Expression and purification of the recombinant HBcAg core particles derived from methyltrophic Pichia pastoris, and TEM and AFM of the core particles and their natural aggregates

The recombinant hepatitis B virus core antigen (rHBcAg) core particles derived from Pichia pastoris were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicate that the purified rHBcAg particles with HBcAg antigenicity mainly locate at the densities of 1.2576 and 1.3013 g.mL(-1), respectively. After purification, a portion of purified sample of rHBcAg particles was immediately subjected to detection using transmission electron microscopy (TEM) and atomic force microscopy (AFM), the remainder were kept in -20 degrees C for 1 month or longer. After 30 days, the sample of rHBcAg particles previously frozen was imaged by TEM and AFM. The detection results indicate that the stored rHBcAg particles aggregated into a string of beads. The above results suggested that the rHBcAg particles expressed and self-assembled in P. pastoris, which were stored at -20 degrees C, can gradually and naturally aggregate with storage time.     

The application of the atomic force microscope to studies of medically important protozoan parasites

Both living and fixed specimens of the medically-important parasitic protozoa, Trypanosoma cruzi, Toxoplasma gondii, Giardia lamblia, Entamoeba histolytica, and Acanthamoeba spp. were studied by atomic force microscopy (AFM). The preparation of fixed specimens was similar to methods used for either scanning or transmission electron microscopy. AFM scanning was performed using both contact and tapping modes. A classical fixation procedure utilizing glutaraldehyde followed by ethanol dehydration was not suitable for all parasite species. AFM images could not be obtained from fixed samples of T. cruzi, T. gondii or E. histolytica. However, excellent topographic images could be obtained from specimens of G. lamblia and Acanthamoeba under identical conditions. Critical point drying permitted AFM imaging of both trypomastigote and epimastigote stages of T. cruzi. Phase imaging of T. cruzi elucidated unique surface details at a level of resolution not visible using any other imaging modalities. AFM elasticity map imaging of T. cruzi-infected and T. gondii-infected cells demonstrated that both parasites were markedly firmer than the surrounding host cell cytoplasm. The parasitophorous vacuole surrounding replicating T. gondii tachyzoites was also visualized by elasticity map imaging. These data suggest that although much remains to be learned about preparing parasitic protozoa for AFM imaging, the technique has the potential of providing unique and important insights into these disease causing organisms.     

Structure of rice starch granules in nanometre scale as revealed by atomic force microscopy

Atomic force microscopy (AFM) was performed for the analysis of the fine structure of rice starch granules in the nanometre scale which were prepared by a physical destruction method. The present study directly demonstrated that fine particles of approximately 30 nm in diameter were present inside each granule and occasionally formed straight chain arrangements. We considered that these fine particles correspond to the individual single cluster in the cluster model which has been proposed in previous studies on the starch granule structure.     

PC12细胞凋亡及其细胞膜表面超微结构的原子力显微镜形态学观察

本文采用原子力显微镜(AFM)对PC12细胞的凋亡及其膜表面超微结构进行了实验研究，研究表明AFM可直接观察PC12细胞的生长情况，凋亡细胞膜发生皱缩、凹陷，并形成微绒毛消失的凋亡小体等现象。AFM有望发展成为一种研究细胞凋亡的工具。     

Preparation of ultrasmooth and defect-free 4H-SiC(0001) surfaces by elastic emission machining

In this work, elastic emission machining (EEM), which is a precise surface-preparation technique using chemical reactions between the surfaces of work and fine powder particles, is applied to the flattening 4H-SiC (0001) surface. Prepared surfaces are observed and characterized by optical interferometry, atomic force microscopy (AFM), and low-energy electron diffraction (LEED). The obtained images show that the processed surface has atomic-level flatness, and the subsurface damage and surface scratches of the preprocessed surface are almost entirely removed.     

