受激辐射损耗显微术的研究和应用

发布时间：2018-04-05 13:00

本文选题：远场超分辨显微镜　切入点：受激辐射损耗显微镜　出处：《中国科学院研究生院(上海应用物理研究所)》2017年博士论文

【摘要】：荧光显微镜由于其无损非侵入性的探测能力,是生物学和化学实验室常用的实验设备。传统的显微镜在最优条件下,使得光学显微镜的分辨率最高可以达到200nm。然而由于阿贝衍射极限的限制,使得光学显微镜的分辨率不能够进一步提高,许多200nm以下的生物学信息都无法被观测到。为了观测衍射极限以下的信息,无数的科研工作者提出了多种观测衍射极限以下信息的方法。无论是扫描隧道显微镜、电子显微镜、原子力显微镜,还是近场光学显微镜都是各行各业的研究者共同提出的获取微观信息的方法。然而这些显微术都存在无法观察活细胞样品,无法获得厚样品的内部信息等问题。科学研究还急需一种可无损获得样品内部信息的高分辨率显微术。超分辨技术很好的解决了这一问题。目前,发展比较成熟的超分辨显微术有受激辐射损耗显微术(STimulated Emission Depletion,STED)、结构光照明显微术(Structured Illumination Microscopy,SIM)、单分子定位显微术(STochastic Optical Reconstruction Microscopy/Photo Activated Localization Microscopy,STORM/PALM)。STED显微术作为超分辨显微术其中的一员,由于它是基于传统的共聚焦显微镜,因而不仅具有共聚焦系统的所有优点,也是其中唯一一种能够直接在物理层获得超分辨显微图像的技术。本文从STED超分辨显微镜的搭建入手,对STED显微镜的搭建和应用进行了一系列的研究。本论文主要内容如下:1)脉冲光STED的搭建。本论文使用荧光滤片从超连续激光中截取两段激光,一段作为激发光,一段作为损耗光进行脉冲光STED显微镜的搭建。在进行STED显微镜的搭建过程中,本文解决了一系列工程技术问题,如光纤耦合、空间滤波、4f系统,脉冲同步系统等。最终基本在已搭建的STED显微镜上看到了荧光小球的同物理大小相差无几的像。在60nm的微管染色体系中也获得了80nm的分辨率。并成功实现了DNA Origami的超分辨成像。2)连续光STED的搭建。相对于脉冲光,连续光STED不需要进行精细的脉冲同步,搭建和维护的成本相对来说都比脉冲光STED要低。在搭建的连续光STED上,本文实现了多种荧光染料、GFP和p-dots的荧光超分辨成像,获得了生物样品70nm的分辨率。另外,本文还根据已有的理论,模拟了三维STED Z轴方向的点扩散函数,根据计算的参数制作出了0/pi相位板,并最终获得了Z轴方向的点扩散函数。获得了三维STED的搭建能力。3)FRET与STED联用的理论计算。基于已经搭建的仪器,本文提出了一种提高STED分辨率降低STED损耗光的方法。我们使用MATLAB软件计算了FRET与STED联用的各种可能情况。最终的出结论,在所有的技术方案中,将损耗光同时作用在受体和供体上是效果最好的。本文基于此设计了高效FRET对,并使用设计的FRET对进行了小球实验验证了理论的准确性。4)STED多色实验。STED显微镜使用激发光和损耗光两束激光,而这两束激光都必须和所使用的荧光分子的激发谱和发射谱相匹配,因此,STED技术实际上并不太适合进行多色成像。基于此,本文提出了一种基于分步染色的STED双色成像策略。在成像过程中,本文使用了合成荧光标记的一抗进行现场快速染色,基于MATLAB编写自相关算法程序进行多个蛋白靶标之间的叠加,进行多个区域的成像以提高获得良好成像效果的概率。最终获得了线粒体和微管的双色STED成像。5)荧光漂白超分辨成像。现行的超分辨技术都需要对显微镜的硬件或者软件进行改造。本文利用荧光漂白原理实现了一种全新的超分辨成像方法。这种超分辨显微术仅需要一台传统的共聚焦显微镜,荧光漂白超分辨显微术与传统共聚焦的不同仅是提高分激发光的强度。本文从理论方面对这种显微术进行了建模仿真,从小球实验方面对仿真结果进行确认,最终在生物样品上获得了多种荧光分子的超分辨成像结果。
[Abstract]:Fluorescence microscopy due to its non - invasive detection ability, experimental equipment of chemical and biological laboratory. The traditional microscope under optimal conditions, the resolution of optical microscopy can reach the highest 200nm. because of Abbe's diffraction limit, so that the optical microscope resolution can be further improved, many biological information below 200nm can not be observed. In order to observe the information below the diffraction limit, many researchers proposed various methods of observation information below the diffraction limit. Both scanning tunneling microscopy, electron microscopy, atomic force microscopy and near-field optical microscopy are methods to obtain the microscopic information put together researchers from all walks of life however. These are not observed live cell microscopy samples, unable to obtain thick samples inside information. Scientific research has also need a lossless high resolution microscopy sample information. Super resolution technology is a good solution to this problem. At present, the development of more mature nanoscopy has stimulated emission depletion microscopy (STimulated Emission, Depletion, STED), the structure of light microscopy (Structured Illumination was Microscopy, SIM), single molecule localization microscopy (STochastic Optical Reconstruction Microscopy/Photo Activated Localization Microscopy, STORM/PALM.STED) as a member of the super resolution microscopy microscopy which, because it is based on the traditional confocal microscope, so it not only has all the advantages of confocal system, it is the only one which can directly obtain superresolution image technology in physics this paper starts from the STED layer. To build a super resolution microscope, STED