal length lenses, the point spread function in custom built stochastic optical reconstruction microscopy instruments has been measured. A method to evaluate the cylindrical lens was developed based on linear region and localization error. The results show that 90nm axial resolution can be achieved with suitable focal length. The third part is about the SIM imaging mode of STORM-SIM system. Firstly introduced the SIM imaging process and algorithm in detail. The grating is generated by digital micro-mirror device(DMD). With fourier transformation, 1-order laser beams are generated. Then, the two beams of laser interference occur in the focal plane and generate cosine fringes to illuminate samples. At last, the SIM-images of actin are demonstrated. In last part, the paper made a summary. The shortcomings of our experiment were analyzed. Then possible solutions were proposed. Finally, some prospects for super-resolution microscopy were introduced.
语种	中文
文献类型	学位论文
条目标识符	http://ir.ciomp.ac.cn/handle/181722/49326
专题	中科院长春光机所知识产出
推荐引用方式 GB/T 7714	张世超. 应用于三维STORM超分辨率成像的轴向纳米定位技术研究[D]. 中国科学院大学,2015.

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