| 题名 | 连续光受激发射损耗显微系统的研制及超分辨光学显微术研究 |
| 作者 | 邓素辉 |
| 学位类别 | 博士 |
| 答辩日期 | 2010 |
| 授予单位 | 中国科学院上海光学精密机械研究所 |
| 导师 | 李儒新 ; 刘力 |
| 关键词 | 突破光学衍射极限 连续光受激发射损耗显微成像系统 高数值孔径聚焦的矢量理论 像差 光瞳滤波器 |
| 其他题名 | Stimulated emission depletion microscopy with continuous wave beams and the study of super-resolution techniques in optical microscopy |
| 中文摘要 | 远场光学显微镜可以实现对活体生物组织的无损伤、非侵入及实时的探测和成像，是生命科学研究中的重要手段。但由于光的波动性，光学系统存在衍射极限，即类似点源的物体成像为横向尺寸约半个波长大小的光斑，而不是一个像点。对于可见光，普通的远场光学显微镜的横向分辨率约为200nm，纵向分辨率约为400-700nm。 人们迫切期望拥有一套分辨率为几十纳米的显微系统去回答更多的生命科学问题。最近二十年来，涌现了一批新型的提高远场光学分辨率甚至突破衍射极限的光学显微方法。而受激发射损耗显微术(Stimulated emission of depletion microscopy, STED )是第一个被提出的突破衍射极限的远场光学显微技术，是超分辨率荧光显微镜的典型代表。除了提供高空间分辨率外，STED显微镜还具备了很多优点：(1) STED 系统能够在已经很成熟的共焦显微镜上进行改装实现；(2) STED系统实现了对普通光学显微镜无法探测的神经生物学，神经系统科学，细胞膜生物学和细胞内动力学行为的观测；(3) STED提供了实时的光学分辨率，无需对数据进行下一步的类似于去卷积运算，图像复原，统计分析等数学方法处理即可得到即时的超分辨效果；(4) STED显微成像可以通过进一步的去卷积和图像复原处理来改善图像质量。 在国家自然科学基金的资助下，本论文对连续光受激发射损耗显微成像系统(CW-STED microscopy) 进行了研制，着重开展了三个方面的研究工作：一，受激发射损耗显微系统的理论研究。二，连续光受激发射损耗扫描成像系统的研制。三，光瞳滤波器的超分辨技术研究。概括起来，本论文取得了以下几个方面的创新性进展： 1. 首次研究了各类初级像差包括球差，彗差和象散对受激发射损耗显微系统中常用的三种损耗模式的焦斑形状改变。这三种损耗模式可通过在高斯光路中插入螺旋型相位板，半圆半波长延迟相位板和中心半波长延迟相位板而获得。利用高数值孔径聚焦的矢量理论分析了一定量大小的此类像差对损耗模式的最大强度，中心暗点强度，形成的焦孔直径的影响。 2. 研究了显微系统聚焦穿过多层样品时由于折射率不匹配而引入的球差对受激发射损耗显微镜的损耗光焦斑分布的变形和对STED系统分辨率的影响。其计算结果与无像差情况的光强分布进行了比较。 3. 建立了连续光受激发射损耗显微成像系统。设计了整套装置包括照明系统，荧光收集系统和扫描系统部分，完成了部分光学和机械元件的设计，扫描成像系统程序的编写，激发光和损耗光空间同步的调节，扫描系统和探测系统的调试，系统参数优化，减小背景噪音，数据处理等过程。使用80nm金颗粒扫描出激发光斑和中心零光强的损耗光焦斑分布。利用该装置，在20nm直径的深红色荧光小球和染料Atto647N荧光团样品上成功观察到了受激发射引起的荧光猝灭现象，并完成了对以上两类样品的CW-STED荧光扫描成像，和对数据进行高斯拟合确定了系统的实际分辨率。该装置获得了突破衍射极限的分辨率，其大小为相同条件下共焦系统的2-3.7倍。 4. 首次将多种超分辨技术结合起来提高光学成像系统分辨率，即提出变迹术与改善分辨率的成像结构(theta显微镜)结合起来的方法，利用双色双光子激发的特点，在双色双光子Theta显微镜中插入两个超分辨光瞳滤波器实现了低旁瓣强度的三维分辨率提高。我们的计算对多种超分辨技术结合起来使用的方案具有一定的启发作用。 |
| 英文摘要 | The far-field light microscope is a valuable scientific instrument in life sciences for the noninvasive or time-resolved imaging of the interior of transparent objects, like cells in three dimensions. However, its resolution is limited by diffraction, a fundamental physical law. The diffraction barrier states that far-field optics cannot focus light to an infinitely small spot, rather its size is limited in lateral dimension to about half the wavelength of the light used. The resolution of a conventional far-field light microscope applying visible light is about 200nm in the lateral direction and 400-700nm in the axial directions. For the reason that microscope with imaging resolution in the range of a few tens of nanometers will have a dramatic impact on a large variety of key questions in life science research, in the recent years, new methods have emerged aiming to improve the resolution of far-field microscopes and eventually overcome the diffraction barrier. Stimulated emission depletion (STED) microscopy was the first concept of breaking the diffraction barrier in the optical far-field microscopy verified in biological applications. Besides the ability of providing the resolution of a few tens of nanometers, STED microscopy has several advantages: (1) STED is compatible with most advanced confocal microscopy techniques. (2) STED will allow scientists to study structures previously inaccessible to light microscopy in the fields of Neurobiology and Neuroscience, Membrane biology and Intracellular transport. (3) STED microscopy provides real, optical resolution. No mathematics at all (e.g. deconvolution, image restoration, statistical analysis, etc) is needed. (4) STED images can benefit from further deconvolution and image restoration techniques. It means that researchers can enhance STED image quality