HeLa细胞突起中微丝束的纳米分辨荧光成像

在Matlab编程环境下模拟了单分子定位显微的纳米分辨成像,在传统实验参数条件下,对不同间隔分子带模型进行了模拟成像.模拟结果表明,单分子定位显微方法可以区分中心相隔20 nm的两个分子带.同时,也分析了不同像元大小对单分子定位精度的影响.此外,通过单分子定位显微方法在IX71倒置荧光显微镜上实现了纳米分辨,系统极限分辨率,即半高全宽为48 nm.在该系统上,获得了HeLa细胞突起中微丝束结构的纳米分辨图像.从重构获得的图像中可以看到微丝束的直径为75—200 nm.     

正交像散高密度三维单分子定位显微的数值模拟

单分子定位显微(single molecule localization microscopy, SMLM)成像技术利用荧光分子的稀疏发光、探测及定位,实现了纳米级空间分辨率的超分辨成像.为了提高其时间分辨率,需要提高同时发光的荧光分子密度.但随着分子密度的提高,不同分子的点扩散函数(point spread function, PSF)在探测器上将发生严重的重叠现象,导致空间分辨率降低,尤其是在进行三维SMLM成像时.为了解决这一问题,本文提出了一种基于正交像散的高密度三维单分子定位超分辨成像方法,并对该方法进行分析和数值模拟研究.该方法的核心是在单分子定位显微镜中将采集的荧光分成两束成像在同一个探测器的两个区域,并在两个通道中各引入一个光学参数相同但取向相互正交的柱透镜,实现对同一个荧光分子正负两个像散PSF图像的同时探测,然后建立该成像过程的线性投影模型,利用压缩感知算法求解出荧光分子的三维定位信息.结果表明,由于两个正交柱透镜产生的一组正交像散PSF对作为一个分子的系统响应时具有较低的相关性,该方法的高密度三维定位准确性可显著优于采用单个柱透镜的传统像散方法,且离焦程度越大两个...     

超分辨荧光显微成像染料结构与生物应用（特邀）

荧光显微成像技术的生物医学应用离不开荧光染料的设计与开发。有机小分子荧光染料因其易于修饰、生物相容性好、光物理性质优异等特点,在细胞生物成像领域受到了广泛关注。随着超分辨荧光显微镜的发展和技术的进步,使得荧光显微成像突破了光学衍射极限,可以获得更为精准的生物分子学信息,观察纳米尺度下亚细胞器之间的相互作用。根据不同的成像原理,科学家开发出了单分子定位成像技术、受激辐射损耗成像技术、结构光照明技术等超分辨荧光显微技术。这些技术在细胞荧光显微成像领域的应用与发展,同时对有机小分子荧光染料的设计与开发提出了新要求。本文介绍了主流超分辨荧光显微技术的原理,总结已发表的超分辨荧光显微成像荧光染料的结构和光物理性质特点,归纳了其设计要求,旨在为新型荧光染料的设计提供参考。     

高效双螺旋点扩展函数相位片的设计与实验研究

双螺旋点扩展函数具有随离焦连续旋转变化的特性, 结合单分子定位方法可用于厚样品三维超分辨成像及分子定位追踪研究. 但双螺旋点扩展函数不足之处是光能利用率低, 对于光子数受限的荧光显微成像而言其应用受限. 本文通过对双螺旋点扩展函数在空域、频域和拉盖尔-高斯模式面等三个不同域中进行约束优化. 模拟结果表明, 优化后的双螺旋点扩展函数的光能效率提高了30多倍. 同时, 基于最优设计方案制备了相位片, 并实验验证了该设计的正确性. 与文献报道相比较, 本文结果在成像深度和光能利用率方面都有所改善. 关键词： 双螺旋点扩展函数 超分辨成像 轴向定位 优化算法     

