Negative staining across holes: application to fibril and tubular structures

The negative staining technique, when used with holey carbon support films, presents superior imaging conditions than is the case when samples are adsorbed to continuous carbon films. A demonstration of this negative staining approach is presented, using ammonium molybdate in combination with trehalose, applied to several fibrillar and tubular samples. Fibrils formed from the amyloid-beta peptide and the protease inhibitor pepstain A spread very well unsupported across holes and the different polymorphic fibril forms can be readily assessed. However, tubular forms of amyloid-beta have a tendency to be flattened, due to surface tension forces prior to and during specimen drying. Sub-fibril assembly forms and D-banded rat tail type 1 collagen fibres are presented. The air-dried collagen images produced are shown to contain almost as much detail as those obtainable by cryo-negative staining. Fragile DNA and DNA-protein nanotubes are also shown to yield superior quality images to those produced on continuous carbon films. The iron-storage protein, frataxin, creates elongated oligomeric assemblies, containing bound ferrihydrite microcrystals. The iron particles within these flexuous oligomers can be defined in the presence of ammonium molybdate, but they are more readily demonstrated if the frataxin is spread across holes in the presence of trehalose alone. The samples used here serve to show the likely benefit obtainable from negative staining across holes for a range of other fibrillar and tubular samples in biology, medicine and nanobiotechnology.     

Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: a review of applications

Several representative examples are given of the successful application of negative staining across the holes of holey carbon support films using 5% (w/v) ammonium molybdate solution containing trehalose. The inclusion of 0.1% (w/v) trehalose is considered to be most satisfactory, although good data have also been obtained in the presence of 0.01 and 1.0% (w/v) trehalose. The examples given fall into the following groups: protein molecules in the absence of polyethylene glycol (PEG), protein molecules in the presence of PEG (Mr 1000), lipoproteins, lipids and membranes, filaments and tubules, viruses in the absence of PEG, viruses in the presence of PEG, aqueous polymer solutions, and finally for comparison purposes, four unstained samples studied in the presence of trehalose alone. In all these cases, and many others not documented here, successful spreading of the sample across holes has been achieved, with the sample embedded within a thin film of air-dried ammonium molybdate+trehalose. These specimens can be rapidly produced and provide an alternative to negatively stained specimens on carbon support films. Specimen stability in the electron bean is good and such specimens can usually generate superior negatively stained TEM images without flattening and adsorption artefacts. The formation of 2-D arrays/crystals of protein molecules and viruses, suspended across holes in the presence of ammonium molbybdate+trehalose, and trehalose alone, is also demonstrated.     

金膜上亚波长小孔阵列表面等离激元颜色滤波器偏振性质

金属薄膜上制备的表面等离激元颜色滤波器具有很强的颜色可调性. 在200 nm厚的金膜上, 通过聚焦离子束刻蚀, 制备一系列周期逐渐变化的圆形、方形、矩形亚波长尺寸小孔方阵列表面等离激元颜色滤波器, 改变入射光的偏振方向, 观察其超透射滤波现象. 研究发现: 对于矩形小孔阵列, 其透射光颜色随入射光偏振方向的变化而改变; 而对于圆形、方形的小孔阵列, 其透射光颜色对入射光的偏振方向并不敏感. 分析表明, 对于金膜上刻蚀的小孔结构, 虽然结构的周期性导致的表面等离激元极化子会对透射光的颜色变化产生一定影响, 但是随小孔形状变化的局域表面等离激元共振才是影响透射光颜色的决定性因素. 如果入射光没有在小孔中激发出局域表面等离激元, 则表面等离激元极化子对透射光的影响也会消失. 根据不同形状小孔周期结构透射光颜色随入射光的偏振变化特点, 制备出了包含两种小孔形状的复合周期结构. 随着入射光偏振方向的改变, 该结构会显示出不同的颜色图案. 关键词： 表面等离激元极化子 局域表面等离激元 颜色滤波器 亚波长小孔阵列     

