已经成熟建立起来的超高分辨率荧光显微技术大致可分为3类：受激发射损耗技

已经成熟建立起来的超高分辨率荧光显微技术大致可分为3类：受激发射损耗技术及数种衍生方法。结构光照明技术和随机光学重构单分子技术。早在1989年William E. Moerner 就在世界上首次实现了超低温下单分子分光吸收的测量（1）并在1997年与Roger Y . Tsien(因绿色荧光蛋白获得2008年诺贝尔化学奖)合作发现了绿色荧光蛋白荧光的闪烁和切换行为。Eric Betzig 在1993年利用近场成像率先实现了常温下的单分子荧光观测，随后在1995年提出了基于单分子信号实现超高分辨率成像的思想。二者的早期研究都为日后超高分辨率成像技术的发展奠定了坚实的基础。
1994年，Stefan W. Hell 在芬兰图尔库大学最先提出了受激辐射损耗（stimulated emission depletion，STED ）理论，用来打破光学衍射极限，并最终于2000年在哥根廷大学得以真正实现。STED技术利用了类似与产生激光的“受激辐射”原理，将一束空心的光斑套在激发光光斑之外，这个环状激光覆盖的荧光分子会发生受激辐射，而环中心的荧光分子则发生自发辐射。因为波长不同，环中心的荧光可以被分开并单独检测。这样，通过增加环形光的强度来不断缩小环形光的孔径就可以获得一个小于衍射极限的荧光激发光斑，并通过扫描最终获得一幅超高分辨率的图像。STED技术，使其更加使用与生物研究。另外，他还通过相似原理发明了一系列的超高分辨率技术，统称为可逆饱和荧光跃迁（RESOLFT）,为超分辨率荧光显微成像技术的发展做出了巨大贡献。
2000年，美国科学家Mats Gustafsson开发了基于结构照明原理的超高分辨率技术（structured illumination microscopy，SIM），适用于快速的活细胞成像研究。SIM技术基于2个高空间频率团重叠可以形成低频率莫尔条纹的原理，通过对莫尔条纹的解析实现超高分辨率成像，可惜分辨率只达到了约100nm。
2006年，超分辨率荧光显微镜技术领域展开了一场科研竞赛，几乎同时出现了3种基于单分子定位随机重构原理的超分辨率光学成像技术：由哈佛大学的庄小威团队发明的随机光学重构显微技术（stochastic optical reconstruction microscopy，STORM）技术、这次的诺贝尔奖得主Betzig团队发明的光敏定位显微技术（photo-activated localization microscopy，PALM）以及缅因大学Samuel Hess发明的荧光活化定位显微技术（fluorescence photoactivation localization microscopy，fPALM）。它们在原理上非常相似，都是基于荧光分子（有机染料或是荧光蛋白）的光转化能力和单分子定位，通过对激活光的调制，因为对单个荧光分子中心的定位精度远远超过衍射极限，所以把同一区域的多张图片叠加就可重构出一幅超高分辨率图像。这种“以时间换空间”的巧妙方法把荧光成像的分辨率提高了20倍，达到10nm左右。
自荷兰科学见、显微镜创制者Antonie van Leeuwenhoek在17世纪第一次将光线通过透镜聚焦制成光学显微镜并用它观察微生物以来，显微镜就一直是生物学家从事研究工作、探寻生命奥秘必不可少的利器。而现代生物学研究迫切需要超高分辨率荧光显微技术，因为很多亚细胞结构都在微米到纳米尺度，衍射极限的存在限制了我们使用光学显微镜观察这些微结构和过程。超高分辨率荧光显微技术从方法实现到科学研究中大显身手虽然不过几十年时间，以对多个领域产生显著推动，并且可以预言在未来将给生命科学研究带来巨大的变化。

結果 (英文) 1:

Is ripe for the establishment of super high-resolution fluorescence microscopy can be divided into 3 categories: stimulated emission depletion techniques and a number of derivatives. Structured light illumination and stochastic optical reconstruction of single molecule techniques. As early as 1989, William e. Moerner under ultra low temperature throughout the world's first single molecule measurement of optical absorption (1), and in 1997, with Roger y. Tsien (won the 2008 Nobel Prize in chemistry for green fluorescent protein) to cooperate in discovery of green fluorescent protein fluorescence of blinking and switching behavior. Eric Betzig in 1993 using the near-field spectra of single molecule fluorescence at room temperature first observation, then in 1995 based on signal Super-resolution imaging of single molecules. Their early research for the future development of super high resolution imaging technology has laid a solid foundation.In 1994, Stefan w. Hell in Finland University of Turku was first proposed by stimulated emission depletion (stimulated emission depletion,STED) theory, used to break up the optical diffraction limit, and finally in 2000 at the University of gegenting to true. STED technology uses a laser-like "stimulated emission" principle, a hollow beam of light spot light spot fires outside the annular laser cover fluorescent molecule stimulated emission occurs, while fluorescent molecule Center, spontaneous emission occurs. Because different wavelength, fluorescence can be divided and a separate detection Center. Thus, by increasing the intensity of the ring light to shrink annular aperture can get a little Yu Yan the diffraction limit of the fluorescence excitation beam and scan ends up with a super high resolution image. STED technology, making it more use and biological research. In addition, he also adopted similar principles developed a series of ultra high resolution technique, known collectively as the reversible saturated fluorescent jump (RESOLFT) for Super-resolution fluorescence microscopy imaging technology has made great contribution to the development.In 2000, the United States scientist Mats Gustafsson had developed ultra-high resolution based on structure lighting technology (structured illumination microscopy,SIM), suitable for high-speed imaging of living cells. 2 high spatial frequency-based mass of overlapping of SIM technology can form the principle of low-frequency moire, through the analysis of moire fringes for ultra high resolution imaging, but the resolution only at about 100nm.In 2006, the Super resolution fluorescence microscopy technology launched a research competition, almost at the same time there were 3 kinds of positioning based on single molecule Super-resolution optical imaging techniques for stochastic reconstruction principle: invented by the Williams team at Harvard University the stochastic optical reconstruction microscopy (stochastic optical reconstruction microscopy,STORM), The Nobel Laureate Betzig team invented photosensitive localization microscopy (photo-activated localization microscopy,PALM) as well as the University of Maine, Samuel Hess invented fluorescence-activated localization microscopy techniques (fluorescence photoactivation localization microscopy,fPALM). They are similar in principle, is based on fluorescent molecules (organic dye or fluorescent protein) and single molecular orientation ability to transform, through the modulation of light is activated because the positioning accuracy for single fluorescent molecule Center far exceeds the diffraction limit, superimposing multiple images of the same area is a super high resolution image can be reconstructed. This "space-for-time" the ingenious method of high-resolution fluorescence imaging of 20 times, up to 10nm or so.Netherlands scientific findings, and created a microscope by Antonie van Leeuwenhoek in the 17th century for the first time the light is focused by a lens made of optical microscope used it since the Microbe, microscope has been a biologist doing research, to explore the mysteries of life essential to the weapon. Modern biology there is an urgent need for a high-resolution fluorescence microscopy, because a lot of subcellular structures in the micro to the nano-scale, diffraction limited our use of an optical microscope observation of these micro-structures and processes. Ultra high-resolution fluorescence microscopy methods to show their talents in scientific research, but decades in order to have a significant push on a number of fronts, and predicted that in the future will bring great changes to the life sciences.