microscope setup and Application A series of studies. The main contents of this thesis are as follows: 1) to build a STED light pulse. This paper use fluorescence filter from supercontinuum laser interception two laser as a excitation light, as a loss of light pulse light microscope STED were built. In the process of building the STED microscope in this paper, to solve a series of problems such as engineering technology, optical fiber coupling, spatial filtering, 4f system, synchronization system. Finally the basic STED microscope has built on to see the same physical size as the fluorescent beads in 60NM. Not much difference between the microtubule staining system also obtained 80nm and successful resolution. Achieve super-resolution imaging.2 DNA Origami) to build a continuous light STED. Compared with pulsed light, continuous light STED does not need synchronization fine, build and maintenance costs are relatively lower than the pulsed light STED in the building of. Continuous light on STED, this paper implements a variety of fluorescent dyes, fluorescent GFP and p-dots super resolution imaging of biological samples, obtained the resolution of 70nm. In addition, according to the existing theory, to simulate the STED Z axis point spread function, according to the calculation parameters for making a 0/pi phase plate, and finally get the the Z axis direction of the point spread function. The 3D STED ability to build.3) calculation of FRET combined with STED theory. The instrument has been built based on, this paper proposes a method to improve the resolution of STED STED reduce the loss of light. We use MATLAB software to calculate the various FRET and STED combined with the possible situation. The final conclusion, the technical proposal of all the loss of light at the same time in the acceptor and donor is the best effect. Based on the design of high performance FRET, and use FRET to design the ball experiment The theoretical accuracy of.4 STED experiment using.STED microscopy) polychromatic excitation light and optical loss of two laser beams, excitation spectrum and emission spectrum, and the two laser beams must be used fluorescent molecules and therefore, STED actually is not suitable for multicolor imaging. Based on this, this paper presents a step by step dyeing the STED dual color imaging based on strategy. In the process of imaging, the synthesis of fluorescent labeled anti site fast staining for the use of this, MATLAB prepared superposition between multiple protein targets auto correlation based algorithm, imaging of multiple regions in order to improve the probability to obtain good imaging effect. Finally obtained the mitochondria and microtubules double color STED imaging.5) fluorescence bleaching super-resolution imaging. Super resolution technology the current need for the transformation of the microscope hardware or software. In this paper, using fluorescence bleaching principle. Present a new method of super resolution imaging. This nanoscopy only needs a conventional confocal microscopy, fluorescence microscopy and traditional bleaching super-resolution confocal difference is only to increase the intensity of the excitation light. This paper built the simulation of the microscopy from theory, from the aspects of the simulation experiment of ball to confirm the results, finally obtained the super-resolution imaging results of different fluorescent molecules in biological samples.

【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3

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