Optical microscopy using a single-molecule light source

Rapid progress in science on nanoscopic scales has promoted increasing interest in techniques of ultrahigh-resolution optical microscopy. The diffraction limit can be surpassed by illuminating an object in the near field through a sub-wavelength aperture at the end of a sharp metallic probe. Proposed modifications of this technique involve replacing the physical aperture by a nanoscopic active light source. Advances in the spatial and spectral detection of individual fluorescent molecules, using near-field and far-field methods, suggest the possibility of using a single molecule as the illumination source. Here we present optical images taken with a single molecule as a point-like source of illumination, by combining fluorescence excitation spectroscopy with shear-force microscopy. Our single-molecule probe has potential for achieving molecular resolution in optical microscopy; it should also facilitate controlled studies of nanometre-scale phenomena (such as resonant energy transfer) with improved lateral and axial spatial resolution.     

敲击模式扫描近场光学显微镜

为了实现敲击模式的扫描近场光学显微镜 ,采用以石英音叉为灵敏探测器件 ,将光纤探针垂直音叉侧臂粘接 ,使探针在垂直样品的方向上振动 ,以“敲击”方式探测样品表面的信息 ,实现近场范围的高度控制。仪器结构紧凑 ,操作方便 ,灵敏度高 ,可用于液体环境下的样品探测 ,在对样品形貌进行探测的同时可实现对光信号的调制。将这种探测模式应用于实验 ,获得 2 40 0线 /mm光栅、光盘母盘和生物细胞的形貌图像。实验证明 :这种探测方式分辨率优于 2 0 0 nm,适用于不同硬度的样品 ,可以作为材料学、生物学等领域的有力工具。     

反义HSP90诱导HeLa细胞凋亡的研究

目的 探讨反义ＨＳＰ９０降低ＨｅＬａ细胞恶性度的机制。方法 经荧光染色观察细胞形态,ＤＮＡ Ｌａｄｄｅｒ及流式细胞仪检测凋亡细胞。结果 转染有反义ＨＳＰ９０的ＨｅＬａ细胞经荧光染色细胞呈不均一亮蓝色,荧光染色观察可见细胞变小,染色质浓缩并产生凋亡小体,ＤＮＡ Ｌａｄｄｅｒ可见明显的梯形条带,流式细胞仪检测有凋亡的细胞。结论 反义ＨＳＰ９０可诱导细胞凋亡。     

Adaptive subwavelength control of nano-optical fields

Adaptive shaping of the phase and amplitude of femtosecond laser pulses has been developed into an efficient tool for the directed manipulation of interference phenomena, thus providing coherent control over various quantum-mechanical systems. Temporal resolution in the femtosecond or even attosecond range has been demonstrated, but spatial resolution is limited by diffraction to approximately half the wavelength of the light field (that is, several hundred nanometres). Theory has indicated that the spatial limitation to coherent control can be overcome with the illumination of nanostructures: the spatial near-field distribution was shown to depend on the linear chirp of an irradiating laser pulse. An extension of this idea to adaptive control, combining multiparameter pulse shaping with a learning algorithm, demonstrated the generation of user-specified optical near-field distributions in an optimal and flexible fashion. Shaping of the polarization of the laser pulse provides a particularly efficient and versatile nano-optical manipulation method. Here we demonstrate the feasibility of this concept experimentally, by tailoring the optical near field in the vicinity of silver nanostructures through adaptive polarization shaping of femtosecond laser pulses and then probing the lateral field distribution by two-photon photoemission electron microscopy. In this combination of adaptive control and nano-optics, we achieve subwavelength dynamic localization of electromagnetic intensity on the nanometre scale and thus overcome the spatial restrictions of conventional optics. This experimental realization of theoretical suggestions opens a number of perspectives in coherent control, nano-optics, nonlinear spectroscopy, and other research fields in which optical investigations are carried out with spatial or temporal resolution.     

三尖杉酯碱诱导HeLa细胞凋亡的研究

报道了三尖杉酯碱 (harringtonine ,HT)可以诱导HeLa细胞凋亡 .采用Heochst33342荧光染色、琼脂糖凝胶电泳及流式细胞光度术 (FCM)的方法 ,研究了HT对HeLa细胞凋亡的影响 .利用细胞同步化技术和斑点杂交法研究发现 ,HT影响了HeLa细胞c myc和bcl 2基因的表达并且与细胞周期密切相关 .初步探讨其凋亡诱导的机制 ,认为HT通过在G1和G2 期下调细胞凋亡抑制基因bcl 2的诱导凋亡 ,以及下调c myc癌基因阻滞细胞增殖 ,并延迟凋亡发生 .这些结果对于了解HT的药物作用机制和提高临床化疗疗效具有重要意义 .     

