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.     

病毒性肝炎窦内皮细胞损伤的超微结构观察

应用电镜技术对光学显微镜下不易观察清楚的肝窦内皮细胞的形态作进一步的分析研究。重点观察了窦内皮细胞受损时的超微结构特征。对窦内皮细胞的形态、位置变异、及枯否氏细胞与窦内皮细胞受损的密切关系,做了较详尽的观察。窦内皮细胞的损伤可直接影响到肝细胞的营养及物质交换,肝窦毛细血管化倾向是一个值得进一步深入研究的课题。     

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.     

Three-dimensional structural changes in living hippocampal neurons imaged using magnetic AC mode atomic force microscopy

We developed the magnetic AC (MAC) mode atomic force microscopy (AFM) to image the 3D ultrastructure of living hippocampal neurons under physiological conditions. Initially, the soma, the dendrites and the growth cones of hippocampal neurons were imaged. The imaging force was adjusted to a small value for the long-term observation. The neural spines were damaged when the tip produced a large force; the spines regenerated after the force was reduced. Subsequently, we explored the relationship between structural changes in hippocampal neurons and Alzheimer's disease by employing the new imaging technique. Time-lapse image acquisition (10 min intervals) showed that the growth cone collapsed after the addition amyloid peptide fragment beta(25-35), which is thought to initiate Alzheimer's disease. In addition, we found substantial changes in mechanical properties and in the volume of individual growth cone. This study suggested that MAC mode AFM may be a powerful tool for observing long-term structural changes in living neural cells under physiological conditions.     

Morphological and histochemical analyses of living mouse livers by new 'cryobiopsy' technique

A new 'cryobiopsy' (CB) technique has been invented for freezing the functioning livers of living mice in vivo without stopping their blood circulation. Livers of anesthetized mice were pinched off with pre-cooled CB forceps and immediately plunged into isopentane-propane cryogen. They were routinely freeze-substituted in acetone containing paraformaldehyde for light microscopy (LM) or osmium tetroxide for scanning electron microscopy (SEM). By freeze-fracturing some of them with a scalpel in liquid nitrogen before the freeze-substitution, well-preserved tissue areas were exposed only for SEM. They were either embedded in paraffin wax for LM or infiltrated with t-butyl alcohol followed by freeze-drying for SEM. Serial paraffin sections were stained with hematoxylin-eosin (HE) or histochemical periodic acid-Schiff (PAS) reaction. By HE-staining, the tissue surface areas were often compressed with the CB forceps and sinusoidal erythrocytes became aggregated side by side. In slightly deeper tissue areas, however, hepatic sinusoids were widely open with flowing erythrocytes. Lots of PAS-reaction products were well preserved in hepatocytes of the CB specimens. On the contrary, they were unevenly distributed in hepatocytes of conventionally quick-frozen specimens, and often lost in those of the conventionally dehydrated specimens. By SEM, some cell organelles, such as mitochondria and endoplasmic reticulum, and also dilated fenestrae of endothelial cells, open Disse's spaces and bile canaliculi appeared to be under normal blood circulation in the prepared CB samples. The new CB technique would be easy and useful for repeated examination of functioning organs of a living animal.     

Control of interaction force in constant-height contact mode atomic force microscopy

Contact mode is a versatile and widely used technique for imaging samples using the Atomic Force Microscope (AFM). When contact mode imaging is performed in constant-height mode, it enables linear and faster response but leads to uncontrolled tip-sample forces. Here, a control strategy based on magnetic actuation is proposed to achieve high-bandwidth control of the tip-sample forces in constant-height contact mode AFM. A magnetic particle attached to the AFM probe is actuated by an external solenoid and employed for force regulation. A quasi-static model has been proposed and employed to develop the control strategy. Likewise, the contact natural-frequency, which decides the limit of achievable speed, has been shown to be significantly higher relative to the free probe and to be relatively insensitive to the particle size. Subsequently, a setup is developed to validate the control strategy and demonstrate reduction of tip-sample force variation by over a factor of 12 compared to conventional constant-height mode operation. Likewise, in comparison with conventional contact mode AFM, an improvement of linearity by over a factor of 9 and improvement in response speed by a factor of 100 have been demonstrated while imaging hard samples. The system has been shown to image topography at speeds of 2.44 frames per second while regulating the interaction force. Finally, the stiffness of a sample has also been characterized using the developed system and simultaneous estimation of topography has also been demonstrated. They are shown to agree well with theoretical expectations.     

