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Green emitted CdSe@ZnS quantum dots for FLIM and sted imaging applications

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Journal of Innovative Optical Health Sciences 2019年第5期12卷 79-85页

作者： Mengjie Zhao Shuai Ye Xiao Peng Jun Song Junle Qu Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province College of Physics and Optoelectronic Engineering Shenzhen University Shenzhen 518060P.R.China

Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(sted)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by sted laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under sted laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-sted experiments.

关键词： CdSe@ZnS QDs FLIM sted living cells

Fusion of clathrin and caveolae endocytic vesicles revealed by line-switching dual-color sted microscopy

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Journal of Innovative Optical Health Sciences 2021年第6期4卷 71-80页

作者： Hefei Ruan Jianqiang Yu Yayun Wu Xiaojun Tang Jinghe Yuan Xiaohong Fang Beijing National Laboratory for Molecular Sciences Key Laboratory of Molecular Nanostructure and Nanotechnology Institute of Chemistry Chinese Academy of SciencesBeijing 100190P.R.China University of Chinese Academy of Sciences Beijing 100049P.R.China

Clathrin-and caveolae-mediated endocytosis are the most commonly used pathways for the internalization of cell membrane receptors.However,due to their dimensions are within the diffraction limit,traditional fluorescence microscopy cannot distinguish them and little is known about their interactions underneath cell membrane.In this study,we proposed the line-switching scanning imaging mode for dual-color triplet-state relaxation(T-Rex)stimulated emission depletion(sted)super-resolution microscopy.With this line-switching mode,the cross-talk between the two channels,the side effects from pulse picker and image drift in frame scanning mode can be effectively eliminated.The dual-color super-resolution imaging results in mixed fluorescent beads validated the excellent performance.With this super-resolution microscope,not only the ring-shaped structure of clathrin and caveolae endocytic vesicles,but also their semi-fused structures underneath the cell membrane were distinguished clearly.The resultant infor-mation will greatly facilitate the study of clathrin-and caveolae-mediated receptor endocytosis and signaling process and also our home-built dual-color T-Rex sted microscope with this line-switching imaging mode provides a precise and convenient way to study subcellular-scale protein interactions.

关键词： Super-resolution microscopy sted dual-color endocytosis line-switching

ELECTROCHEMICAL CORROSION BEHAVIOR OF STEEL IN THE JUNCTURE AREA OF SEA CLAY AND SAND

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Chinese Journal of Oceanology and Limnology 1993年第2期11卷 171-175页

作者：张经磊侯保荣郭公玉杨芳英谢肖勃 Institute of Oceanology Academa Sinica Qingdao 266071

Corrosion behavior of steel in the juncture area of sea clay and sand was studied. The results show that galvanic corrosion occurred between steel samples buried in sea clay and sand, and that the polarity of the galv... 详细信息

关键词： corrosion sted juncture area clay sand

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几种超分辨率荧光显微技术的原理和近期进展

生物化学与生物物理进展 2009年第12期36卷 1626-1634页

作者：吕志坚陆敬泽吴雅琼陈良怡北京市科学技术情报研究所北京100044 中国科学院生物物理研究所北京100101

在生命科学领域,人们常常需要在细胞内精确定位特定的蛋白质以研究其位置与功能的关系.多年来,宽场/共聚焦荧光显微镜的分辨率受限于光的阿贝/瑞利极限,不能分辨出200nm以下的结构.近年来,随着新的荧光探针和成像理论的出现,研究者开发... 详细信息

在生命科学领域,人们常常需要在细胞内精确定位特定的蛋白质以研究其位置与功能的关系.多年来,宽场/共聚焦荧光显微镜的分辨率受限于光的阿贝/瑞利极限,不能分辨出200nm以下的结构.近年来,随着新的荧光探针和成像理论的出现,研究者开发了多种实现超出普通共聚焦显微镜分辨率的三维超分辨率成像方法.主要介绍这些方法的原理、近期进展和发展趋势.介绍了光源的点扩散函数(point spread function,PSF)的概念和传统分辨率的定义,阐述了提高xy平面分辨率的方法.通过介绍单分子荧光成像技术,引入了单分子成像定位精度的概念,介绍了基于单分子成像的超分辨率显微成像方法,包括光激活定位显微技术(photoactivated localization microscopy,PALM)和随机光学重构显微技术(stochastic optical reconstruction microscopy,STORM).介绍了两大类通过改造光源的点扩散函数来提高成像分辨率的方法,分别是受激发射损耗显微技术(stimulated emission depletion,sted)和饱和结构照明显微技术(saturated structure illumination microscopy,SSIM).比较了不同的z轴提取信息的方法,并阐述了这些方法与xy平面上的超分辨率显微成像技术相结合所得到的各种三维超分辨率显微成像技术的优劣.探讨了目前超分辨率显微成像的发展极限和方向.

