DNA单分子近场光学成像与荧光探测

介绍了扫描近场光学(SNOM-Scanning Near-Field Optical Microscope)／原子力显微镜（AFM－Atomic Force Microscope)系统（SNO／AM）的工作原理。在AFM模式和SNOM模式下对DNA分子进行成像和荧光探测,得到了清晰的DNA单分子的形貌像和荧光像。由形貌圆像得到的DNA分子尺寸横向为20nm,高度为2nm,其中包含了探针形貌的影响。实验中采Tapping模式的AFM成像,样品经多次搜索扫描无明显损坏。AFM模式的分辨率优于1nm。SNOM模式下DNA分子形貌像和荧光像清晰,由近场荧光分布可以确定分子取向和浓度。用YOYO－1染料对λDNA分子进行染色和荧光探测。通过对DNA分子多个截面进行测量,分析染料 与DNA结合状态。     

近场扫描光学显微镜及其在生物学领域的应用

评述了近场扫描光学显微镜（near-field scanning optical microscopy,NSOM）的仪器构造、工作原理及其在生物学领域的应用成果。对NSOM目前存在的主要问题进行了讨论,并展望了NSOM在该领域的发展潜力。     

近场扫描光学成像结合量子点标记的纳米技术在细胞生物学中的应用

近场扫描光学显微镜（NSOM）对传统的光学分辨极限产生了革命性的突破,可在超高光学分辨率下无侵人性和无破坏性地对生物样品进行观测。量子点（QDs）具有极好的光学性能,如荧光寿命长、激发谱宽、生物相容性强、光稳定性好等优点,适合先进的生物成像。NSOM结合QDs标记的纳米技术被应用在细胞生物学中。通过纳米量级NSOM免疫荧光成像（50nm）对特定蛋白分子在细胞表面的动态分布进行可视化研究和数量化分析,阐明了蛋白分子在不同细胞过程中的作用机制。因此,NSOM／QD基成像系统提供了单个蛋白分子最高分辨率的荧光图像,为可视化研究蛋白分子机制的提供了一种强有力的工具。     

原子力显微术在生物研究中的应用

原子力显微术不仅能够提供样品表面纳米级别分辨率的三维图像数据,而且能够对pN级微小力进行测量,同时将两者结合发展出的TREC（topography and recognition）显微术还能够在进行高分辨成像的同时实现对特定分子的定位。原子力显微术的这些特点使之成为生物化学、细胞生物学等生物研究的有利工具。本文主要介绍了原子力显微镜高分辨成像和检测生物分子识别的原理,以及TREC显微术在生物学上的应用。     

同时应用近场光学显微镜的透射和反射模式研究单个细胞的光学性质

通常认为．在近场光学显微技术的光收集模式中,观察透光性好的样品时采用透射模式．研究不透明样品时采用反射模式。本文同时采用透射和反射两种模式观察透明性较好的PCI2细胞和淋巴细胞样品．初步研究单个细胞的反射、吸收、透射和荧光等光学性质,以促进组织光学和激光生物医学等领域的进一步发展。细胞光学的时代就要到来。     

生物单分子研究的进展

大多数分子生物学实验都代表一种集体平均的测量,所记录的都是复杂系统中大量分子的行为,随着技术的发展,目前已能够对单分子观察、探测、操纵并研究其动态与构象变化。从单分子研究可得到新的信息,这些信息隐藏在集体测量之中,或已被平均掉。这一领域代表着本世纪结构生物学的一个新方向。     

量子点的近场激发荧光特性研究

近场光学显微镜具有nm量级的空间分辨率,量子点(quantum dots,QDs)荧光探针具有激发谱宽、发射谱线窄、荧光强度高、抗光漂白和稳定性高等优点,两者结合用于生物大分子的成像探测和识别具有广泛的应用前景。用近场光学显微镜对链霉亲和素偶联的QDs进行近场荧光激发,并对其荧光发射特性和光稳定性进行研究,结果表明:近场光学显微镜nm量级的空间分辨率,可以同时观察到了QDs的单体、二聚体和三聚体;QDs的荧光发射强度高,近场荧光像对比度好,单量子点的荧光半高宽达到25nm;对一定入射波长的单色激发光,QDs的近场荧光强度随着激发功率密度的增加线性增加,并很快趋于稳定。与传统的荧光染料如异硫氰酸荧光素相比,QDs的稳定性非常好,在激发功率密度为300W/cm2的近场辐射下,量子点的荧光强度超过6h基本保持不变,其抗光漂白能力远远高于普通荧光染料。     

光学显微镜技术在活细胞单分子检测中的应用

单分子检测是一门以高度的时间以及空间分辨率研究生物单分子的技术。近来,科学技术的探索发展使我们可以观察、检测甚至操纵单个分子并且研究它们的构象变化和动力学行为。这一发展使得以前被传统系综研究体系平均化所隐藏的新信息被揭示出来。单分子检测技术的发展已经揭开了生命科学研究的新篇章。在本文中,我们将介绍有关活细胞中单分子检测技术的发展以及活细胞内单分子检测的现状。     

分子动态模拟及其在生物大分子研究中的应用

生物分子动念模拟技术是运用计算机对生物大分子的结构、功能、质子运动轨迹以及生物分子间的相互作用进行预测,是研究生物分子结构和功能的重要手段.该文介绍分子动态技术的原理及其在生命科学研究中的应用和研究进展,分析目前存在的问题,并提出对未来工作的展望.     