Coercive field enhancement in microstructured (La<Subscript>0.4</Subscript>Pr<Subscript>0.6</Subscript>)<Subscript>0.67</Subscript>Ca<Subscript>0.33</Subscript>MnO<Subscript>3</Subscript> thin films

The perovskite material (La_0.4Pr_0.6)_0.67Ca_0.33MnO₃ (LPCMO) has complex electronic and magnetic behavior based on phase competition between ferromagnetic metallic (FMM) and insulating phases with similar free energies. Experimental evidence has indicated that in-plane stress anisotropy influences these phases and can affect electronic and magnetic properties. Here we investigate the roles that both stress and shape anisotropies may play in controlling the coercive field of the material. LPCMO thin films of various thicknesses (20, 25, and 30 nm) were deposited on (110) NdGaO₃ (NGO) substrates using pulsed laser deposition and the coercive fields were measured. Photolithography was then used to fabricate microstructured arrays of LPCMO on the NGO substrates for each of the films. The coercive fields of these arrays of LPCMO were compared to the behavior of the corresponding unpatterned LPCMO thin films across a range of temperatures. Microstructure arrays for the thicker (25 and 30 nm) films showed a substantial increase in the coercive field after forming the arrays, whereas a thinner film (20 nm) showed almost no change in the coercive field. Stress anisotropy continues to play a dominant role in the behavior of LPCMO thin films and dimensionality of the magnetic domains also influences the results. The films show 2D behavior when film thickness approaches the size of the critical radius for single-to-multidomain transitions. Making thicker films allows for 3D behavior and a role for shape anisotropy to influence the coercive fields.     

Formation of two-dimensional crystals of icosahedral RNA viruses

The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. SYNOPSIS: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods.     

Comparison of the sensitivity of extraordinary transmittance peaks with the surrounding media for hole and slit arrays

In this paper we compare the sensitivity of the Extraordinary Transmittance (ET) peak position with the surrounding media for two types of metallic structures: arrays of holes and arrays of slits recorded in Au films. Both types of array were fabricated using Interference Lithography (IL) with a period of 700 nm and an Au thickness of 150 nm. The transmission spectra measurements were performed at normal incidence using a spectrophotometer. The results show that an array of slits presents a higher sensitivity for the surrounding media than the array of holes. Theoretical TE and TM simulations of the transmission spectra for the slit arrays agree very well with the experimental results, confirming the better sensitivity of the slit arrays.     

Coercive field behavior of permalloy antidot arrays based on self-assembled template fabrication

High-density magnetic antidot arrays have been fabricated by deposition of Fe₂₀Ni₈₀ thin films on self-assembled nanoporous alumina membranes (NAM) with high-order hexagonal symmetry. The magnetic properties induced by the size and the geometry configuration of the holes introduced in a Fe₂₀Ni₈₀ thin film are discussed based on hysteresis loops measured as a function of temperature. The precursor NAMs have pore diameters ranging between 35 and 95 nm (55 and 75 nm after the film deposition) and a lattice parameter of 105 nm. An enormous increase of coercitivity, as compared with the corresponding continuous films, was observed for temperatures between 2 and 300 K. This effect depends on the size and surface density of holes in the Fe₂₀Ni₈₀ antidot arrays. Rutherford backscattering spectrometry (RBS) measurements were performed in order to better clarify the magnetic material that was eventually deposited within the NAM pores.     

Electronic conduction through 2D arrays of nanometer diameter metal clusters

An experimental study of electrical conduction through arrays of nanometer-diameter metallic clusters linked by organic molecules is presented. Gold clusters, having diameters of ∼4 nm and encapsulated by a monolayer of dodecanethiol, are deposited from solution on to specially prepared substrates to form a close-packed cluster monolayer. Nearest-neighbors in this 2D array of encapsulated clusters are then covalently linked using a conjugated organic molecule approximately 2.2 nm in length having isocyanide groups at both ends. In order to allow both electrical characterization and TEM imaging, the cluster arrays are deposited in 500 nm wide gaps between gold contacts on a free standing, insulating SiO₂ film. Electronic conduction through linked 2D arrays approximately 80 clusters in length has been observed at room temperature. The structure of the arrays and current-voltage relationships for the linked arrays are presented.     