双色荧光辐射差分超分辨显微系统研究

为了拓展荧光辐射差分(Fluorescence Emission Difference,FED)显微术的应用,使得该方法可以同时对生物样品的不同组织结构进行超分辨成像,本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像,以此获得超分辨显微图像。在对单色FED显微系统进行研究后,本文提出了一种可行的双色FED显微成像系统方案。实验结果表明,在488 nm和640 nm激发光下,该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率,另外也能对生物样品的不同组织进行多色同时超分辨显微成像,满足了实际应用的要求。     

超分辨率成像荧光探针材料应用进展

为了进一步认知复杂环境中的细胞生物学过程,研究人员发展了各种各样的生物成像技术。在这些技术中,生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而,传统的荧光成像技术受到了光学衍射极限的限制,无法分辨低于200 nm的空间结构,阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制,能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外,目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。     

基于压缩感知的三维单分子定位显微成像方法研究

本文建立了一种三维压缩感知模型以实现对高密度荧光分子图像的快速三维定位。首先,根据荧光显微的三维点扩展函数成像理论,设计测量矩阵,并建立压缩感知模型。接着,对荧光显微成像过程进行了模拟,并采用凸优化方法(CVX)、正交匹配追踪(Orthogonal Matching Pursuit,OMP)算法和同伦算法对建立的压缩感知模型中模拟生成的图像进行了定位分析,分别从恢复率、定位精度、重构时间几方面进行了对比。最后,采用同伦算法对模拟的生物样品和实验室采集的细胞进行了三维定位,并获得了三维超分辨图像。对比结果表明:在重构密度和定位精度接近的情况下,同伦算法比CVX方法的重构速度快2个数量级。同伦算法较OMP算法的定位精度要高一倍。采用同伦算法来实现三维的超分辨荧光显微成像在节约计算时间、实现实时成像方面具有一定的意义。     

介质微球超分辨成像薄膜

针对现有基于微球的超分辨成像系统中液体浸没方式的不稳定性和繁琐性,提出采用介质层来替代液体层,制成含有单层密排微球的薄膜.研究折射率较低的二氧化硅微球和折射率较高的钛酸钡微球浸没在三种不同液体中时的成像特性,设计并制备了一种由单层密排的钛酸钡微球和聚二甲基硅氧烷(PDMS)软膜构成的薄膜,并开展了相应的超分辨成像实验.结果表明:当液体折射率在1.33~1.548之间时,二氧化硅微球只有在半浸没时才能分辨出小于衍射极限的样品特征,而钛酸钡微球则需要全浸没才能实现超分辨成像.在600nm中心波长的照明下,利用该薄膜可以清晰地分辨出周期为278nm,占空比为1:1的硅结构光栅.     

单分子定位超分辨显微成像技术研究进展及展望(特邀综述)

随着新型荧光探针、先进激光、高灵敏光电探测器等相关领域的不断发展,突破衍射极限的超分辨光学显微技术为现代生物医学研究提供了新的有力工具,其中的单分子定位技术利用荧光分子的光开关效应,实现了亚细胞结构的纳米精度超分辨成像.本文介绍了单分子定位超分辨显微技术的基本原理与实现,例举了其在细胞生物学、组织生物学以及神经科学等方面的应用,讨论了该技术目前的发展趋势及可能的改进方向,为相关领域科学研究提供参考.超分辨光学显微技术的不断创新将推动生命科学的新发展.     

结构光照明相位/荧光双模式显微技术

本文提出了一种基于结构光照明的高分辨相位/荧光双模式显微成像方法.该方法利用一数字微镜阵列(DMD)产生条纹结构光,并记录样品在结构光照明下的全息图像和荧光图像,最终可以重建出样品的定量相位图像和超分辨荧光图像.此外,还提出了一种补偿环境扰动对相位成像影响的数值方法,提高了成像系统的抗干扰能力.在该双模式成像系统中,定量相位成像和荧光成像的空间分辨率分别为840 nm和440 nm,为同一样品提供互补信息.该方法有望被广泛应用于生物医学、工业和化学等诸多领域.     

