小鼠胚胎干细胞超薄切片的AFM观察

对Balb／c小鼠胚胎干(ES)细胞的超薄切片进行AFM成像，以获得ES细胞内部结构的信息。尝试把AFM作为ES细胞形态学鉴定的一种手段。方法：利用超薄切片技术，将ES细胞样品制成厚约60nm的切片，固定在盖玻片上，用AFM在大气中观察。结果：观察到不同时期的单个ES细胞以及分裂时期ES细胞核中高度盘曲和浓聚的染色体。利用AFM的高分辨成像能力，观察了线粒体亚显微结构，细胞基质和核质的超微结构。     

电镜样品的多糖特异性染色－PA－TCH－SP反应

电镜样品的多糖特异性染色－ＰＡ－ＴＣＨ－ＳＰ反应罗玉英（首都师范大学生物系，北京１０００３７）在光镜水平对组织和细胞进行多糖定位的细胞化学技术目前已经非常成熟。特别是近年来利用塑料包埋的半薄切片进行染色（ＰＡＳ反应）已获得非常清晰的图像￣［１］。但在...     

基于AFM检测信息存储介质表面结构的研究

介绍了原子力显微镜(AFM)的工作原理、组成、特点及其应用领域。利用AFM对光盘和软盘表面结构进行了三维检测,使用CSPM2000Imager软件对扫描得到的图像进行了计算和分析,由所得数据可以看出,DVD光盘将取代CD光盘成为外存的主流,而软盘将被淘汰;利用原子力显微镜对单晶硅进行基于金针的纳米加工,刻蚀出“河南理工大学”的字样。     

一种新的双重标记免疫组化图像自动分割方法

本文描述一种自动分割双重标记免疫组化图像的方法,此方法直接从双重标记的两种阳性组织细胞着色特征对其进行分离。与常用的彩色反卷积方法相比,此方法提高了分割准确度且需要样品切片的数量从3张减少到1张。通过细胞角蛋白和S-100蛋白双重标记,分别经二氨基联苯胺(DAB)与3-氨基-9-乙基咔唑(AEC)显色的舌鳞癌样品图像的实验证明,此方法可以有效地将鳞癌与神经组织细胞两种不同的染色区域分割出来,与手动分割的结果作比较其准确度和精确度都有较大的提高。此方法也适用于分割其他用DAB和AEC双重染色的免疫组化图像。     

基于亚取样分形插值预测的混合图像编码方法

提出了一种基于亚取样分形插值预测的混合图像编码方法。将原始图像在水平方向和垂直方向皆作１／２抽取得到一幅“亚抽图像”，对亚抽图像进行分形编码得到亚抽图像的分形码，采用分形插值方法由亚抽图像的分形码解码得到原始图像的分形预测图像，然后对预测误差图像进行基于ＤＣＴ的自适应补偿编码。实验表明，这种方法的编码速度有了很大提高，而且恢复图像的质量具有较高的保真度。     

原子力显微镜在细胞力学特性研究中的进展

原子力显微镜(AFM)能够以高分辨率和高灵敏度实现液体环境下活细胞的操作和检测,利用AFM力曲线测量功能还可以获得细胞力学特性的相关信息。在简要介绍AFM力曲线测量原理的基础上,从细胞的杨氏模量、硬度和配体-受体之间相互作用力三个方面,详细阐述了近年来AFM在细胞力学特性研究中的发展状况,并着重介绍了其在细胞杨氏模量研究中的进展,表明AFM已经成为细胞力学特性研究中重要的生物显微技术。最后,针对AFM的不足及局限性,提出了目前存在的问题,并对AFM在细胞力学特性研究中的前景进行了展望。     

牛膀胱上皮细胞形态结构及AUM蛋白的定位

本文以牛膀胱变移上皮为材料,利用石蜡切片、免疫荧光及多种电镜技术,对其形态结构进行了较为系统的研究,并对上皮细胞膜蛋白 Uroplakin I 的定位做了初步的探讨。在显微与亚显微水平上,牛膀胱变移上皮细胞大致可分为三类,即富含膜泡的表层细胞,富含线粒体的中间层细胞和其下的基底层细胞。在超薄切片样品中其腔面细胞质膜由扇形凹陷区与冠状突起区相间排列而成,细胞质膜的暗一亮一暗单位膜结构可以清晰地分辨出来,靠腔面厚约80A,靠细胞质面厚约40A,表现出明显的不对称性(图1),因而称之为不对称单位膜。冰冻断裂复型样品中在细胞质膜纵断面上,表层细胞腔面膜的形状与超薄切片中类似,亦由扇形凹陷区与冠状突起区相间排列而成。扇形凹陷区即为斑区,分布着许多的颗粒状结构,颗粒大小约11—13nm(图1左     

