| 题名 | 超分辨非线性光学显微成像术的研究及应用 |
| 作者 | 肖繁荣 |
| 学位类别 | 博士 |
| 答辩日期 | 2005 |
| 授予单位 | 中国科学院上海光学精密机械研究所 |
| 导师 | 徐至展 |
| 关键词 | 光学超分辨 双光子激发荧光显微成像 相干反斯托克斯Raman散射显微成像 变迹术 光瞳滤波器 非线性光学 |
| 其他题名 | The study and applications of superresolving non-linear optical microscopy |
| 中文摘要 | 生物医学领域正在以日新月异的速度发展，亚细胞尺寸钙离子浓度的测定、病毒入侵细胞的视见以及从生物大分子水平上对各种生命现象的再认识等课题在近几十年取得了重大的进展。各种成像检测技术也因此得到了前所未有的发展。现代光学成像技术，不仅继承了传统光学成像技术无损、大信息量的优势，而且比传统光学成像技术具有更高的空间和时间分辨能力。在生物活体成像研究应用中，远场空间超高分辨技术有着非常重要的作用。本论文将利用基于光学变迹术的光学超分辨技术和非线性光学技术，研究提高远场光学显微成像系统空间分辨率的途径。在实验上，提出并研制了一套新型的多模式共焦扫描显微成像系统，包括系统的设计、探测系统的调试、扫描系统的调试、部分光学和机械元件的设计、系统的软件控制以及软件实现的图像重组等。角军决了系统研制中遇到的问题。该系统不仅可以实现普通共焦扫描显微成像和双光子激发荧光显微成像，而月＿还可以实现相干反斯托克斯Roman散射显微成像。在理论上，分析了位相型光瞳滤波器和振幅型光瞳滤波器在超分辨应用中优缺点设计了几种在实际中易于制作、且具有超分辨特性的光瞳滤波器，首次提出了双Torald。光瞳滤波器的共焦成像系统，通过增加光瞳滤波器的数量大大降低了单个光瞳滤波器制作上的困难；结合环形光瞳滤波器，首次研究了具有光学超分辨特性双色双光子激发荧光显微系统。概括起来，本论文主要取得了以厂几个方面创新性的进展：1．首次将超分辨技术应用于双色双光子激发荧光显微系统中。在其中一束激发光路加入超分辨光瞳滤波器，提高了双色双光子激发荧光显微系统的横向分辨率，同时利用双色双光子激发本身的乘积特性，在不加小孔的情况下，有效的压制了超分辨所引起的横向旁瓣增强效应。另外，利用这种方法也大大减弱了通常横向超分辨所无法避免的轴向加宽效应。2．理论上证明了，如果不考虑能量的限制，纯振幅型光瞳滤波器的超分辨效果将好于纯位相型光瞳滤波器。考虑到光瞳滤波器由于本身制作困难对其应用的影响，本论文中讨论了两种改进的方法：1．设计了一种易于制作的三卜＿复振幅光瞳滤波器。在相同第一零点比情况下，该三区复振幅光瞳滤波器的横向旁瓣强l度比优于三区位相型光瞳滤波器50％，并且有效的降低了后者的轴向加宽效应。11．首次提出了双Toraldo光瞳滤波器的共焦成像系统，通过增加光瞳滤波器的数量大大降低了单个光瞳滤波器制作上的困难。本论文中从衍射光学的角度出发，详细分析了以上几种光瞳滤波器的超分辨特性。3．首次提出了利用单轴晶体制作可调节位相型光瞳滤波器。基于线偏振光穿过单轴晶体的位相偏移依赖于其与单轴晶体光轴夹角的特性，通过旋转光轴方向可以调节光瞳滤波器中单轴晶体区域与其它区域之间的位相差，这种效应有两个作用：一是实现不同的超分辨效果，二是弥补制作中的误差。4．多模式共焦扫描显微成像系统的研制。该系统不仅可以进行普通的反射式共焦扫描成像和双光子激发荧光显微成像，而且集成了CARS成像的功能。具有同时进行双光子荧光和CARS成像的能力。解决了系统研制中遇到的问题：1．图像重构困难。通过分析找出了导致图像重构困难的因素-步进电机的折返误差，并通过软件处理的方法消除了该误差对图像重组的影响。II探测系统的优化。，分析了在弱信号探测时，计数噪声的来源。给出了抑制计数噪声的方法。III收集透镜的选择。分析了光电倍增管的光阴极响应特性，据此给出了收集透镜的最佳参数。本论文中给出了利用该系统，对4Onm的纳米小球、纸纤维和转染GFP的CHO细胞的两维和三维双光子激发荧光成像效果。根据CARS产生的原理，在理论上证明了所得到的二澳甲烷和肝癌组织切片的光谱为样品的CARS光谱。利用手动和自动两种扫描方法，实现了对二澳甲烷和肝癌组织切片样品的两维CARS显微成像。上述研究的相关文章子经发表在ApPI．OPt．和OPt．Comm．等杂志上，并子快了已被同行引用。 |
| 英文摘要 | Biomedical science has seen remarkable progress in recent years, especially measurement of Calcium, tracking virus in living cell and recongnization of bio-phenomena on biological large molecular level. Compared with other measurement techniques, optical imaing techniques can work for the vivo body and pursue more information of the sample. It is crucial for the biomedical science. A series of new ideas have been growing to break down the spatial resolution limit of conventional optical microscopy. Among them, superresolution in far-field is more important for the vivo imaging. The research in this thesis focuses on the superresolution in far-field based on optical apodization for superresoltion and nonlinear optics. In experiments, we have developed a multi-mode optical microsocope. Conventional scanning confocal imaging, two-photn excitation fluorescence imaging and a coherent anti-Stokes Raman scattering imaging can be realized in this system. The properties of phase-only and transmittance pupil filters have been analyzed in theory. To overcome the obstacles of fabrication of pupil filters, several new types of pupil filters have been designed. And the application of annular pupil filter in two-color two-photon excitation fluorescence microscopy was also