浅析高数值孔径对STED超分辨显微成像的影响

数值孔径是显微成像和光纤应用领域的重要概念,孔径的大小对传统光学显微镜成像效果具有重要作用。在其他技术参数一定的情况下,数值孔径越大,显微镜成像分辨率越高。因为受到光的衍射作用的限制,所以传统光学显微镜的分辨率不可能无限制增大,最多只能分辨到200 nm。为了突破这一限制,STED超分辨率显微镜被研制并广泛应用,将分辨能力提高到50 nm。数值孔径对STED超分辨率显微镜的影响不能简单套用传统显微镜的相关研究成果,必须从STED超分辨率显微镜本身的性质出发,有针对性地进行分析。该文以高数值孔径对STED超分辨率显微成像的影响为主题,从简要论述STED超分辨率显微镜概况入手,从有利影响和不利影响2个层面进行深入分析,并将该领域的研究从定性分析拓展到定量研究领域,理论上可以达到纳米级甚至更小的程度。

LeicaSP8STED3X激光扫描共聚焦显微镜的使用研究

本文主要介绍上海交通大学分析测试中心配置的Leica SP8 STED 3X激光扫描共聚焦显微镜的组成、技术参数和工作原理,它在Leica TCS SP8共聚焦显微镜基础上搭载了超分辨模块、活细胞工作站和双光子模块,既可以开展纳米水平的亚细胞结构和动力学的研究,也可以观察活细胞中亚细胞器的运动特性及变化,是一个具备多种检测功能的超高分辨共聚焦显微系统。超高分辨率荧光显微技术主要包括基于调制照明光斑以缩小系统的点扩散函数的超分辨技术和基于单分子定位的超分辨技术。同时也简述了样品的制备方法和图像采集的参数设置原则,以及日常维护和管理,具有临床借鉴意义。

STED超高分辨显微成像技术原理及其在医学研究中的应用

受激发射损耗(STED)超高分辨率成像技术是一种突破光学衍射极限的显微镜技术,可以达到纳米级分辨率,适合于对200 nm以内的细胞细微结构进行观察和分析。STED技术的优势包括超高分辨率、快速扫描、多色观察、活细胞监测、厚组织标本3D重建、共定位分析等,是现代医学和细胞生物学研究的理想工具。目前STED技术成功实现了对于荧光纳米颗粒的成像,更多的亚细胞结构细节以及活细胞器的生理活动可以被STED技术解析出来,得到了越来越广泛的应用。文章主要介绍了STED超高分辨率成像技术的主要原理及其在医学细胞生物学研究领域的主要应用。

STED microscopy based on axially symmetric polarized vortex beams

A stimulated emission depletion （STED） microscopy scheme using axially symmetric polarized vortex beams is pro- posed based on unique focusing properties of such kinds of beams. The concept of axially symmetric polarized vortex beams is first introduced, and the basic principle about the scheme is described. Simulation results for several typical beams are then shown, including radially polarized vortex beams, azimuthally polarized vortex beams, and high-order axi- ally symmetric polarized vortex beams. The results indicate that sharper doughnut spots and thus higher resolutions can be achieved, showing more flexibility than previous schemes based on flexible modulation of both phase and polarization for incident beams.

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

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.

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

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.

A Low Cost and Versatile STED Superresolution Fluorescent Microscope

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.

试论中国石化石油科学研究院STED苯乙烯生产技术在新疆运用的成熟可靠性分析

苯乙烯是化学工业中重要的材料,对我国生活生产都有着重大的意义。新疆地域辽阔,有着丰富的石油和天然气资源,因此新疆苯乙烯生产既能够有效的缓解市场供应紧张,又能够给当地带来经济效益。文章从新疆地理环境等因素考虑,分析苯乙烯技术在新疆运用的可行性。

受激发损耗(STED)显微术及在生物邻域的应用

受激发射损耗(STED)显微成像通过引入一束损耗光以受激发射的方式减小有效荧光的发光面积,激发光激发样品的荧光基团形成一个艾里斑,损耗光生成中空型的面包圈抑制艾里斑周围的荧光强度,使我们仅看到中心的强度。基于StefanW.Hell第一次提出受激发射损耗显微术,本文介绍了受激发射损耗(STED)显微术的发展过程,并阐述了近几年来,STED在生物领域的应用。最后,总结和展望了该技术的应用前景。

STED Imaging of Vesicular Endocytosis in the Synapse

Endocytosis is a fundamental biological process that couples exocytosis to maintain the homeostasis of the plasma membrane and sustained neurotransmission.Super-resolution microscopy enables optical imaging of exocytosis and endocytosis in live cells and makes an essential contribution to understanding molecular mechanisms of endocytosis in neuronal somata and other types of cells.However,visualization of exo-endocytic events at the single vesicular level in a synapse with optical imaging remains a great challenge to reveal mechanisms governing the synaptic exo-endocytotic coupling.In this protocol,we describe the technical details of stimulated emission depletion(STED)imaging of synaptic endocytosis at the single-vesicle level,from sample preparation and microscopy calibration to data acquisition and analysis.

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

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.

Superresolution imaging of DNA tetrahedral nanostructures in cells by STED method with continuous wave lasers

DNA tetrahedral nanostructures are considered to be uew nanocarriers because they can be precisely controlled and hold excellent penetration ability to the cellular membrane. Although the DNA tetrahedral nanostructure is extensively studied in biology and medicine, its behavior in the cells with nanoscale resolution is not understood clearly. In this letter, we demonstrate superrcsolution fluorescence imaging of the distribution of DNA tetrahedral nanostructures in the cell with a simulated emission depletion （STED） microscope, which is built based on a conventional eonfocal microscope and can t）rovide a resolution of 70 nm.