Structural analysis of red blood cell membrane with an atomic force microscope

To evaluate the usefulness of the atomic force microscope (AFM) for structural analysis of biomedical samples and to determine suitable sample preparation methods for AFM observation, the membrane of human erythrocytes prepared by various methods for electron microscopy was examined by the AFM. Strand-like elevations with 20-50 nm in width, 30-80 nm in length and 3-5 nm in height were observed, which formed networks composed of squares, pentagons and hexagons on the cytoplasmic or back surface of the erythrocyte membrane. Using colloidal gold labelled antibody, this network was found to contain spectrin molecules. Therefore it was very likely that the undercoat molecules of the plasma membrane were imaged by AFM. A large number of gentle elevations 300-400 nm in diameter and 2 nm in height were found to be distributed uniformly on the extracellular or true surface of intact erythrocyte, presumably reflecting the presence of undercoat membrane skeleton on the cytoplasmic surface. However, no structure that seemed to be derived from glycocalyces was discernible on the true surface. Structure corresponding to the unit membrane or lipid bilayer structure observable by electron microscopy was not demonstrated in the cross-section of the membrane. In freeze-fractured samples, a large number of small particles that corresponded to the intramembranous particle were also demonstrated on the membrane halves. Since AFM allows depiction of the fine structures of biological samples with very simple sample processing at a resolution comparable to or exceeding that of SEM, imaging technology using AFM can be applied to obtain biomedical information. However, several problems have to be solved in future development of the equipment.     

粉末样品的原子力显微镜研究方法

本文提出了用纯净水对粉末样品充分湿润以后,把粉末样品置于空气中,待其自然蒸发到样品表面只有分子结合水以后再进行AFM扫描成像的粉末样品原子力显微镜研究方法。通过分析实验过程发现,原始粉末样品颗粒表面大量的过剩电荷被分子结合水层中和,解决了扫描过程中颗粒易于粘到针尖上的问题;颗粒间存在的分子结合水与毛细管水,使颗粒之间结合牢固,解决了扫描过程不稳定的问题。实验结果表明,该法获得的粉末样品形貌图清晰真实,且具有简单易行,适用范围广的特点。另外,该方法提出了一种针尖在大气中振动．而被扫描样品在液体中的复合扫描环境,具有进一步深入研究和推广的价值。     

Au/SiO_2纳米复合薄膜的结构表征及光致发光特性

采用磁控溅射和退火技术制备出Au/SiO2纳米复合薄膜。利用扫描电子显微镜(SEM),X射线衍射(XRD)和原子力显微镜(AFM)对上述纳米复合薄膜进行了结构表征。实验结果表明,纳米复合薄膜的表面上均匀分布着直径在100～300nm的金纳米颗粒。金纳米颗粒的大小随着退火时间的增加而增大。用荧光光谱仪(PL)对薄膜的光致发光特性进行了研究。结果表明,在激发波长为325nm时,分别在525nm和560nm处出现两个发光峰;在激发波长为250nm时,在325nm处出现发光峰,这一发光峰可能与非晶SiO2的结构缺陷有关。     

On-substrate lysis treatment combined with scanning probe microscopy revealed chromosome structures in eukaryotes and prokaryotes

The proper function of the genome largely depends on the higher-order architecture of the chromosome. To understand the detailed chromosome structure in a native state, we developed an on-substrate procedure of subcellular fractionation suitable for the observation by atomic force microscopy (AFM). HeLa cells on a coverslip were successively treated with a detergent and a high-salt solution to remove the cytoplasmic and nucleoplasmic materials. A closer observation of the nucleus by AFM revealed that the interphase chromosome is composed of a granular unit of approximately 80 nm in diameter. Subsequent mild treatment with deoxyribonuclease I (10 U ml(-1)) exposed these units more clearly, which enabled us to uncover the 80-nm granules forming a fibre of approximately 80 nm width. In the cytoplasmic regions, cytoskeletal fibres with varying widths (10-70 nm) were observed. These observations suggest that the 80 nm granular fibre is a fundamental structural unit of the interphase chromosome. This on-substrate procedure was also applied to Escherichia coli. Cells attached on a coverslip were successively treated with lysozyme and detergent to partially release the nucleoid onto the substrate. The AFM observation revealed that the approximately 80 nm fundamental structural unit forms a granular fibre similar to that of HeLa cells. These results suggest that the fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.     