原子力显微镜在病毒学研究中的应用

1982年德裔物理学家G.Binnig和H.Rohrer发明了具有原子级分辨率的扫描隧道显微镜(scanning tunneling microscope,STM),使人类第一次能够实时地观察单个原子在物质表面的排列状态和相关的理化性质,两位科学家因此荣获1986年诺贝尔物理学奖[1].在STM基础上发展起来的利用探针扫描技术的一类显微镜统称为扫描探针显微镜(SPM),包括扫描隧道显微镜、原子力显微镜、摩擦力显微镜、磁力显微镜、近场光学显微镜和弹道电子发射显微镜等.档     

Near-field scanning optical microscopy in cell biology

Near-field optics has produced the highest optical resolution that has ever been achieved. The methods involved lie at the interface of far-field optical microscopy and scanned probe microscopy. This article describes the principles behind near-field scanning optical microscopy (NSOM) and highlights its potential in cell biology.     

Near-field optical imaging of abasic sites on a single DNA molecule

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.     

低温电镜技术及其在植物（动物）材料研究中的应用

通过一些实例介绍了高压冷冻,冷冻置换和冷冻超薄切片等低温电镜样品制备技术,并且与传统方法对照,说明低温电镜技术的优越性,其中,发菜（Nostoc flagelliforme)营养细胞的冷冻超薄切片（未经化学固定,脱水）所显示的超微结构更客观地反映了生物样品的自然生理状态。此外,应用高压冷冻和冷冻置换的免疫标记电镜技术,首次对发菜营养细胞中的DNA进行定位,明确了核区的位置及范围。     

Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy

A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100 pg/ml. The minimum streptavidin detection limit using this technique is 1 pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.     

同步辐射圆二色谱及其在糖生物学及生物分子中的应用

同步辐射圆二色谱与普通圆二色谱相比,特点在于向真空紫外波段（〈200nm）拓展,以及同步辐射所提供的高强度紫外和真空紫外光源。糖的圆二色谱结构主要在200nm以下。蛋白质和核酸在200nm以下的真空紫外范围,也具有丰富的光谱结构。因此向真空紫外拓展,伴随新的电子跃迁,对应新的光谱结构,包含更丰富的结构信息,确定的结构种类就越多和越精确。同步辐射高强度的真空紫外光源,是获得高质量真空紫外圆二色谱数据的保证,为糖及糖蛋白、蛋白质和核酸研究提供了溶液中结构探测新的实验方法。综述同步辐射圆二色谱特点及其在结构生物学中的应用,以及新发展的蛋白质圆二色谱数据库（PCDDB）。介绍已对外开放的北京同步辐射实验室同步辐射圆二色谱探测,及其在蛋白质、糖和核酸研究中的应用,以及基于微流控混合芯片的亚毫秒动态探测发展。     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Confocal microscopy via reciprocal optical fibre image detection

We describe a compact form of confocal scanning microscope using a semiconductor laser. Confocal operation is ensured by the use of a single mode optical fibre for both launching the light into the microscope and collecting the signal from the object. The collected light is allowed to re-enter the laser and the image is detected as a modulation on the signal from the laser power monitor diode. Images are compared with those obtained from traditional point detectors. The alignment tolerances of the reciprocal scheme are found to be greatly reduced over conventional confocal systems.     

Diffraction imaging of single particles and biomolecules

Theory predicts that with a very short and very intense X-ray pulse, the image of a single diffraction pattern may be recorded from a large macromolecule, a virus, or a nanocluster of proteins without the need for a crystal. A three-dimensional data set can be assembled from such images when many copies of the molecule are exposed to the beam one by one in random orientations. We outline a method for structure reconstruction from such a data set in which no independent information is available about the orientation of the images. The basic requirement for reconstruction and/or signal averaging is the ability to tell whether two noisy diffraction patterns represent the same view of the sample or two different views. With this knowledge, averaging techniques can be used to enhance the signal and extend the resolution in a redundant data set. Based on statistical properties of the diffraction pattern, we present an analytical solution to the classification problem. The solution connects the number of incident X-ray photons with the particle size and the achievable resolution. The results are surprising in that they show that classification can be done with less than one photon per pixel in the limiting resolution shell, assuming Poisson-type photon noise in the image. The results can also be used to provide criteria for improvements in other image classification procedures, e.g., those used in electron tomography or diffraction.