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。     

振动模式扫描极化力显微镜及其应用

振动模式扫描极化力显微镜采用一种新的扫描探针显微成像方式，它可以在极化力介导的非接触方式和轻敲方式之间自由切换。在极化力介导的非接触方式中，极化力叠加在范德华力上，克服了一般的原子力显微镜(AFM)非接触模式中因成像力程太短而不容易稳定的缺点；通过调节针尖的高度，从极化力介导的非接触方式进入到极化力介导的轻敲方式，又能部分消除AFM轻敲模式中毛细力的干扰，还可以用比AFM轻敲模式中最小稳定成像力更小的力进行成像。针尖的高度可以通过调节Asp(Amplitude setpoint)或插入扫描高度参数(lift scan height)来控制，这一方法简单易行。利用这一模式对肢体金颗粒和DNA的高度进行测量，在一定程度上证明了轻敲模式中针尖压力的确会造成柔软生物样品的变形。     

Quantitative analyses of topography and elasticity of living and fixed astrocytes

The topography and elasticity of living and fixed astrocytes cultured from the rat cerebra were studied quantitatively by atomic force microscopy (AFM). Ridge-like structures reflecting F-actin beneath the cell membrane were prominent in the contact-mode images of living astrocytes. Many of these ridges became unclear after fixation (2% glutaraldehyde). In addition, the ridge-like structures were invisible in the topography of living cells observed at zero-loading force in the force mapping mode, which is considered to show the real cell surface not pressed down by an AFM tip. The topography of fixed cells observed both in the contact mode and at zero-loading force in the force mapping mode was similar to that of living cells observed at zero-loading force in the force mapping mode, although some deformed areas were detected in the fixed cells. The elasticity map images of living astrocytes showed that the cell membrane above the nucleus was softer (2-3 kPa) than the surroundings, and that the cell membrane above F-actin was stiffer (10-20 kPa) than the surroundings. In the elasticity map images of fixed astrocytes, on the other hand, the elasticity of the cells was found to be relatively uniform (200-700 kPa) irrespective of the inner structures of cells. These results show that images observed by AFM should be carefully examined in consideration of the force introduced to specimens and the elasticity of specimens to find out the real surface topography.     

固液界面纳米气泡的研究

在经典热力学理论中，室温下水中纳米气泡被认为是不能稳定存在的。近年来随着对疏水表面研究的深入，越来越多的现象暗示固液界面存在纳米气泡，并引起疏水长程作用力。目前直接探测固液界面纳米气泡的最有力手段是AFM，但它只能观察纳米气泡的形貌，无法对其进行直接定性，很有必要提供纳米气泡存在和来源的更直接证据。在云母表面进行乙醇和水替换形成纳米气泡，从成像条件和脱气对纳米气泡的影响两方面进行系统研究。不同成像模式和成像条件下AFM观察到的差异在一定程度上证明了观察到的就是纳米气泡。脱气实验结果表明，经脱气后乙醇和水形成纳米气泡的数量和概率明显降低，表明乙醇和水中溶解的气体是纳米气泡的来源，并为固液界面存在纳米气泡提供了更直接的证据。     

Ghost cancellation algorithms for MRI images

Ghost cancellation algorithms for restoring magnetic resonance imaging (MRI) images in medical applications are described. The approach is to model the effect of the time delays and the asymmetry of the sinusoidal readout gradient for even and odd lines with two phase functions relating the actual object density to even and odd parts of the observed image. A priori information about the phase functions is exploited in order to estimate the true object density. Examples of applying this ghost cancellation approach to liver and heart images is presented.     