Investigation of the degradation of smooth SiGe epitaxial layer on Si substrate

The degradation of smooth SiGe epitaxial layer was investigated by transmission electron microscopy (TEM), X-ray reflectivity (XRR) and atomic force microscopy (AFM). It was shown from AFM results that the crosshatch was formed with increasing annealing temperature, which indicated the degradation of smooth surface. The surface degradation was caused by the internal dislocations, which were observed by plan-view TEM (PTEM) and cross-sectional TEM (XTEM). From XTEM, the sharp interface between SiGe top layer and Si substrate was broadened and there were a lot of 60° dislocations formed in SiGe top layer, which resulted in the crosshatch on the surface. The crosshatch was also verified by PTEM.     

Co/BaTiO3纳米复合薄膜的PLD法制备及其Raman光谱

采用脉冲激光气相沉积（PLD）技术在MgO（100）衬底上生长了Co/BaTiO₃纳米复合薄膜,利用原子力显微镜、透射电镜、X射线衍射（XRD）及Raman光谱等测试分析手段对Co/BaTiO₃纳米复合薄膜进行测试分析。结果表明：薄膜表面均匀、致密,具有原子尺度的光滑性;Co纳米晶粒呈单分散、均匀分散在沿C轴呈单取向生长的BaTiO3单晶基体中;随着掺杂Co含量的增高,BaTiO3的Raman峰的峰强随之增大。     

Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit

Genome function is closely linked to the higher-order chromatin structures. To reveal a structural basis for the interphase chromatin organization, the 'on-substrate' lysis procedure was applied to nuclei isolated from human HeLa cells, chicken erythrocyte cells and yeast Schizosaccharomyces pombe, which possessed different intrinsic properties of the genomes such as histone composition and inter-nucleosomal distance. The isolated nuclei on a coverslip were successively treated with a detergent and a high-salt solution to extract the nuclear membrane and the nucleoplasm, and therefore, atomic force microscopy (AFM) visualized the structural changes in response to the lysis procedure. After the nucleoplasm was extracted, AFM clarified that chromatin fibers, approximately 40 nm in width, were partially released out of the nuclei and that the other chromatin still remaining in the nuclei was composed of granular structures with diameter of 80-100 nm. Thus, these results suggest that the approximately 40 nm fiber would be a stable structural unit and fold the 80-100 nm granules into a one-step higher unit. A common mechanism could be implied regardless of the intrinsic properties of the eukaryotic genomes.     

Quantitative measurement of channel temperature of GaAs devices for reliable life-time prediction

The channel temperature of Gallium Arsenide (GaAs) devices was quantitatively measured using scanning thermal microscopy (SThM), which is a variation of atomic force microscopy (AFM). The temperature of the devices was also characterized by infrared (IR) imaging and thermal modeling. The measured SThM temperature values were close to the calculated values from the model, and were higher than those found by IR, as predicted. In contrast to most published AFM results which have reported only qualitative and indirect semi-quantitative thermal information about the sample, the results presented here can be used directly to determine accurately the device-temperature. These results are useful to the reliability community in that they help to predict a more accurate semiconductor device lifetime. By careful calibration of an AFM thermistor probe tip, a quantitative temperature measurement of the channel temperature of the GaAs PHEMTs and MESFETs can be made. The result of the measurement can be substantiated by applying a suitable thermal calculation, such as the Cooke model. A secondary measurement technique, such as IR microscopy, can also be useful in providing further information about the thermal response of the device. Published results using AFM techniques have been unable to determine the channel temperature quantitatively. The method in this paper applies to other types of electronic devices for which the channel (or junction) temperature can be probed from the top surface of the device.     

Investigation of the Surface Removal Process of Silicon Carbide in Elastic Emission Machining

Elastic emission machining (EEM) is a precise surface preparation technique, which uses chemical reactions between the surfaces of the workpiece and fine powder particles. The purpose of this study is to clarify the surface removal process of silicon carbide (SiC) in EEM. A SiC sample with a periodic step-bunched structure was prepared as the initial surface and was flattened by EEM. Optical interferometer and atomic force microscopy (AFM) observations show that the topmost areas on the periodic step-bunched structure in contact with the powder particles are preferentially removed and surface protrusion is gradually reduced as removal depth increases. Moreover, power spectral density analyses reveal that the surface is smoothed in the spatial wavelength range from 0.07 μm to 10 μm.     