Fast Fourier domain localization algorithm of a single molecule with nanometer precision

We present an algorithm to determine the location of a fluorescent molecule with nanometer-scale accuracy. A Fourier domain localization scheme based on zero-padded fast Fourier transform and phase gradient operators is used to obtain a powerful mathematical model for localizing the molecule without numerical fitting. Compared with conventional algorithms, our position estimator does not require prior background information or initial parameter estimation. Numerical simulations indicate that the proposed method exhibits high localization precision and small bias while executing almost as fast as the fluoroBancroft algorithm.     

Single molecule spectroscopy on photosynthetic pigment-protein complexes

Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of the coupling with the protein environment and by the efficiency of the triplet state quenching. Based on single molecule and hole burning data, we envisage the dimeric nature of the strongly phonon coupled red-most Chl state.     

Tracking of fluorescent molecules diffusing within membranes

A new method is proposed for tracking fluorescing single molecules diffusing within a two-dimensional membrane. It is based on a confocal microscopy setup with a constantly rotating laser focus, which follows the position of the molecule. The optimization and efficiency of the method are theoretically studied for a broad range of experimentally realistic conditions. The proposed method allows for a long-time observation of diffusing molecules while allowing the application of fast spectroscopic techniques such as fluorescence decay time determination or fluorescence anisotropy measurements. Received: 14 January 2000 / Published online: 13 September 2000     

Photocleavage reaction of bromine substituted aromatic acyl compounds studied by CIDEP and transient absorption spectroscopy

The photocleavage of the CBr bond in bromoacetylnaphthalene is investigated by transient absorption and time resolved EPR spectroscopy. In the transient absorption of 2-bromo-2′-acetylnaphthalene, the absorption band observed at λ_max ~440 nm is assigned to the triplet state of the parent molecule. After decay of the triplet absorption, a long lived absorption band is observed at λ_max ~380 nm, which is assigned to naphthoylmethyl radical. The yield of this radical is not dependent on the concentration of oxygen even though the absorption band of the triplet state was quenched by addition of oxygen. Thus we conclude that the spin multiplicity of the precursor molecule is singlet. The CW time resolved EPR spectrum shows a typical E?/A CIDEP pattern of three hyperfine lines of the naphthoylmethyl radical. This result suggests some contribution from triplet precursor molecules. However, a careful analysis of the time profile of the CIDEP intensity observed by FT-EPR revealed that the polarization is generated from the radical pair mechanism (RPM) from the encountered pair of two free naphthoylmethyl radicals and the radical-triplet pair mechanism. RPM polarization by the geminate radical pair, formed by the Br atom and the naphthoylmethyl radical, is not observed. This fact indicates that large spin-orbit coupling (Δg and/or fast spin relaxation by g anisotropy) spoils the RPM polarization. The finding is in contrast to the recent observation of RPM polarization in the Cl cleavage reaction of 1-(chloromethyl)naphthalene.     

麻黄素和左旋咪唑显微拉曼测试分析

在可见光激发下，利用显微拉曼技术得到了黄素和左旋旋咪唑的拉曼谱，并将它们的拉曼谱和甲基安非地明做了对比分析，麻黄素和甲基安非他明同属单取代苯，其分子结构十分相似，由于取代基的成分不同一样，因而拉曼谱中一些峰的相对强度和位置发生了变化。左旋咪唑由于取代基上为咪唑亚塞唑，其拉曼谱中主要谱线位置和强度明显不同于麻醉黄素和甲基安非他明。实验结果表明，利用显微拉曼技术在短时间内，对致幻药物进行准确快速的检测分析，将这一技术应用于运动比赛中，有助于对参赛人员服用违禁药物的检测和分析。     