Preparation of defined structures on very thin foils for characterization of AFM probes

In atomic force microscopy (AFM), knowledge of the probe (tip) geometry is a critical factor for obtaining reproducible images. This is particularly important for measurements in the contact mode, in which a certain amount of wear of the probe always occurs affecting the image quality of small, flat and/or larger surface structures. In addition to probe geometry, the slope of the probe with respect to the sample is of importance. In this work, probe geometry is determined by the use of structured foils obtained using focused ion beam (FIB). In this manner, we demonstrate the possibility of determining the AFM probe geometry and the slope on the basis of differently-sized structures. An established algorithm was implemented for the reconstruction of the probes. The shape of FIB structured foils was determined separately by scanning electron microscopy (SEM).     

纳米操纵辅助的原子力显微镜原位定位观察

在原子力显微镜（atomic force microscope,AFM）原位定位观察中,时常会遇到因失去标记物而无法定位的情况。本文介绍了一种在表面标记物被覆盖后,运用原子力显微镜的操纵功能,将标记物上的覆盖物“扫”开,重新找到标记物并用于精确定位的方法。以对高序热解石墨（highly ordered pyrolytic graphite,HOPG）表面牛血清白蛋白（bovine serum albumin,BSA）吸附的原位观察为例,在BSA膜覆盖HOPG表面的原子台阶后．采用接触模式AFM扫描,将BSA“扫”开,露出HOPG原子台阶作为标记,对图像上的结构进行精确定位。通过调节设置点、扫描范围、扫描速率、扫描线数、偏置值等戍像参数及扫描时间,可以控制“清扫”的力度和范围。     

Structural characterisation of CdS layers deposited on porous p-type GaAs

The purpose of this paper is to investigate the initial stage of cadmium sulphide (CdS) layer deposited on porous p-type GaAs substrate by vacuum evaporation technique. The deposited CdS layer was investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD). SEM imaging shows that the CdS was penetrated deeply in the porous structure down to the bottom and reaching the interface GaAs/porous GaAs. The AFM image demonstrates that the CdS deposited are grains of several nanometres and XRD patterns exhibit that the deposited layer has a hexagonal prominent phase.     

POSS聚合物微观结构的原子力显微镜研究

采用轻敲及接触模式原子力显微镜(AFM)对不同POSS含量下的甲基丙烯酸丁酯-甲基丙烯酸甲酯-苯乙烯基-POSS聚合物的表面微观形态及聚集相的分布进行了考察.实验结果表明:轻敲模式及接触模式的AFM均可应用于POSS聚合物的微观结构研究,接触模式下对样品表面有一定损伤;聚合物表面的聚集相与本体聚合物具有相异的微观物理性质,为POSS分子聚集在一起形成的无机相;随着POSS含量的增加,POSS聚集体数量增多、尺寸增大;添加POSS后聚合物表面粗糙度减小.     

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.     

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。     

碳纳米管原子力显微镜针尖的研究

本文研究了制作碳纳米管原子力显微镜针尖的方法和过程。在光学显微镜下，通过两个微工作台操纵将纯化后的多壁碳纳米管粘结在传统的原子力显微镜的Si针尖上。运用电蚀的方法优化碳管针尖的长度使其达到高分辨率的要求。我们运用制作的碳纳米管针尖在敲击模式下时G型免疫球蛋白进行扫描成像，结果显示了其典型的Y形结构，这是传统AFM的Si针尖无法获得的。     

扫描探针显微镜进行细胞扫描时探针对于细胞活性的影响

扫描探针显微镜(scanning probe microsope，SPM)是近几年发展很快的一种形貌表征仪器。它的一个突出优点是，可在溶液中以很高的分辨率对细胞活体进行观察。不同于光学显微镜，SPM是利用探针和样品之间的相互作用来扫描成像，探针对细胞有力的作用。这种作用力会在扫描过程中直接影响细胞的状态。为了研究SPM成像过程中探针作用力对于细胞的影响，我们用SPM的接触模式(contact mode)和敲击模式(tapping mode)对培养液中的生物活细胞进行了较长时间的扫描观察。结果显示，尽管接触模式SPM成像清楚，但长时间的扫描会造成细胞凋零；敲击模式SPM进行长时间扫描时也会造成细胞变形，而细胞会以新的形态来适应外力的影响。