Nanofabrication of a space-variant optical transmission filter

A space-variant optical transmission filter is demonstrated for which a simplified process is used to tailor the spatial response of the filter across the surface of a single wafer. A multilayer stack, of alternating high or low refractive index dielectric materials, was used to produce a narrow transmission notch in the center of a wide stop band. Subsequent patterning and etching of arrays of holes through the volume of the dielectric stack was performed to control the fill factor of the dielectric in the layers. The position of the transmission notch within the reflection spectrum was varied across the device surface by adjusting the hole diameter of the hole arrays. Experimental and numerical simulation were used to confirm the space-variant transmission characteristics of a single-wafer sample with two zones of different hole diameter arrays in the 1550 nm wavelength regime.     

Angle-resolved transmission measurements through anisotropic two-dimensional plasmonic crystals

We report on high-accuracy angle-resolved optical transmission measurements through anisotropic 2D plasmonic crystals made of gold films with large-area rectangular arrays of nanoscale square holes, deposited on GaAs substrates. The measurements reveal the dispersion relations of air-gold and gold-GaAs surface plasmon polaritons. The crystal anisotropy induces a separation between plasmonic modes propagating in different directions. Their symmetry and dispersion properties are discussed.     

Formation, TEM study and 3D reconstruction of the human erythrocyte peroxiredoxin-2 dodecahedral higher-order assembly

The production of a higher-order assembly of peroxiredoxin-2 (Prx-2) from human erythrocytes has been achieved during specimen preparation on holey carbon support films, in the presence of ammonium molybdate and polyethylene glycol. TEM study suggested that this assembly is a regular dodecahedron, containing 12 Prx-2 decamers (Mr 2.62 MDa, external diameter approximately 20 nm). This interpretation has been supported by production of a approximately 1.6 nm 3D reconstruction from the negative stain TEM data, with automated docking of the available X-ray data of the Prx-2 decamer. Comparison with other known protein dodecahedral and viral icosahedral structures indicates that this arrangement of protein molecules is one of the fundamental macromolecular higher-order assemblies found in biology. Widespread biotechnological interest in macromolecular "cage" structures is relevant to the production of the Prx-2 dodecahedron.     

BRD96N光调制吸收增强现象的实验研究

通过光谱响应特性实验和记录／读出图像实验，研究了基因变异型细菌视紫红质（BR_D96N）分子膜对单色光的光调制特性.发现BR_D96N分子膜在550nm—600nm范围内对调制光有吸收增强的现象，且对此范围内不同波长的单色光其调制程度有差异.利用曲线拟和方法发现550nm—600nm吸收增强的变化过程分为快过程和慢过程，其对应的时间常数分别为30s和5min.利用强度调制器的吸收强度与图像灰度之间的关系，分析了560nm—600nm范围内出现图像反转的实验现象. 关键词： 细菌视紫红质D96N分子膜 光谱响应 吸收增强现象 图像反转现象     

Charge carrier photogeneration in thin films of iodoform

The photogeneration of electrons and holes in thin polycrystalline iodoform films has been investigated as a function of the electric field. Equal values of the primary rates of production of electrons and holes support the idea of an intrinsic photogeneration mechanism. The experimental results are fitted by the Onsager model. The fraction of absorbed photons that produce thermalized electron-hole pairs is found to be close to unity, with an initial pair separation of ca. 3 nm.     

Deposited metamaterial thin film with negative refractive index and permeability in the visible regime

Thin films are fabricated from arrays of silver nanorods with thicknesses of 160?nm and 200?nm, to function as a metamaterial. The negative refractive index and negative permeability are retrieved from measured reflection and transmission coefficients using walk-off interferometer in the visible regime. A negative-index-material thin film with negative permittivity or (and) permeability can be produced by glancing angle deposition.     