Absorption and emission lines of a mercury molecule in a mercury discharge lamp

Absorption lines are revealed in spectra of a mercury discharge lamp (Planck’s radiator). Some of the lines were identified by Lord Rayleigh as the absorption lines of a Hg₂ molecule. New absorption lines at 253.29, 253.53, 253.76, and 254.20 nm, as well as emission lines at 264.80, 254.96, 265.11, 279.89, 288.82, 301.50, 301.74, and 301.95 nm are discovered, which enabled the construction of a diagram of the lower excited electronic states of a mercury molecule. It is demonstrated that the dips in the intensity profiles of the atomic mercury lines at 253.65, 435.83, 404.65, and 546.07 nm arise from the radiation absorption in vibronic transitions of diatomic Hg₂ molecules. The concentration of mercury molecules in the plasma of mercury discharge lamps is determined to be of the order of 10⁸ cm^?3. However, their contribution to the formation of the emission line profiles is essential due to the multiple radiation passage through the emitting plasma.     

AOTF在光谱分析中的应用研究4.FSD用于提高AOTF原子发射光谱仪的光谱分辨率

本文报道了利用傅里叶自去卷积 (FSD)来提高AOTF原子发射光谱仪的光谱分辨率。选择合适的卷积参数 :半峰高宽度和剪截长度 ,可使模拟重叠峰半峰高宽度变窄约 3 5倍 ,峰高增加约 5倍 ,而峰的位置不变。将其应用于处理AOTF ICP AES扫描重叠峰 ,可使La 4 0 7 735nm与La 4 0 8 6 72nm的自身重叠峰以及Ca 393 36 6nm与Al394 4 0 1nm的干扰重叠峰达到基线分离 ;对于Eu Sr系列混合样和Sc Sr系列混合样(Sr浓度不变 ,Eu和Sc的浓度改变 )的扫描图 ,经FSD处理后 ,原来Eu 4 2 0 5nm与Sr 4 2 1 6nm以及Sc4 2 4 7nm与Sr4 2 1 6nm重叠峰均可达到基线分离 ,校准曲线的斜率提高了 2～ 3倍。     

绝对探测大气温度的纯转动拉曼激光雷达系统

纯转动拉曼激光雷达是探测大气温度廓线的重要手段之一,其正常工作需要配置其他并行校正设备,制约其在气象及环境监测领域中的实用化进程.基于大气氮气分子的纯转动拉曼谱型对温度的依赖性,提出并设计了绝对探测大气温度廓线的纯转动拉曼激光雷达系统.系统采用波长532 nm且脉冲能量300 m J的激光激励源和口径250 mm卡塞格林望远镜的接收器,设计了衍射光栅和光纤Bragg光栅结合的多通道并行纯转动拉曼光谱分光系统;仿真分析氮气和氧气分子的纯转动拉曼散射谱线间关系,优化选择了6条氮气分子的纯转动拉曼谱线以直接反演大气温度,设计了两级滤光器间转接光纤阵列的结构;基于最小二乘原理推导了绝对探测大气温度的反演算法,并结合标准大气模型,分析了纯转动拉曼激光雷达绝对探测大气温度的探测性能.结果表明,所设计纯转动拉曼激光雷达系统可直接反演大气温度廓线,在测量时间17 min内,温度偏差小于0.5 K的探测高度达2.0 km.     

A dynamic approach to the sudden jump model in single molecule spectroscopy

Various theories that use the sudden jump model to explain the time dependence of the optical band of a single molecule in a fluctuating environment are analyzed. It is shown that the dynamic theory of the optical band, based on the Hamiltonian of the system, should necessarily include the initial conditions for the quantum system. We show how these initial conditions should be chosen for the dynamic theory to properly describe the quantum jumps of the optical band of a single molecule. Unlike stochastic theories based on the sudden jump model, the dynamic theory predicts several absorptances of a single molecule and directly connects the absorptance fluctuations with this fact. The theory is used to account for the time dependence of the broadening of experimentally measured lines of individual molecules.