SIM显微镜照明结构光场优化与系统点扩散函数表征

	SIM显微镜照明结构光场优化与系统点扩散函数表征
其他题名	Optimization of Structured Illumination Light Field and Characterization of System Point Spread Function in Structured Illumination Microscopy (SIM)
	鹿伟民
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	机械电子工程
关键词	荧光显微镜超分辨率结构光衍射极限数字微镜阵列
摘要	荧光显微镜具有可特异性标记、对活体细胞实时动态成像等优势，在细胞生物学研究中获得广泛的应用。然而，由于衍射极限的存在，传统荧光显微镜的分辨率受到限制。这严重阻碍了传统荧光显微镜在亚细胞水平研究中的进一步应用。为了突破衍射极限的限制，近年来，研究人员提出了一系列超分辨荧光显微技术。尽管如此，长时间活细胞成像对这些方法仍然存在着较大的挑战。结构光照明超分辨荧光显微成像技术(Structured Illumination Microscopy，SIM)是根据莫尔效应提出的一种技术。其原理为：利用空间有结构的光束来激发荧光，激发图形和荧光团密度的混合频率，将样品中通常不可见的高频信息携带到显微镜的可见低通频带；通过改变图案的方向和相位，记录荧光结果并对得到的多个图像数据集进行适当的处理，提取携带的高频信息并重建出超分辨率图像。SIM横向分辨率理论上可以达到传统荧光显微镜的两倍，且其成像速度快，对荧光标记没有特殊要求。这些特点使得SIM在细胞生理学、细胞动力学等亚细胞水平的生命科学研究中有更广阔的应用前景。本文基于LED光源和可编程数字微镜阵列(Digital Micro-mirror Device, DMD)，设计和搭建了结构光照明超分辨荧光显微镜。利用设计的结构光生成模块与奥林巴斯X73显微镜结合做成了SIM超分辨成像系统,并设计了SIM超分辨重构算法。对使用LED、DMD和物镜结合产生余弦结构光进行了理论分析以及仿真实验。通过理论分析、仿真以及实验，提出了使用荧光微球精确测量系统分辨率的新算法。使用自搭建显微镜(40倍、数值孔径0.75的物镜)对细胞肌动蛋白丝(激发/发射波长：650nm /668nm)成像，测量得到SIM系统分辨率为307nm，传统宽场荧光显微镜系统分辨率为545nm，较传统荧光显微镜SIM系统分辨率提高了1.78倍；使用自搭建结构光生成模块和奥林巴斯显微镜(100倍、数值孔径1.4的物镜)，对直径200nm的荧光微球(激发/发射波长：660nm /680nm)成像，测量宽场成像和SIM成像中荧光微球半高宽(Full Width at Half Maximum, FWHM)分别为328nm、203nm，利用本文设计的系统分辨率测量算法，得到宽场显微镜系统和SIM超分辨成像系统的分辨率分别为312nm、170nm，较传统宽场荧光显微镜，SIM系统的分辨率提高了1.84倍。实验表明，本文搭建的基于LED和DMD的结构光照明超分辨荧光显微镜系统突破了衍射极限的限制，实现了超分辨荧光成像。
其他摘要	Fluorescent microscope with the particular function of real time dynamic imaging living cells with special marker is widely used in cell biological study. However, due to the existence of the diffraction limit, resolution of traditional fluorescent microscope is limited. Hence, it cannot be used in sub-cellular level research. In order to break the diffraction limit and improve the resolution, a series of super-resolution fluorescent microscope have been proposed. Even so, long time research of living cells is still difficult. Structured Illumination Microscopy (SIM), based on Moiré effects, works by the follow steps. Firstly, it excites fluorescence with geometric light beam. Secondly, it modulates the invisible high-frequency signal of experimental sample to low-frequency band of fluorescence. Thirdly, it changes the orientations and phases of the geometrics and records multiple sets of data. Finally, it extracts information carrying the high-frequency signals from the data recorded. and rebuilds the super-resolution image. Theoretically, the lateral resolution of SIM can achieve twice of traditional fluorescent microscope. What‟s more, SIM has traits of fast imaging and no special requirements for fluorescent marker. It has widely application prospects in sub-cellular level research such as stoichiology and cyto-dynamics with these traits. In this paper, a structured illumination super-resolution fluorescent microscope with LED illuminant and programmable digital micro-mirror device (DMD) has been designed and set up, via combining the designed microscope with an Olympus X73 microscope. The design of the super-resolution rebuilt algorithm is very important. We have carried on the theoretical analysis about using LED, DMD and objective to generate cosine structure illumination, and put forward the new algorithm of measuring system resolution using fluorescent microspheres. The results of imaging of cell action filament, which excitation wavelength is 650nm and emission wavelength is 668nm, with magnification 40 and numerical aperture 0.75, shows the system resolution is 307nm. It is 1.78 times of the traditional fluorescent microscope resolution which was measured 545nm. The measurement of fluorescent beads, with 200nm in diameter, with magnification 100 and numerical aperture 1.4, shows resolutions are 328 nm and 203 nm respectively. The system resolutions respectively are 312nm and 170nm. It is 1.84 times of traditional fluorescent microscope resolution. Experiments show that the structured illumination fluorescence microscopy system that based on LED and DMD has been break through the diffraction limit, and has realized the super resolution imaging.
语种	中文
文献类型	学位论文
条目标识符	http://ir.ciomp.ac.cn/handle/181722/49261
专题	中科院长春光机所知识产出
推荐引用方式 GB/T 7714	鹿伟民. SIM显微镜照明结构光场优化与系统点扩散函数表征[D]. 中国科学院大学,2015.

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