纳米银六角阵列在掺氧氮化硅中的局域表面等离激元共振特性仿真

通过COMSOL Multiphysics 和 Lumerical FDTD solution对不同尺寸纳米银六角阵列在非晶态掺氧氮化硅(a-SiN_x:O)介质中的局域表面等离激元共振(LSPR)特性进行仿真, 计算结果表明半径为25 nm的纳米银六角阵列形成的局域表面等离激元(LSP)与厚度为70 nm的a-SiN_x:O的蓝光发射(460 nm)的共振效果最为显著, 随着纳米银颗粒尺寸的增大其消光共振峰红移. 在460 nm波长激发下半径为25 nm的纳米银阵列在a-SiN_x:O中的极化强度和表面极化电荷的分布模拟证明了该阵列在460 nm激发下形成的LSP为偶极子极化模式, 通过对该尺寸的纳米银阵列的LSP 在a-SiN_x:O中的最强垂直辐射空间计算, 获得了银颗粒上方a-SiN_x:O的最佳厚度为30 nm, 仿真结果对硅基蓝光发射器件(450–460 nm)的设计提供了重要的理论参考.     

Relation between molecule ionization energy,film thickness and morphology of two indandione derivatives thin films

Nowadays most organic devices consist of thin (below 100 nm) layers. Information about the morphology and energy levels of thin films at such thickness is essential for the high efficiency devices. In this work we have investigated thin films of 2-(4-[N,N-dimethylamino]-benzylidene)-indene-1,3-dione (DMABI) and 2-(4-(bis(2-(trityloxy)ethyl)amino)benzylidene)-2H-indene-1,3-dione (DMABI-6Ph). DMABI-6Ph is the same DMABI molecule with attached bulky groups which assist formation of amorphous films from solutions. Polycrystalline structure was obtained for the DMABI thin films prepared by thermal evaporation in vacuum and amorphous structure for the DMABI-6Ph films prepared by spin-coating method. Images taken by SEM showed separate crystals or islands at the thickness of the samples below 100 nm. The ionization energy of the studied compounds was determined using photoemission yield spectroscopy. A vacuum level shift of 0.40 eV was observed when ITO electrode was covered with the thin film of the organic compound. Despite of the same active part of the investigated molecules the ITO/DMABI interface is blocking electrons while ITO/DMABI-6Ph interface is blocking holes.     

Depth dependence of Néel wall pinning on amorphous CoxSi1−x films with diluted arrays of elliptical antidots

Diluted arrays of elliptical antidots have been fabricated by optical lithography, electron beam lithography and plasma etching on amorphous Co₇₄Si₂₆ magnetic films with a well-defined uniaxial anisotropy. The magnetic behavior of two identical antidot arrays but with different hole depth in comparison with film thickness has been studied by transverse magneto-optical Kerr effect. Significant differences appear in the coercivity depending on whether the magnetic film is completely perforated or not, indicating a much more effective domain wall pinning process when the depth of the holes is smaller than the magnetic film thickness.     

激光加速实验超薄类金刚石碳靶的制备

采用过滤阴极真空弧法,制备了满足激光稳相加速机制要求的超薄自支撑类金刚石碳靶。室温下沉积,基片偏压为-32 V,薄膜沉积速率约为每脉冲0.002 nm。选取NaCl膜作脱膜剂,采用漂浮法进行脱膜。打捞板孔径为1 mm时,自支撑厚度范围为5~50 nm,自支撑成功率约为70%。利用拉曼光谱仪及原子力显微镜等仪器,测量了薄膜的结构、表面粗糙度等关键参数。     

激光加速实验超薄类金刚石碳靶的制备

采用过滤阴极真空弧法,制备了满足激光稳相加速机制要求的超薄自支撑类金刚石碳靶。室温下沉积,基片偏压为-32 V,薄膜沉积速率约为每脉冲0.002 nm。选取NaCl膜作脱膜剂,采用漂浮法进行脱膜。打捞板孔径为1 mm时,自支撑厚度范围为5~50 nm,自支撑成功率约为70%。利用拉曼光谱仪及原子力显微镜等仪器,测量了薄膜的结构、表面粗糙度等关键参数。