多层铜布线表面CMP后颗粒去除研究

针对目前清洗技术存在的问题进行了详细分析,研究了微电子材料表面污染物的来源及其危害,并介绍了表面活性剂在颗粒去除方面的作用。研究了化学机械抛光(CMP)后Cu布线片表面的颗粒吸附状态,分析了铜片表面颗粒的吸附机理。采用非离子表面活性剂润湿擦洗方法,使Cu表面的颗粒处于易清洗的物理吸附状态。利用金相显微镜和原子力显微镜(AFM)在清洗前后进行对比分析,实验采用聚乙烯醇(PVA)刷子分别对铜片和铜布线片进行清洗,发现非离子界面活性剂能够有效去除化学机械抛光后表面吸附的杂质,达到了较好的清洗效果。     

Chemical-Mechanical polishing of parylene- n films: evaluation by X-Ray photoelectron spectroscopy and atomic force microscopy

The surface quality of parylene-N(PA-N) films, as determined by x-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), after chemical-mechanical polishing (CMP), is influenced mostly by two factors: quality of the as-deposited film and the slurry composition. The higher the quality of the as-deposited film (more specifically, less oxygen content), the higher the quality of the polished film. The XPS and AFM results show that PA-N film polished in 1% A1₂O₃ abrasive (0.3 Μm particles), NH₄OH (2% by volume), and water, has better quality compared to the other slurries investigated. With high quality PA-N films, the film surface quality affected by CMP is relatively independent of polishing time, indicating that changes in surface chemistry occur in the initial seconds of polishing.     

原子力显微镜在生殖支原体研究中的应用

为了确定生殖支原体在活性条件下的结构形貌,并在较高的分辨水平上观察其三维形貌结构,本研究采用原子力显微在常温常压下对生殖支原体标准株及分离株进行形态学的初步观察,将标本固定于云母上,在ｔａｐｐｉｎｇ模式下扫描成像,结果显示：生殖支原体多呈烧瓶状或鸭梨状,有突出的颈产及膨大的头端,与电镜观察结果类似,其大小亦与银镜测量结果类似。     

六方氮化硼薄膜制备及其压电响应的研究

采用射频(RF)磁控溅射的方法,通过改变工艺参数在n型Si(100)片上制备六方氮化硼(h-BN)薄膜。通过傅立叶红外(FTIR)光谱仪,X射线衍射(XRD)仪进行结构表征,原子力显微镜(AFM)进行表面形貌和压电性能表征。测试结果表明,在射频功率为300 W、衬底温度为500℃、工作压强在0.8Pa、N2与Ar流量比为4∶20和衬底偏压在-200V时制备的六方BN薄膜具有高纯度、高c-轴择优取向,颗粒均匀致密,粗糙度为2.26nm,具有压电性并且压电响应均匀,符合高频声表面波器件基片高声速、优压电性要求。薄膜压电性测试研究表明,AFM的PFM测试方法适用于纳米结构半导体薄膜的压电性及其压电响应分布特性的表征。     

硅衬底上CdS纳米晶和纳米碳管极化特性的动态电场力显微术观察

在现有的商用原子力显微镜上实现了用动态电场力显微术来研究单个纳米颗粒的极化特性。将AOT（bis(2-ethylhexyl)sulfosuccinate disodium)分子包覆的CdS纳米晶和Au纳米晶共同沉积在n型硅片表面,以分析导电探针对其诱导极化,同时研究了纳米碳管和碳纳米颗粒的不同极化特性,对样品的原位观察表明：其半导体和金属介电特性的差别,CdS,Au粒子呈现较大的极化反差,在同一纳米碳管不同位置也能观察到类似反差。通过比较在半导体和金属粒子上探针对外加交变电场的响应幅度,可以估计纳米半导体纳米粒子的介电常数。     

人舌鳞癌组织超薄切片的AFM成像和切割

利用一种基于电镜超薄切片法改进的制样方法，将人舌鳞状细胞癌病理组织以环氧树脂包埋并切片后，将薄片平整地贴附在云母上，用原子力显微镜(AFM)对切片表面进行研究，可以得到高分辨率的细胞超微结构图像，局部的亚细胞水平的形态结构可以与电镜下得到的图像相比拟。在此基础上，利用AFM针尖对肿瘤细胞核内特定区域进行切割和操纵，形成生物分子的堆积，从而为拾取(pjck—up)和进一步用分子生物学手段在亚细胞基因水平研究人舌鳞癌的病理学奠定了基础。