关键词：超分辨率荧光显微技术点扩散函数 PALM STORM sted SSIM

Super-resolution microscopy: successful applications in centrosome study and beyond

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Biophysics Reports 2019年第Z1期5卷 235-243页

作者： Jingyan Fu Chuanmao Zhang State Key Laboratory of Agrobiotechnology College of Biological Sciences China Agricultural University Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education College of Life SciencesPeking University

Centrosome is the main microtubule-organizing center in most animal cells. Its core structure, centriole,also assembles cilia and flagella that have important sensing and motility functions. Centrosome has long been recognized as a highly conserved organelle in eukaryotic species. Through electron microscopy, its ultrastructure was revealed to contain a beautiful nine-symmetrical core 60 years ago, yet its molecular basis has only been unraveled in the past two decades. The emergence of super-resolution microscopy allows us to explore the insides of a centrosome, which is smaller than the diffraction limit of light. Super-resolution microscopy also enables the compartmentation of centrosome proteins into different zones and the identification of their molecular interactions and functions. This paper compiles the centrosome architecture knowledge that has been revealed in recent years and highlights the power of several super-resolution techniques.

关键词： Centrosome Super-resolution microscopy SIM sted SMLM

Coherent optical adaptive technique improves the spatial resolution of sted microscopy in thick samples

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Photonics Research 2017年第3期5卷 176-181页

作者： WEI YAN YANLONG YANG YU TAN XUN CHEN YANG LI JUNLE QU TONG YE Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province College of Optoelectronic EngineeringShenzhen University Department of Bioengineering and the COMSET Clemson University State Key Laboratory of Transient Optics and Photonics Xi’an Institute of Optics and Precision MechanicsChinese Academy of Science Department of Regenerative Medicine and Cell Biology Medical University of South Carolina

Stimulated emission depletion(sted) microscopy is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the sted microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of a specimen's optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the severe distortion of the depletion beam profile may cause complete loss of the superresolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is difficult to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique. The full correction can effectively maintain and improve spatial resolution in imaging thick samples.

关键词： sted is Coherent optical adaptive technique improves the spatial resolution of sted microscopy in thick samples of in

Self-aligned fiber-based dual-beam source for sted nanolithography

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Chinese Optics Letters 2021年第7期19卷 82-86页

作者：陈建陈国梁詹其文 School of Optical-Electrical and Computer Engineering University of Shanghai for Science and TechnologyShanghai 201800China

A fiber-based source that can be exploited in a stimulated emission depletion(sted) inspired nanolithography setup is presented.Such a source maintains the excitation beam pulse, generates a ring-shaped depletion beam, and automatically realizes dual-beam coaxial alignment that is critical for two beam nanolithography.The mode conversion of the depletion beam is realized by using a customized vortex fiber, which converts the Gaussian beam into a donut-shaped azimuthally polarized beam.The pulse width and repetition frequency of the excitation beam remain unchanged, and its polarization states can be controlled.According to the simulated point spread function of each beam in the focal region, the full width at half-maximum of the effective spot size in sted nanofabrication could decrease to less than 28.6 nm.

关键词： nanolithography vortex fiber direct laser writing sted controlled fabrication

A Low Cost and Versatile sted Superresolution Fluorescent Microscope

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Modern Instrumentation 2013年第3期2卷 41-48页

作者： Daniel McBride Chin Su Jun Kameoka Stanislav Vitha Electrical and Computer Engineering Department Texas A&M University College Station USA Microscopy & Imaging Center Texas A&M University College Station USA

A versatile and inexpensive super-resolution fluorescent microscope that functions as easily as a conventional confocal microscope is described. Components of the microscope were designed on a platform which was placed atop a surplus microscope frame. All optical components and equipments used are given. The excitation and depletion beams are extracted from a compact low-cost supercontinuum light source. The focal spot of the depletion beam at the focal plane is studied and imaged by a 100 nm aperture near-field fiber tip. The collinear excitation and depletion beam focused by a 0.9 numerical aperture microscope objective produce a 90 nm lateral super-resolution as verified by imaging 100 nm diameter fluorescent beads.