Probing the electromagnetic field of a 15-nanometre hotspot by single molecule imaging

When light illuminates a rough metallic surface, hotspots can appear, where the light is concentrated on the nanometre scale, producing an intense electromagnetic field. This phenomenon, called the surface enhancement effect, has a broad range of potential applications, such as the detection of weak chemical signals. Hotspots are believed to be associated with localized electromagnetic modes, caused by the randomness of the surface texture. Probing the electromagnetic field of the hotspots would offer much insight towards uncovering the mechanism generating the enhancement; however, it requires a spatial resolution of 1-2?nm, which has been a long-standing challenge in optics. The resolution of an optical microscope is limited to about half the wavelength of the incident light, approximately 200-300?nm. Although current state-of-the-art techniques, including near-field scanning optical microscopy, electron energy-loss spectroscopy, cathode luminescence imaging and two-photon photoemission imaging have subwavelength resolution, they either introduce a non-negligible amount of perturbation, complicating interpretation of the data, or operate only in a vacuum. As a result, after more than 30 years since the discovery of the surface enhancement effect, how the local field is distributed remains unknown. Here we present a technique that uses Brownian motion of single molecules to probe the local field. It enables two-dimensional imaging of the fluorescence enhancement profile of single hotspots on the surfaces of aluminium thin films and silver nanoparticle clusters, with accuracy down to 1.2?nm. Strong fluorescence enhancements, up to 54 and 136 times respectively, are observed in those two systems. This strong enhancement indicates that the local field, which decays exponentially from the peak of a hotspot, dominates the fluorescence enhancement profile.     

云芝多糖影响人宫颈癌HeLa细胞的增殖和凋亡的研究

研究云芝多糖(CVP)对宫颈癌HeLa细胞系的增殖、凋亡及产生途径。采用MTT法测定了CVP对HeLa细胞增殖的体外抑制作用,流式细胞仪检测凋亡细胞的比例,qRT-PCR检测P53、Bcl-2和Fas基因在细胞处理前后表达量的变化。结果显示：CVP以时间和剂量依赖的方式抑制HeLa细胞的生长。作用时间在24 h,48 h和72 h时对细胞抑制的抑制率分别为92%、95%和98%;当CVP浓度达到1.25 mg/L时,作用24 h、48 h和72 h时的IC50值分别为0.2507±0.01 mg/L、0.2720±0.04 mg/L和0.2736±0.03 mg/L;当CVP浓度为0.5 mg/L时,作用24 h和48 h后细胞凋亡率分别为26.36% 和63.81%;CVP处理HeLa细胞24 h后,Bcl-2基因的表达被显著下调。研究表明：CVP促进HeLa细胞凋亡,其作用机制与Bcl-2基因的表达下调有关,推测是通过Bcl-2介导的途径促进细胞凋亡。图4表1参30     

新型光纤探针的研制与应用

采用熔拉与腐蚀相结合的方法，制作出了能用于近场探测的新型光纤探针。该制作方法不需要复杂的仪器设备，具有费用低廉、重复性好等优点。镀金膜的直锥型光纤探针首次用于扫描隧道显微镜，由于针尖的曲率半径比铂铱针更小，因此可获得更精细的信息，并可提高图像的分辨率。弯曲型光纤探针固定于特别设计的支架后，可作为悬臂探针用于原子力显微镜和近场光学显微镜。最后讨论了针尖的不同形状及不同镀膜对实验结果的影响。     

The changes in telomerase activity and telomere length in HeLa cells undergoing apoptosis induced by sodium butyrate

The changes in telomerase activity and telomere length during apoptosis in HeLa cells as induced by sodium butyrate (SB) have been studied. After a 48 h SB treatment, HeLa cells demonstrated characteristic apoptotic hallmarks including chromatin condensation, formation of apoptotic bodies and DNA Laddering which were caused by the cleavage and degradation of DNA between nucleosomes. There were no significant changes in telomerase activity of apoptotic cells, while the telomere length shortened markedly. In the meanwhile, cells became more susceptible to apoptotic stimuli and telomere became more vulnerable to degradation after telomerase activity was inhibited. All the results suggest that the apoptosis induced by SB is closely related to telomere shortening, while telomerase enhances resistance of HeLa cells to apoptotic stimuli by protecting telomere.     