用ESI技术检测病毒中的磷元素

ESI(electron spectrum imaging)技术的创立和展[1][2],使在亚显微水平上对生物体中的特定元素进行定性、定量、和定位检测成为可能。P是构成生命物质的重要元素,我们取有囊膜的Sindbis病毒,初步提纯[3](仍带有细胞碎片)滴在微筛碳膜上,1％醋酸双氧铀负染,使用有计算机图像处理系统的电镜,在P元素L2,3离化能(132eV)前后对样品作ESI成像。用能量损失为E=139eV的图像减去E=117eV的图像得到样品的净磷分布[4]。以病毒核心(core)富含P元素(含于核酸中)为判据,鉴定图1(a)中箭头所指为病毒颗粒。本实验应用乔宝义建立的超薄微筛碳膜技术和ESI技术相结合,用于检测生物样品中的元素分布,具有普遍的应用价值。     

石蜡切片的扫描电镜观察方法

电子显微镜使人们能够用眼睛直观的看到从亚细胞结构的细节到微小的病毒,甚至原病毒颗粒,从核酸蛋白质的生物大分子到小分子甚至原子的形态结构。但美中不足的是,由于微小样品在高放大时的视野局限性以及透射电镜对样品厚度的超薄要求,使得电镜(透射)照片所形成的微小视野平面图像给人们直观了解生物亚显微结构的三维联系和形态与功能的相关分析带来了困难。为补尝这个不足,我们对石蜡切片的扫描电镜观察方法进行了新的探讨。     

高分辨率扫描类电镜图像处理和分析测量系统

我们研制成功一套高分辨率的扫描类电镜用的图像处理系统,可直接连接到任一台扫描电镜(SEM)、电子探针(EPMA)、透射电镜(TEM)的扫描附件和扫描隧道显微镜(STM)等扫描类电镜(简称电镜)上采集电镜图像信号和样品成份的X射线面分布像的信号存入计算机。经系统的硬件软件作图像处理后,图像的清晰度,信噪比可得到提高(如图1所示)。本系统的图像分辨率为1024×1024像素点阵,具有256个灰度级的高质量电镜图像,达到国外高档电镜     

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.     

A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells

Atomic force microscopy (AFM) has been used to image a wide variety of cells and has proven to be successful in cellular imaging, by comparing results obtained by AFM with SEM or TEM. The aim of the present study was to investigate further the conditions for AFM imaging of living cells and compare the results with those obtained by SEM. We chose to image skin fibroblast and liver sinusoidal endothelial cells of two different sources, because these cells have been well described and characterized in earlier studies. AFM imaging of living cells mainly reveals submembranous structures, which could not be observed by SEM. This concerns the visualization of the overall cytoskeletal architecture and organelles, without the necessity of any preparative steps. The AFM study of living cells allows a time lapse study of dynamic changes of the actin cytoskeleton under the influence of the cytoskeleton-disturbing drug cytochalasin B in cells that can be followed individually during the process. However, softer samples, such as the fenestrated parts of living rat liver sinusoidal endothelial cells in culture could not be visualized. Apparently, these cell parts are disrupted due to tip-sample interaction in contact mode. To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was applied, resulting in images of higher quality. Still, endothelial fenestrae could not be visualized. In contrast, contact imaging of immortomouse liver sinusoidal endothelial cells, which are devoid of fenestrae, could easily be performed and revealed a detailed filamentous cytoskeleton.     

人外周血淋巴细胞体外刺激活化的原子力显微镜研究

本文利用原子力显微镜,对分离的人外周血淋巴细胞和经多克隆刺激剂佛波醇酯(PDB)和离子霉素(Ion)刺激活化的淋巴细胞形态和膜表面超微结构的形态学变化进行了研究。结果表明:淋巴细胞在受刺激后形貌发生明显变化,尺寸变大,同时细胞表面超微结构也趋复杂,出现免疫突触。这些形态变化是与淋巴细胞的功能相适应的,AFM是研究淋巴细胞形态学变化和细胞结构功能之间关系的有力工具。     

Replication of cancer cells using soft lithography bioimprint technique

Replication of biological cells for the purpose of imaging and analysis under electron and scanning probe microscopy has facilitated the opportunity to study and examine some molecular processes of living cells in a manner that was not possible before. The difficulties faced in direct cellular analysis when using and operating atomic force microscopy (AFM) in situ for morphological studies of biological cells has lead to the development of a novel method for biological cell studies based on nanoimprint lithography. The realisation of the full potential of high-resolution AFM imaging has revealed some very important biological events such as exocytosis and endocytosis. In this work, a soft lithography bioimprint replication technique, which involved simple fabrication steps, was used to form a hard replica of the cell employing a newly developed biocompatible polymer that has fast curing time at room temperature essential for this process. The structure and topography of the endometrial (Ishikawa) cancer cell was investigated in this study. Cells were cultured and incubated in accordance with standard biological culturing procedures and protocols approved by the Human Ethics Committee, University of Otago. An impression of the cell profile was created by applying a layer of the polymer onto the cells attached to a substrate and rapidly cured under UV-light. Fast UV radiation helps to lock cellular processes within minutes after exposure and replicas of the cancer cells exhibit ultra-cellular structures and features down to nanometer scale. Elimination of the AFM tip damping effects due to probing of the soft biological tissue allows imaging with unprecedented resolution. High-resolution AFM imagery provides the opportunity to examine the structure and topography of the cells closely so that any abnormalities can be identified. Craters that resemble granules may be observed. These represent steps on a transitional series of sequential structures that indicate either an endocytotic or exocytotic processes, which were evident on the replicas. These events, together with exocytosis, play a very significant part in the tumorigenesis of these cancer cells. By forming cell replica impressions, not only have they the potential to understand biological cell conditions, but may also benefit in synthesizing three dimensional (3-D) scaffolds for natural growth of biological cells and provide an improvement over standard cell growth conditions.     