discussed. The research progress is shown as follows: First, optical superresolution technique was applied to two-color two-photon excitation fluorescence microscopy. By inserting a pupil filter in one of the two excitation beams, the side-lobes increase was suppressed efficiently without the present of pinhole. And the axial expanding effect also was decreased. Second, it has been proved that transmission pupil filter is better than phase-only pupil filter in superresolution application if energy loss can be ignored. To overcome the obstacles in fabrication, two new types of pupil filter have been proposed. I. A three zones hybrid amplitude-phase pupil filter was designed. The lateral side-lobe ratio is better 50% than the phase-only pupil filter. II. A confocal system with two Toraldo pupil filters was given. The pupil filter is easy to be fabricated by increasing in the quantity of pupil filters. The superresolution properties of these pupil filters were discussed ui detail. Third, Phase-only pupil filers with uniaxial crystal were analyzed firstly. Based on the phase offset which depends on the angle between the orientation of input light polarization and the optical axis, phase difference can be changed by rotating the angle of the filter. There are two effects of this filter. First the different types of superresolution can be realized. Second it can be used to make up the distortions in fabrication. Finally, a multi-mode scanning confocal imaging system has been built. Conventional scanning confocal imaging and two-photn excitation fluorescence imaging can be realized in this system. Coherent anti-stokes Raman scattering imaging was also combined in the system. The development details of the system are given in the thesis, including hardware and software for data collection and image reconstruction. Main consideration and solutions are aiming at the following aspects. I. Problems in picture construction. By analyzing the system in detail, we noticed that foldback error is the mainly obstacle. This problem has been solved by software programme. II. Optimize detection equipments. The origin of noise in wesk signal detection was analyzed. And methods were given to suppress it. III. Choose the parameter of collective lens. By analyzing the response of photocathode of photomultiplier tube (PMT), proper parameters of the collective lens are given. The TPEFM system has been used to image nanometer sphere (40nm), paper filament and CHO cell translated by GFP. Two-dimensional and three-dimensional image was shown in this dissertation. CARS spectrum of dibromomethane and cancer tissue was gotten by a single fs-laser. The experiment data was confirmed by theory numerical simulation. Two-dimensional image of the sample was given by manual and/or automatic scanning. Related papers have been published in Appl. Opt., Opt. Comm. etc. Some of them have been cited by other researchers. |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.siom.ac.cn/handle/181231/15573]  |
| 专题 | 上海光学精密机械研究所_学位论文 |
| 推荐引用方式 GB/T 7714 | 肖繁荣. 超分辨非线性光学显微成像术的研究及应用[D]. 中国科学院上海光学精密机械研究所. 2005. |
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