further by mathematics, optionally. With support from National Natural Science Fund of China, the work in this thesis is focused on the study of stimulated emission depletion microscopy with continuous wave beams (CW-STED microscopy). My work has three parts. The first part is the theoretical study of the STED. Second part is about the experimental setup of a CW-STED imaging system. The third part is the study of super-resolution techniques in optical microscopy. The main progresses are shown as follows 1. For the first time, effects of primary aberrations including spherical aberration, coma and astigmatism on the three fluorescence depletion patterns mainly used in stimulated emission depletion (STED) microscopy were investigated by using vectorial integral. The three depletion patterns are created by inserting a vortex phase plate, a central half-wavelength plate or a semi-circular half-wavelength mask within Gaussian beam respectively. Attention is given to the modification of the shape, peak intensity, the central intensity of the dark hole and the hole size of these depletion patterns in the presence of primary aberrations. 2. Aberration is generated when focusing beams through an interface between the media of the mismatched refractive indices. By use of the vectorial integral, the effects of such aberration on the shape of depletion patterns and the size of fluorescence emission spot in the STED microscopy are studied. Results are presented as a comparison between the aberration-free case and the aberrated cases. 3. A CW-STED imaging system has been built. We designed the experimental setup including the illumination system, the fluorescence-collecting system and the scanning system. Also, we finished lots of work like writing the software for scanning system, finishing the adjustment of the alignment of the excitation and STED beams, decreasing the noise of environment as low as possible, optimizing the system and data processing. For the first time, the focal intensity distributions of the excitation beam and STED beam were obtained by scanning the sample of 80-nm gold bead in reflectance mode. For testing our setup, two samples of 20nm fluorescent beads and the fluorophore Atto647N adherent to a glass cover-slip were chosen. The depletion of fluorescence through stimulated emission and the enhancement of resolution of the system were successfully observed. The resolution of CW-STED system was obtained by fitting the data with gaussian function. And the data shows that our CW-STED system provides a resolution enhancement of 2-3.7 on the focal plane, compared with the resolution of corresponding confocal system. 4. For the first time, we proposed a new method of combining several superrresolving technologies to increase the resolution. For example, superresolving beam-shaping elements were designed to realize a three-dimensional (3D) superresolution in two-color excitation (2CE) fluorescence microscope with theta illumination method. Taking the advantages of 2CE fluorescence microscope and theta microscope, we proposed to use two leaky filters being inserted in the two illumination arms reducing the three-dimensional (3D) PSF main-peak width with low sidelobes. Our numerical study is helpful to provide a new sight for the use of combination of multi-superresolving techniques. |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.siom.ac.cn/handle/181231/15302]  |
| 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 | 邓素辉. 连续光受激发射损耗显微系统的研制及超分辨光学显微术研究[D]. 中国科学院上海光学精密机械研究所. 2010. |
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