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.     

原子力显微镜磁驱动轻敲模式在活细胞成像中的应用研究

应用MI公司最新发展的磁驱动轻敲模式(MAC mode)时体外培养成纤维细胞系3T3细胞进行在位成像研究.分别用力常数为0.95 N/m及0.03 N/m的微悬臂进行磁驱动轻敲模式成像,并与接触模式进行比较.同时研究了固定细胞与活体细胞之间的形貌差异.结果显示,利用上述两种微悬臂探针,磁驱动轻敲模式均可获得高分辨像.与接触模式相比,磁驱动轻敲模式对活细胞的影响较小,在细胞膜表面微结构及细胞内亚结构成像方面,有明显优势.而接触模式由于其施力方式,使活细胞应力纤维应激性绷紧,更适合于对活体细胞应力纤维的成像研究.固定细胞与活细胞表面形貌存在较大差异,在生理环境下,进行活细胞检测更能了解细胞的真实状况.     

表面性质对扫描力显微术成像的影响

利用微接触印刷的方法制备了图形化的自组装膜,考察了膜的表面性质对接触式原子力显微镜（ＡＦＭ）,摩擦力显微镜（ＦＦＭ）和剪切力显微镜（ＳＦＭ）的成像的影响。发现ＡＦＭ能反映样品的表面形貌,而ＦＦＭ和ＳＦＭ的以表面性质的影响较大,在表面性质差别很大的图形表面上会出现图像衬底与表面形貌反转的现象。     

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.     

海马神经元的原子力显微成像

原子力显微镜(AFM)对完整的细胞成像并同时进行微细结构观察尚有一定困难。本实验改进了标本制备的过程．用原子力显微镜对戊二醛固定的海马神经元进行扫描，建立了方便、实用的完整海马神经元自完整胞体至超微结构的原子力显微镜成像技术，并用改进的方法获得了完整海马神经元及其超微结构的清晰的三维成像，并发现了一些其它显微技术所不能发现的微细结构。这些结构包括：①海马神经元胞体的亚细胞部分及这些亚细胞部分所具有的不同功能；②神经突触的完整形态；③损伤细胞膜表面出现的孔洞；④通过神经突触所形成的神经网络结构。     

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。     

原子力显微镜对生理溶液中活细胞成像条件的研究

本文研究用原子力显微技术(AFM)在生理条件下对活细胞成像的基本方法,并对各种影响成像因素如针尖与细胞表面的非特异性相互作用、AFM悬臂弹性系数及细胞表面柔性等问题提供相应的解决方案。从而为AFM在成像的基础上对活细胞其它性质的研究提供基础。用本文方法清晰地显示了固定细胞与活细胞膜表面所具有的明显差别：活细胞膜完整平滑,固定细胞表面粗糙,边缘不整。     

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。     

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.     

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.     

Observation on Surface and Cross Section of Thin Film Solar Cells Using Atomic Force Microscope

Atomic force microscope (AFM) is able to produce thrce-dimensional digital data in both force-mode and heightmode and its applications are not limited to map the surfaces of conducting materials. It can use the force-mode to image the repulsive and attractive force patterns. The cross sections of polycrystalline CdS/CdTe and amorphous silicon heterojunction solar cells are observed with AFM. In case of short circuit, the microstructures of different layers in the samples are clearly displayed. When the cells are open circuit, the topographical images are altered, the potential outline due to the space charge in junction region is observed. Obviously, AFM can be employed to investigate experimentally built-in potential in junction of semiconductor devices, such as solar cells.     

宫颈上皮脱落细胞的微尺度力学性质及形貌学研究

目前临床中对宫颈脱落细胞的检查多局限于大体细胞形态学的观察.本文应用原子力显微镜及环境扫描电子显微镜对5例临床宫颈炎患者的宫颈脱落细胞进行了微区力学性质表征以及细胞表面微观形态的成像.结果显示,患者正常形态宫颈上皮脱落细胞在针尖压入深度为700 nm时杨氏模量近似正态分布,峰值在20～30kPa.且细胞表面微嵴明显,微...     

Studies of the micro- and nanostructure of polycrystalline CdTe and CuInSe2 using atomic force and scanning tunneling microscopy

Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) have been used to study the micro- and nanostructure of CdTe and CuInSe₂ thin films used for photovoltaic cells. Topographic images are comparable with those reported previously using conventional scanning electron microscopy (SEM)—to the limit of spatial resolution of the SEM technique. For higher magnifications, nanoscale structures and features have been observed for the first time with AFM and STM, and these observations have implications for the suitability and preparation of these semiconductors for high-efficiency solar cell realization.     

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。     

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...     

原子力显微镜观察虫草多糖分子的结构形貌

本文通过原子力显微镜直接观察虫草多糖分子的三维结构的形貌。虫草多糖分子的稀溶液铺展在Ｎｉ＾２＋处理的云母片上,经干燥,乙醇固定后,获得稳定,重复的图像。结果表明虫草多糖分子具有高度分枝的结构,并且糖链间形成小环或螺旋结构     

原子力显微镜技术在细胞研究中的进展

原子力显微镜(AFM)是生物显微技术的一个重要组成部分,可实现液体环境下活细胞高分辨率的成像与操作,为细胞生物学研究提供了新的方法。近年来,AFM已经逐渐发展成为集样品成像、力测量及操作等功能于一体的多功能生物细胞研究平台,在细胞研究中得到了广泛的应用。在简要介绍AFM组成与工作原理的基础上,详细阐述了近年来基于AFM成像与力谱技术的细胞研究的发展状况。针对AFM本身所存在的不足,介绍了AFM与其他技术相结合的研究成果,并对AFM在生物细胞研究中未来的发展方向进行了展望。     

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。     

Probing individual nanoscale organic light-emitting diodes with atomic force electroluminescence microscopy and bridge-enhanced nanoscale impedance microscopy

Two recently developed atomic force microscopy (AFM) techniques are used to characterize the impedance and charge transport/emission characteristics of individually addressed micro- and nano-scale organic light-emitting diodes (OLEDs). To fabricate independent diodes at this length scale, a suspended silicon nitride membrane shadow mask scheme is employed with semiconductor processing and electron beam lithography. This approach enables the fabrication of individually addressable OLEDs ranging in size from microns down to hundreds of nanometers. Atomic force electroluminescence microscopy (AFEM) and bridge enhanced nanoscale impedance microscopy (BE-NIM) are used to characterize these devices. AFEM offers real-time nanometer-scale spatial resolution mapping of simultaneously acquired current, topography, and light emission data while BE-NIM enables real-time impedance spectroscopy studies of functioning OLEDs. These two AFM techniques are shown to be capable of analyzing device-to-device response variations across a broad range of length scales and to provide unique quantification of intra-array device variations.     

Magnetic Force Microscopy of Iron and Nickel Nanowires Fabricated by the Matrix Synthesis Technique

Iron and nickel nanowires are grown by the matrix synthesis technique in the pores of the track membrane fabricated based on polyethylene terephthalate (PET). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to image the pores on the surface of the specimens and determine the 3d metal nanowires in a polymer bulk. The magnetization curves of the arrays of the nanowires are obtained. Magnetic properties of the nanowires are studied by the magnetic force microscopy (MFM) methods. The influence of the interposition and magnetostatic interaction of the nanowires of the magnetic metals in the polymer membrane, as well as the magnitudes and orientation of the applied external magnetic field, on the obtained MFM images is shown. The simulation results of the MFM images are in good agreement with the experimental data.