基于液相外延生长的集成式微探尖

阐述了用于超高密度光存储的集成式扫描近场光学显微术（SNOM）微探尖的液相外延选择生长方案,并给出了实验结果．此种微探尖,可直接生长在微型集成式SNOM传感器的垂直腔表面发射激光器（VCSEL）晶片出光口上,避免了探尖转移带来的探尖损坏、难以对准等技术难题．该制作过程具有重复性较好,设备简单、经济,实用价值较高等优点．     

Tunable nanowire nonlinear optical probe

One crucial challenge for subwavelength optics has been the development of a tunable source of coherent laser radiation for use in the physical, information and biological sciences that is stable at room temperature and physiological conditions. Current advanced near-field imaging techniques using fibre-optic scattering probes have already achieved spatial resolution down to the 20-nm range. Recently reported far-field approaches for optical microscopy, including stimulated emission depletion, structured illumination, and photoactivated localization microscopy, have enabled impressive, theoretically unlimited spatial resolution of fluorescent biomolecular complexes. Previous work with laser tweezers has suggested that optical traps could be used to create novel spatial probes and sensors. Inorganic nanowires have diameters substantially below the wavelength of visible light and have electronic and optical properties that make them ideal for subwavelength laser and imaging technology. Here we report the development of an electrode-free, continuously tunable coherent visible light source compatible with physiological environments, from individual potassium niobate (KNbO3) nanowires. These wires exhibit efficient second harmonic generation, and act as frequency converters, allowing the local synthesis of a wide range of colours via sum and difference frequency generation. We use this tunable nanometric light source to implement a novel form of subwavelength microscopy, in which an infrared laser is used to optically trap and scan a nanowire over a sample, suggesting a wide range of potential applications in physics, chemistry, materials science and biology.     

Phonon-enhanced light matter interaction at the nanometre scale

Optical near fields exist close to any illuminated object. They account for interesting effects such as enhanced pinhole transmission or enhanced Raman scattering enabling single-molecule spectroscopy. Also, they enable high-resolution (below 10 nm) optical microscopy. The plasmon-enhanced near-field coupling between metallic nanostructures opens new ways of designing optical properties and of controlling light on the nanometre scale. Here we study the strong enhancement of optical near-field coupling in the infrared by lattice vibrations (phonons) of polar dielectrics. We combine infrared spectroscopy with a near-field microscope that provides a confined field to probe the local interaction with a SiC sample. The phonon resonance occurs at 920 cm(-1). Within 20 cm(-1) of the resonance, the near-field signal increases 200-fold; on resonance, the signal exceeds by 20 times the value obtained with a gold sample. We find that phonon-enhanced near-field coupling is extremely sensitive to chemical and structural composition of polar samples, permitting nanometre-scale analysis of semiconductors and minerals. The excellent physical and chemical stability of SiC in particular may allow the design of nanometre-scale optical circuits for high-temperature and high-power operation.     

强电脉冲对抗癌药物毒性的提高作用

研究了强电脉冲作用下，抗癌药物环磷酰胺对HeLa细胞和胎儿脐带血细胞的毒性变化。实验发现，当强电脉冲（电压500V，电容10μF， 脉冲5个） 结合环磷酰胺处理HeLa细胞后，细胞凋亡比例和死亡比例都比环磷酰胺单独处理组要高；当强电脉冲（电压1000V，电容10μF，脉冲2个）结合环磷酰胺处理胎儿脐带血细胞，发现染色体畸变和微核比例比单独药物处理组高。说明强电脉冲能明显提高抗癌药物对细胞的毒性。     

快速诱导细胞凋亡方法探讨

[目的]利用流式细胞仪检测细胞凋亡在基础研究、疾病诊断及预后预测及评价中具有重要的应用价值.补偿调节所需的单阳染色管的制备,是流式细胞术检测凋亡过程的一个重要步骤.探讨了快速诱导不同细胞系和原代细胞凋亡的方法,优化用于检测细胞凋亡的流式单阳染色管快速制备.[方法](1)分别用100 μmol/L H2O2、体积分数4％...     

Massively parallel manipulation of single cells and microparticles using optical images

The ability to manipulate biological cells and micrometre-scale particles plays an important role in many biological and colloidal science applications. However, conventional manipulation techniques--including optical tweezers, electrokinetic forces (electrophoresis, dielectrophoresis, travelling-wave dielectrophoresis), magnetic tweezers, acoustic traps and hydrodynamic flows--cannot achieve high resolution and high throughput at the same time. Optical tweezers offer high resolution for trapping single particles, but have a limited manipulation area owing to tight focusing requirements; on the other hand, electrokinetic forces and other mechanisms provide high throughput, but lack the flexibility or the spatial resolution necessary for controlling individual cells. Here we present an optical image-driven dielectrophoresis technique that permits high-resolution patterning of electric fields on a photoconductive surface for manipulating single particles. It requires 100,000 times less optical intensity than optical tweezers. Using an incoherent light source (a light-emitting diode or a halogen lamp) and a digital micromirror spatial light modulator, we have demonstrated parallel manipulation of 15,000 particle traps on a 1.3 x 1.0 mm2 area. With direct optical imaging control, multiple manipulation functions are combined to achieve complex, multi-step manipulation protocols.     

