共聚焦激光扫描光学显微成像关键技术研究

	共聚焦激光扫描光学显微成像关键技术研究
	魏通达
学位类型	博士
导师	唐玉国
	2014-07
学位授予单位	中国科学院大学
学位专业	光学工程
摘要	共聚焦激光扫描光学显微成像(CLSM)，不同于一般的荧光显微成像，它在光路中加入与物点共轭的探测针孔，起到空间滤波作用，能够去除离焦荧光的干扰，并可进行光学层切，CLSM通过逐点逐层的扫描方式，实现对观测样品区域的三维成像。CLSM是无损伤的光学远场成像技术，能够同时提供较高的横向和轴向分辨率，这使得它在生物医学领域有着极高的应用价值，同样也应用在材料科学领域。CLSM本身也具有极高的研究价值，其成像质量和性能上都可以进一步提高，并为新技术的研究提供良好的拓展平台。为此，展开了对共聚焦激光扫描光学显微成像中的关键技术研究，并把这些关键技术应用到CLSM原理样机中。一个完整的CLSM系统极为复杂，包括了许多的关键技术，结合专业特点，本文主要从以下几个关键技术角度展开研究：1. 成像理论研究。从基本理论出发，以Richards-Wolf矢量衍射理论为基础，进行共聚焦光学成像理论研究。推导照明点扩散函数、探测点扩散函数和共聚焦点扩散函数。研究数值孔径、多层介质和针孔因素等对它们的影响。2. 扫描成像技术研究。以扫描振镜的扫描方式为基础，研究扫描光路和对扫描振镜的控制，提出两种不同的扫描校正方式，即扫描透镜校正和反正切振镜驱动信号校正。3. 多光谱成像技术研究。分为两部分，一是多波长激光激发，实现不同波的合束和选通；二是多光谱荧光探测，实现荧光光谱的分光探测。4. 受激辐射损耗技术研究。分析其理论原理，运用切向偏振光作为损耗光形成中空光斑，并通过波前的相位和振幅调制优化，进一步缩小其尺寸，达到突破衍射极限的光学分辨能力。5. 成像系统搭建和成像实验。搭建完整的CLSM成像系统，实现较高分辨率的三维成像，就相关因素对成像质量的影响进行了分析，并完成共聚焦原理样机的研制。最后，对所做的研究内容进行了总结和分析，并对共聚焦激光扫描显微成像进行了展望。
其他摘要	Confocal laser scanning optical microscopy imaging (CLSM) is different from conventional fluorescence microscopy imaging. A point detection pinhole, which is conjugated with the object point, is employed in the optical path. It plays the role of spatial filter that can remove the interference of defocus fluorescence, and do the optical section. In the way of point by point and layer by layer scanning, CLSM achieves the observation of the sample area in three dimensions. CLSM is a kind of far-field optical imaging technology without damage, and also it provides high lateral and axial resolution at the same time, which makes it has a high using value in the biomedical filed, even applied in the materials science filed. CLSM itself has high research value, its image quality and performance can be further improved, also it provides a good expanded platform for the study of new technologies. For these reasons, it is necessary to do some researches of the key technologies in confocal laser scanning microscopy, and to introduce these key technologies into the development of propotype of CLSM.A complete CLSM system is extremely complex, including a number of key technologies. Considering the professional features, in this paper several key technologies were studied as follow: 1. Imaging theory. Based on the Richards-Wolf vector diffraction theory, optical confocal imaging theory was studied from the very beginning. The point spread functions (PSF) of lighting, detection and confocal were derived. Also the effects of numerical aperture, multi-media and pinholes factors on the PSFs were discussed.2. Scanning technology. Applied with galvanometer scanning mirrors, we studied the scanning optical path and the control of galvanometer mirrors, proposed two different scanning correction methods, namely scanning lens correction and arc tangent galvanometer drive signal correction.3. Multispectral imaging technology. It can be divided into two parts: first, multi-wavelength laser excitation, to achieve the combination and selection of different wave beams; second, multispectral fluorescence detection, to achieve the spectral detection of fluorescence.4. Stimulated emission depletion. We analyzed its theoretical principles, and the tangential polarized incident light was used as the depletion beam to shape the hollow focus spot. By optimizing the wavefront with the phase and amplitude, the size of hollow focus spot could be further reduced, and the optical resolution which has broken the diffraction limit could be achieved.5. Imaging system built and imaging experiments. A complete CLSM imaging system has been built which has achieved high-resolution three-dimensional imaging. And the related factors that influenced the quality of imaging were analyzed. The propotype of CLSM was finished.Finally, the research contents were summarized and analyzed. Some prospects for the CLSM were introuduced.
语种	中文
文献类型	学位论文
条目标识符	http://ir.ciomp.ac.cn/handle/181722/41484
专题	中科院长春光机所知识产出
推荐引用方式 GB/T 7714	魏通达. 共聚焦激光扫描光学显微成像关键技术研究[D]. 中国科学院大学,2014.

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