Biocompatible carbon dots with low-saturation-intensity and highphotobleaching-resistance for STED nanoscopy imaging of the nucleolus and tunneling nanotubes in living cells

Many kinds of nano particles and organic dyes as fluorescent probes have been used in the stimulated emission depletion(STED)nanoscopy.Due to high toxicity,photobleaching and non-water solubility,these fluorescent probes are hard to apply in living cell imaging.Here,we reporta new fluorescence carbon dots(FNCDs)with high photoluminescence quantum yield(56%),low toxicity,anti-photobleaching and goodwater-solubility that suitable for live-cell imaging can be obtained by doping fluorine element.Moreover,the FNCDs can stain the nucleolusand tunneling nanotubes(TNTs)in the living cell.More importantly,for STED nanoscopy imaging,the FNCDs effectively depleted backgroundsignals and improved imaging resolution.Furthermore,the lateral resolution of single FNCDs size under the STED nanoscopy is up to 22.1 nm for FNCDs deposited on a glass slide was obtained.And because of their good water dispersibility,the higher resolution of single FNCDs sizein the nucleolus of a living cell can be up to 19.7 nm.After the image optimizati on steps,the fine fluoresce nee images of TNTs diameter with ca.75 nm resolution is obtained living cell,yielding a threefold enhancement compared with that in confocal imaging.Additionally,the FNCDs show excellent photobleaching resistance after 1,000 scan cycles in the STED model.All results show that FNCDs have significant potentialfor application in STED nanoscopy.

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

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.

Superresolution imaging of telomeres with continuous wave stimulated emission depletion (STED) microscope

The significant role of telomeres in cells has attracted much attention since they were discovered.Fluorescence imaging is an effective method to study subcellular structures like telomeres.However,the diffraction limit of traditional optical microscope hampers further investigation on them.Recent progress on superresolution fluorescence microscopy has broken this limit.In this work,we used stimulated emission depletion(STED) microscope to observe fluorescence-labeled telomeres in interphase cell nuclei.The results showed that the size of fluorescent puncta representing telomeres under the STED microscope was much smaller than that under the confocal microscope.Two adjacent telomeres were clearly separated via STED imaging,which could hardly be discriminated by confocal microscopy due to the diffraction limit.We conclude that STED microscope is a more powerful tool that enable us to obtain detailed information about telomeres.

超高分辨率激光扫描共聚焦显微镜的成像技术与应用

超高分辨率激光扫描共聚焦显微镜在生命科学研究领域中的应用日益广泛。该文以Zeiss LSM 880 Airyscan with STEDYCON超分辨率激光共聚焦显微镜为例,介绍了Airyscan技术和基于受激发射现象的超分辨技术(STED)的原理、应用和参数设置,以期能够为科研工作者究竟采用哪种模式进行图像采集提供参考,以获得最佳的成像效果。

基于科研成果转化的超分辨显微成像教学实验设计

为培养创新应用型工程人才,融合专业基础知识与教师科研成果,利用MATLAB GUI强大的计算和人机交互功能构建STED超分辨显微成像仿真实验平台。通过GUI界面可以方便地进行各项参数设置和可视化入射光像差和偏振态对超分辨效果的影响,具有良好的可操作性和交互性。借助该仿真实验摆脱了实验仪器、场地等束缚,解决了实验装置复杂、设备昂贵等问题,为实验教学提供了一种可视化手段,同时还实现了基础理论与前沿技术的融会贯通,使学生充分认识和掌握高新技术,为拔尖创新人才提供了重要的培养途径。

基于受激发射损耗显微术的活细胞和活体超分辨成像

细胞作为生命体基本的结构和功能单元,在生物、医学等领域有着非常重要的研究意义。随着现代科学和技术的发展,科学家们借助电镜对细胞以及细胞器的空间结构已经有非常清晰的认识,但是对它们的功能以及细胞之间的相互作用却了解得非常少,而这恰恰又是疾病治疗和药物开发亟需了解的信息,因此对离体活细胞(简称活细胞)和活体生物组织细胞(简称活体细胞)中亚细胞器的研究变得非常重要。然而细胞中许多细胞器的结构在纳米量级,传统的光学成像技术由于受到光学衍射极限的限制是无法观察到纳米量级的生物结构,因此光学超分辨成像技术是目前研究亚细胞器结构和功能的有效工具。在所有光学超分辨显微技术中,受激发射损耗显微术(stimulated emission depletionmicroscopy,STED)由于具有实时成像、三维超分辨和断层成像的能力,非常适合用于纳米尺度的活细胞和活体细胞成像研究,而且STED超分辨成像技术经过近几十年的发展,已经广泛用于活细胞甚至活体小鼠细胞的超分辨动态观测。本文总结了近年来活细胞和活体小鼠神经元细胞等领域STED超分辨成像的研究进展,介绍了用于活细胞和活体细胞STED超分辨成像的荧光染料和荧光蛋白的发展现状,讨论当前活细胞和活体细胞的超分辨成像面临的问题,以及对未来发展的展望。

玄武岩纤维改善钢渣沥青混合料路用性能试验研究

为改善钢渣沥青混合料的路用性能,并尽可能提升钢渣沥青混合料的体积安定性,基于车辙试验、低温弯曲试验、浸水膨胀实验、疲劳试验、SPT动态模量实验等,对掺与不掺玄武岩纤维的钢渣沥青混合料的性能进行对比试验研究.结果表明:掺入玄武岩纤维后,全钢渣沥青混合料的高温稳定性和低温抗裂性分别提升了20%和7.7%;动态模量提高了18%左右;疲劳寿命有效延长;安定性得到明显改善.

几种超分辨率荧光显微技术的原理和近期进展

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.