子宫内膜异位症细胞微尺度刚度的初步实验研究

目的:基于原子力显微技术,建立子宫内膜异位症细胞力学性质检测方法,比较子宫内膜异位症在位与异位子宫内膜细胞的微尺度刚度,以进一步研究子宫内膜异位症发病机制.方法:采集子宫内膜异位症患者子宫内膜及其异位到卵巢的巧克力囊肿囊皮,并分别进行原代培养,利用原子力显微镜采集细胞力曲线,获取杨氏模量信息,进行统计分析.结果:在位腺...     

Detection of changes in cell membrane structures using the Bioimprint technique

Recent advances in medical sciences have revealed the significance of cellular structures and morphology in biological function. A cell’s membrane represents the boundary between the cells and its environment. The variations in cell surface and morphologies may be used as indications of malfunction or even diseases. If abnormalities such as cancer can be detected at the molecular level this will offer an important means for early diagnosis using small numbers of cells. The use of electron and scanning probe microscopy such as atomic force microscopy (AFM) could facilitates the opportunity to study and examine the molecular processes of living cells in greater details. The difficulties faced in direct cellular analysis when using and operating the AFM in situ for morphological studies of the cells has led to the development of a novel approach called Bioimprint (Traut and Papanicolaou, 1941 [1]). Inspired by the high resolution of nanoimprint lithography processes, Bioimprint has been applied to a new area of biological cell replication for the purpose of imaging and analysis and has revealed some very important biological events when combined with AFM imaging. For this research, the structural features of endometrial cancer cells were investigated. Regulation of selected peptides were examined, especially those which are associated with angiogenic factors that promote the proliferation of nutrient-bearing blood vessels that support tumour growth. Using the Bioimprint technique which is a soft lithography process, an impression of the cell topology was created by applying a layer of monomer mixture onto the cells attached to a substrate and rapidly curing it under UV-light. Fast UV-radiation enables the imprint to lock cellular processes within minutes and replicas of the cancer cells exhibit structures down to nanometer scale. Cancer cells were cultured and incubated in accordance with standard biological culturing procedures and protocols approved by the New Zealand Human Ethics Committee. High-resolution AFM imagery provides the opportunity to examine the structure and topography of the cells closely so that any abnormalities can be identified. To study the replicated imprints, the features that resembled secretory pores on the AFM images were noted. The numbers of pores correlated well with levels of vascular endothelial growth factor (VEGF) that were secreted by the cells. Further experiments were conducted in which anti-VEGF-coated microbeads were observed in the AFM images to be attracted to the areas of the pores.     

Atomic force microscopy analysis of bacteriophages phiKZ and T4.

Bacteriophage phiKZ was investigated by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The well-known phage T4 was used as a reference sample. Reproducible contact mode AFM images of native and partially disintegrated particles of bacteriophage phiKZ in air were obtained. It was demonstrated that the heads of phiKZ and T4 phages were compressed differently and depended on adsorption onto highly ordered pyrolytic graphite and mica. We have established a procedure to partially disintegrate the viral particles after which the internal protein body, which is a helical structure with a cylinder-like form, was easily observable inside the bacteriophage phiKZ head.     

活体细胞骨架的原子力显微成像

为研究原子力显微术在生物医学中的应用,实现对活体细胞骨架纳米尺度的实时观察,采用轻敲模式和接触模式对体外培养的大鼠脑微血管内皮细胞进行成像。结果显示不同模式在分辨细胞超微结构及质膜下细胞骨架纤维束等方面各有特点,可从不同角度获取细胞骨架的信息。     

Investigation of the surface roughness of CdZnTe substrates by different techniques of nanometer accuracy

The results of measurements of the root-mean-square (rms) surface roughness of CdZnTe substrates by confocal microscopy (CM), atomic force microscopy (AFM), and X-ray reflectometry (XRR) are compared. It is determined that CM yields the highest rms roughness values, AFM assumes an intermediate position, and XRR measurements produce results that are an order of magnitude lower than those obtained with the use of the other two techniques. It is demonstrated that CM rms roughness values depend to a considerable extent on the type of the microscope objective used in experiments. Probable reasons for the discrepancy between the obtained results are discussed.