結果 (英文) 3:

Ultra high resolution fluorescence microscopy technology has matured to build up can be divided into 3 categories: stimulated emission depletion technique and several kinds of derivative method. Lighting technology structure and stochastic optical reconstruction of single molecule technology. As early as in 1989, William E. Moerner is the first in the world to achieve the ultra low temperature place an order molecular optical absorption measurements (1) and in 1997 with the Roger Y. Tsien (won the 2008 Nobel prize in Chemistry for green fluorescent protein) Co discovered GFP fluorescence flicker and switching behavior. Eric Betzig in 1993 with the near field imaging to take the lead in realizing the single molecule fluorescence observation under normal temperature,Then the proposed single molecule signals to achieve ultra high resolution imaging based on the ideas in the 1995. Early studies of the two have laid a solid foundation for the future development of ultra high resolution imaging technology.
1994, Stefan W. Hell at the University of Turku in Finland was first proposed by the stimulated radiation loss (stimulated emission depletion, STED) theory, is used to break the diffraction limit, and finally in 2000, the court in the University come true. STED technology uses similar to produce laser "stimulated emission" principle, will be a bunch of hollow spot set in the excitation light spot outside,Fluorescent molecular this ring laser coverage will occur in the stimulated emission, and fluorescence molecular ring center is the occurrence of the spontaneous radiation. Because of different wavelengths, the fluorescence ring center can be separated and separate detection. So, to continue to narrow aperture ring light by increasing the ring light intensity can get a smaller than the diffraction limit of fluorescence excitation beam, and by scanning and eventually get a super high resolution images. STED technology, make it more use and biological research. In addition, he also through a similar principle to create a series of ultra high resolution techniques, collectively referred to as reversible saturated fluorescence transition (RESOLFT),Made a great contribution to the development of super-resolution fluorescence microscopy imaging technology.
2000 years, American scientist Mats Gustafsson developed ultra high resolution technology based on the principle of structure lighting (structured illumination microscopy, SIM), suitable for live cell imaging of fast. Based on the technology of SIM 2 high spatial frequency group overlap can form low frequency principle of moire fringes, and realize the high resolution imaging by analyzing the moire fringe, but resolution reached only about 100nm.
2006, the technical field of super-resolution fluorescence microscopy to start a race,Almost at the same time, there are 3 kinds of super resolution optical imaging technique of single molecule location based on the principle of random reconstruction: microscopic stochastic optical reconstruction technique invented by Zhuang Xiaowei of the Harvard University team (stochastic optical reconstruction microscopy, STORM) photosensitive positioning micro technology, the Nobel laureate Betzig team invented (photo-activated localization microscopy, PALM) and fluorescence University of Maine Samuel Hess invented the activation location microscopy (fluorescence photoactivation localization microscopy, fPALM).They are very similar in principle, is based on the fluorescent molecule (organic dyes or fluorescent protein) ability of light transformation and single molecule localization, by modulating the activation of light, because the localization of single fluorescent molecule center accuracy far beyond the diffraction limit, so the same area of the multiple images superposition can reconstruct a high resolution image. Ingenious method to this "time for space" to the fluorescence imaging resolution is improved by 20 times, reach about 10nm.
from Holland science see,Creator Antonie van Leeuwenhoek microscope in seventeenth Century for the first time since light focused by a lens made of optical microscope and observe microorganisms use it, microscope has been engaged in research work, biologists explore the mysteries of life essential tool. Modern biology research in urgent need of ultra high resolution fluorescence microscopy, because a lot of subcellular structures in micro to nano scale, the existence of the diffraction limit limit our use of optical microscope to observe these micro structure and process. Ultra high resolution fluorescence microscopy from method to realize to display one's skill to the full scientific research though but a few decades,To produce a significant push in many areas, and can be predicted in the future will be to bring about tremendous changes in life science research.