A single ion as a nanoscopic probe of an optical field.

In near-field imaging, resolution beyond the diffraction limit of optical microscopy is obtained by scanning the sampling region with a probe of subwavelength size. In recent experiments, single molecules were used as nanoscopic probes to attain a resolution of a few tens of nanometres. Positional control of the molecular probe was typically achieved by embedding it in a crystal attached to a substrate on a translation stage. However, the presence of the host crystal inevitably led to a disturbance of the light field that was to be measured. Here we report a near-field probe with atomic-scale resolution-a single calcium ion in a radio-frequency trap-that causes minimal perturbation of the optical field. We measure the three-dimensional spatial structure of an optical field with a spatial resolution as high as 60 nm (determined by the residual thermal motion of the trapped ion), and scan the modes of a low-loss optical cavity over a range of up to 100 microm. The precise positioning we achieve implies a deterministic control of the coupling between ion and field. At the same time, the field and the internal states of the ion are not affected by the trapping potential. Our set-up is therefore an ideal system for performing cavity quantum electrodynamics experiments with a single particle.     

PI3K-Akt和JNK信号通路抑制剂对杆状病毒诱导昆虫细胞凋亡影响的初步研究

为了探讨杆状病毒诱导昆虫细胞凋亡通路与细胞内PI3K-Akt和JNK信号通路的关系,应用PI3K的特异性抑制剂Wortmannin和JNK的特异性抑制剂SP600125处理芹菜夜蛾核型多角体病毒(AfMNPV)感染的斜纹夜蛾SL-1细胞,研究了这些抑制剂对杆状病毒诱导昆虫细胞凋亡的影响.分别使用浓度梯度2.5,25,50μmol的SP600125和0.3,3,30μmol的Wortmannin处理感染了SfaMNPV的SL-1细胞,24h后进光镜观察、DAPI荧光染色,流式细胞术分析显示,抑制PI3K-Akt和JNK信号通路后杆状病毒诱导的细胞凋亡受到明显影响,细胞凋亡水平明显降低.研究结果提示AfMNPV诱导斜纹夜蛾SL-1细胞凋亡过程可能涉及细胞PI3K-Akt和JNK信号通路.     

振幅调制与位相畸变光束通过空间滤波-像传递系统的传输

针对有振幅调制和位相畸变的激光束通过空间滤波-像传递系统的传输进行了讨论,通过数值计算,分析了振幅调制和位相畸变大小对激光束传输的影响,并选取适当的滤波孔径来抑制传输光束的高频调制.     

Arylamine N-acetyltransferases: a new inhibitor of apoptosis in HepG2 cells

Objective: To explore how arylamine N-acetyltransferases (NATs) is related to cell apoptosis. Methods: NAT activity in apoptotic HepG2 cells was measured using high performance liquid chromatography (HPLC); the apoptosis rate of HepG2 cells acted upon by an NAT inhibitor was measured using flow cytometry. Results: NAT activity was lowered in apoptotic HepG2 cells; apoptosis rate induced by camptothecin (CAM) increased after inhibition of NAT activity in HepG2 cells. Conclusion: NAT can inhibit apoptosis in HepG2 cells.     

Nanoscale imaging magnetometry with diamond spins under ambient conditions

Magnetic resonance imaging and optical microscopy are key technologies in the life sciences. For microbiological studies, especially of the inner workings of single cells, optical microscopy is normally used because it easily achieves resolution close to the optical wavelength. But in conventional microscopy, diffraction limits the resolution to about half the wavelength. Recently, it was shown that this limit can be partly overcome by nonlinear imaging techniques, but there is still a barrier to reaching the molecular scale. In contrast, in magnetic resonance imaging the spatial resolution is not determined by diffraction; rather, it is limited by magnetic field sensitivity, and so can in principle go well below the optical wavelength. The sensitivity of magnetic resonance imaging has recently been improved enough to image single cells, and magnetic resonance force microscopy has succeeded in detecting single electrons and small nuclear spin ensembles. However, this technique currently requires cryogenic temperatures, which limit most potential biological applications. Alternatively, single-electron spin states can be detected optically, even at room temperature in some systems. Here we show how magneto-optical spin detection can be used to determine the location of a spin associated with a single nitrogen-vacancy centre in diamond with nanometre resolution under ambient conditions. By placing these nitrogen-vacancy spins in functionalized diamond nanocrystals, biologically specific magnetofluorescent spin markers can be produced. Significantly, we show that this nanometre-scale resolution can be achieved without any probes located closer than typical cell dimensions. Furthermore, we demonstrate the use of a single diamond spin as a scanning probe magnetometer to map nanoscale magnetic field variations. The potential impact of single-spin imaging at room temperature is far-reaching. It could lead to the capability to probe biologically relevant spins in living cells.