关键词： Stimulated Emission-Depletion Microscopy sted

基于sted超分辨技术的新型菁染料对细胞线粒体膜结构成像研究

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基于STED超分辨技术的新型菁染料对细胞线粒体膜结构成像研究

作者：田蜜深圳大学

学位级别：硕士

光学显微成像具有非接触、无损伤等优点,是长期以来生物医学领域研究的重要工具。因为光学衍射极限的存在,光学显微镜的分辨率极限自1873年以来止步于200nm这一极值,无法获得尺寸在200nm以内生物结构的清晰图像。超分辨技术的诞生是本... 详细信息

光学显微成像具有非接触、无损伤等优点,是长期以来生物医学领域研究的重要工具。因为光学衍射极限的存在,光学显微镜的分辨率极限自1873年以来止步于200nm这一极值,无法获得尺寸在200nm以内生物结构的清晰图像。超分辨技术的诞生是本世纪光学显微成像领域的重要突破,为生物科学的研究提供了不可替代的有力工具。超分辨光学成像（Super-resolution Optical Microscopy）[1]技术泛指分辨率打破了光学显微系统分辨率极限的显微技术,目前常见的超分辨成像技术主要有两种途径:一是基于点扩展函数工程的方法实现超分辨,例如受激发射损耗显微技术（Stimulated Emission Depletion Microscopy,简称sted）;二是基于单分子定位实现超分辨,例如光激活定位显微技术（Photo activation Localization Microscopy,简称PALM）、随机光学重构成像（Stochastic Optical Reconstruction Microscopy）等。线粒体膜,尤其是内膜在线粒体生理过程中发挥着非常重要的作用,研究线粒体膜结构与功能具有重大意义。荧光成像具有灵敏度高、操作简单、成本低等优点而广受关注,荧光染料作为荧光成像过程中的重要环节具有非常重要的地位,而常规荧光染料,如Rhodamine123和JCT-1等线粒体染色剂,由于自身分子结构与电荷分布等特征优先定位于线粒体基质,对线粒体膜结构有选择性染色的长波长染料比较少见,尤其是能同时超分辨成像线粒体膜结构的荧光探针更是稀缺,因此需要开发新型超分辨荧光探针对线粒体膜结构进行成像研究。多甲川菁染料是常见的长波长荧光染料,例如Cy5,Alexa-647等[2]常用于STORM超分辨成像,但这类染料存在一些问题,如光稳定性差、荧光量子产率不高等,需要开发性能优异的新型菁类荧光染料。与Cy5染料相比,方酸菁染料是一类耐光漂白性好,荧光量子产率高的染料,但其水溶性差、活细胞内染色定位性差等问题。基于上述考虑,开发新型菁荧光染料成为迫切需要,使其同时兼具方酸类染料与甲川类菁染料的优点,更好的用于超分辨荧光成像。本硕士论文工作主要搭建了一套sted超分辨荧光成像系统及开发了一类新型菁染料用于活细胞线粒体内膜结构的特异性荧光标记与sted超分辨成像研究,主要内容包括以下几个方面:1.sted超分辨成像系统的搭建,使用已知纳米荧光材料对sted系统进行相关参数指标的标定;2.新型菁染料（后均简称为探针1）的制备与光谱性能研究,包括吸收,发射光谱,荧光量子产率测试,对细胞进行染色等生物实验等;3.探针1用于活细胞成像实验及sted超分辨成像研究。本硕士论文工作的创新点主要包括以下两点:1.设计制备了一种新型的菁染料探针,可靶向定位共价标记活细胞线粒体膜,其效果优于商用线粒体标识剂罗丹明123;2.搭建了一套sted超分辨成像系统,并获取了用探针标定的RAW-7细胞线粒体膜的超分辨图像,其分辨率与共聚焦成像相比有明显提高。

关键词：超分辨荧光成像 sted 线粒体菁染料