本文選題：遠場超分辨顯微鏡　切入點：受激輻射損耗顯微鏡　出處：《中國科學(xué)院研究生院(上海應(yīng)用物理研究所)》2017年博士論文

【摘要】：熒光顯微鏡由于其無損非侵入性的探測能力,是生物學(xué)和化學(xué)實驗室常用的實驗設(shè)備。傳統(tǒng)的顯微鏡在最優(yōu)條件下,使得光學(xué)顯微鏡的分辨率最高可以達到200nm。然而由于阿貝衍射極限的限制,使得光學(xué)顯微鏡的分辨率不能夠進一步提高,許多200nm以下的生物學(xué)信息都無法被觀測到。為了觀測衍射極限以下的信息,無數(shù)的科研工作者提出了多種觀測衍射極限以下信息的方法。無論是掃描隧道顯微鏡、電子顯微鏡、原子力顯微鏡,還是近場光學(xué)顯微鏡都是各行各業(yè)的研究者共同提出的獲取微觀信息的方法。然而這些顯微術(shù)都存在無法觀察活細胞樣品,無法獲得厚樣品的內(nèi)部信息等問題�？茖W(xué)研究還急需一種可無損獲得樣品內(nèi)部信息的高分辨率顯微術(shù)。超分辨技術(shù)很好的解決了這一問題。目前,發(fā)展比較成熟的超分辨顯微術(shù)有受激輻射損耗顯微術(shù)(STimulated Emission Depletion,STED)、結(jié)構(gòu)光照明顯微術(shù)(Structured Illumination Microscopy,SIM)、單分子定位顯微術(shù)(STochastic Optical Reconstruction Microscopy/Photo Activated Localization Microscopy,STORM/PALM)。STED顯微術(shù)作為超分辨顯微術(shù)其中的一員,由于它是基于傳統(tǒng)的共聚焦顯微鏡,因而不僅具有共聚焦系統(tǒng)的所有優(yōu)點,也是其中唯一一種能夠直接在物理層獲得超分辨顯微圖像的技術(shù)。本文從STED超分辨顯微鏡的搭建入手,對STED顯微鏡的搭建和應(yīng)用進行了一系列的研究。本論文主要內(nèi)容如下:1)脈沖光STED的搭建。本論文使用熒光濾片從超連續(xù)激光中截取兩段激光,一段作為激發(fā)光,一段作為損耗光進行脈沖光STED顯微鏡的搭建。在進行STED顯微鏡的搭建過程中,本文解決了一系列工程技術(shù)問題,如光纖耦合、空間濾波、4f系統(tǒng),脈沖同步系統(tǒng)等。最終基本在已搭建的STED顯微鏡上看到了熒光小球的同物理大小相差無幾的像。在60nm的微管染色體系中也獲得了80nm的分辨率。并成功實現(xiàn)了DNA Origami的超分辨成像。2)連續(xù)光STED的搭建。相對于脈沖光,連續(xù)光STED不需要進行精細的脈沖同步,搭建和維護的成本相對來說都比脈沖光STED要低。在搭建的連續(xù)光STED上,本文實現(xiàn)了多種熒光染料、GFP和p-dots的熒光超分辨成像,獲得了生物樣品70nm的分辨率。另外,本文還根據(jù)已有的理論,模擬了三維STED Z軸方向的點擴散函數(shù),根據(jù)計算的參數(shù)制作出了0/pi相位板,并最終獲得了Z軸方向的點擴散函數(shù)。獲得了三維STED的搭建能力。3)FRET與STED聯(lián)用的理論計算�；谝呀�(jīng)搭建的儀器,本文提出了一種提高STED分辨率降低STED損耗光的方法。我們使用MATLAB軟件計算了FRET與STED聯(lián)用的各種可能情況。最終的出結(jié)論,在所有的技術(shù)方案中,將損耗光同時作用在受體和供體上是效果最好的。本文基于此設(shè)計了高效FRET對,并使用設(shè)計的FRET對進行了小球?qū)嶒烌炞C了理論的準確性。4)STED多色實驗。STED顯微鏡使用激發(fā)光和損耗光兩束激光,而這兩束激光都必須和所使用的熒光分子的激發(fā)譜和發(fā)射譜相匹配,因此,STED技術(shù)實際上并不太適合進行多色成像�；诖�,本文提出了一種基于分步染色的STED雙色成像策略。在成像過程中,本文使用了合成熒光標記的一抗進行現(xiàn)場快速染色,基于MATLAB編寫自相關(guān)算法程序進行多個蛋白靶標之間的疊加,進行多個區(qū)域的成像以提高獲得良好成像效果的概率。最終獲得了線粒體和微管的雙色STED成像。5)熒光漂白超分辨成像�，F(xiàn)行的超分辨技術(shù)都需要對顯微鏡的硬件或者軟件進行改造。本文利用熒光漂白原理實現(xiàn)了一種全新的超分辨成像方法。這種超分辨顯微術(shù)僅需要一臺傳統(tǒng)的共聚焦顯微鏡,熒光漂白超分辨顯微術(shù)與傳統(tǒng)共聚焦的不同僅是提高分激發(fā)光的強度。本文從理論方面對這種顯微術(shù)進行了建模仿真,從小球?qū)嶒灧矫鎸Ψ抡娼Y(jié)果進行確認,最終在生物樣品上獲得了多種熒光分子的超分辨成像結(jié)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.

【學(xué)位授予單位】：中國科學(xué)院研究生院(上海應(yīng)用物理研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：O657.3

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