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。     

扫描离子电导显微镜的研制与实现

在微纳米尺度上对活细胞高分辨率成像对生命科学研究具有重要的意义,其将有助于再现正在发生的生命过程、检测细胞对外界刺激做出的响应,甚至观测某些蛋白簇在细胞膜表面的运动。然而直到今天,仍然没有很好的实现上述目标。扫描离子电导显微镜（SICM）由于其真正的非接触、高分辨、无损独特成像方式,规避了扫描过程中探针与样品表面发生力的接触,得到越来越多的关注和广泛的应用。从系统的角度阐述自制SICM 系统的设计、硬件集成及跳跃模式扫描算法的实现,并通过对聚二甲基硅氧烷（PDMS）栅格成像以及与原子力显微镜（AFM）成像结果的对比,验证了系统功能的正确性和有效性;最后开展了生理环境下活体细胞的原位扫描成像实验,初步获取了活体神经细胞轴突结构的三维形貌图像。SICM 的成功搭建,将为人们深入了解生理条件下活体生物样品表面微观结构与功能机理等提供有效的研究方法与手段。     

非接触式扫描离子电导显微镜技术在探测活体细胞表面微结构中的应用

扫描离子电导显微镜技术是在纳米尺度进行非导电的生物样品成像的一种新型扫描探针显微镜技术。通过成功制备扫描离子电导显微镜扫描探测用纳米尺度玻璃微探针,对其进行了功能性评估;而后通过绘制探针-样品接近曲线,阐述了扫描离子电导显微镜技术实现非接触高分辨率探测的工作原理;最后采用该显微镜技术对导电标准样品及活体肾上皮A6细胞进行了表面形貌扫描成像,并与A6细胞表面形貌的扫描电镜图像进行了对照。结果表明,扫描离子电导显微镜技术不仅可实现导电样品的扫描成像,而且适宜于在生理条件下、非接触式地研究活体细胞表面的三维形貌,从而为人们深入研究细胞表面微观结构与生理功能提供了全新的技术手段。     

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。     

原子力显微镜相位成像模式的设计及研究

相位成像模式是近几年发展起来的一种原子力显微镜的检测模式,该模式可以提供丰富的样品表面纳米尺度信息,是形貌像的有利补充。本文给出了一种应用原子力显微镜轻敲模式的相位检测电路,结构简单,工作可靠稳定。通过实验获得了一些样品的相位像。     

状态激励轻敲模式原子力显微镜的设计与仿真

为轻敲模式原子力显微镜设计了一种激励方式,在带有状态反馈的轻敲模式原子力显微镜基础上,除去原有的外加激励信号而引入一个自动增益控制环节,用于维持状态反馈信号的幅度恒定,并将此信号作为悬臂振动的漱励信号.读激励方式即为状态激励,用于轻跛模式时可称为状态激励轻敲模式原子力显微镜.采用Matlab/Simulink工兵实现了...     

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.     

基于近场光纤探针的活细胞无损测温方法研究

高精度的单细胞温度分布检测对于研究生命活动具有重要意义。为了实现高空间分辨率、高温度灵敏度的细胞测温,本研究团队基于原子力显微镜的扫描成像原理,将对温度敏感的量子点与音叉驱动的近场光纤探针相结合,提出了一种无损伤测量活细胞表面温度分布的方法;并以人脑星形胶质母细胞瘤细胞为研究对象,利用该方法检测了吞金纳米颗粒的固定细胞和未内吞金纳米颗粒的活细胞的温度分布。结果表明：在无金纳米颗粒作为附加热源的情况下,由活细胞内部产热引起的细胞表面最大温差超过0.5℃,而细胞与其外部环境间的温差大于2.5℃。所提活细胞无损测温方法的空间分辨率小于0.2μm,温度分辨率为0.16 nm/℃,为活细胞温度分布成像提供了新思路。     

快速退火气氛对硅片洁净区和表面形貌的影响

使用N2和N2/NH3混合气氛作为快速热退火(RTA)气氛,研究了RTA气氛对洁净区、氧沉淀和硅片表面形貌的影响.在N2/NH3混合气氛下,RTA处理后,硅片表面出现小坑,同时,微粗糙度增加,后续热处理工艺中会出现薄的洁净区(～10μm)和高密度的氧沉淀.经过N2气氛RTA处理的硅片,表面微粗糙度变化不大,后续热处理中获得较厚的洁净区(≥40μm)和较低的氧沉淀密度.     

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.     

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.     

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.     

MEMS based low cost piezoresistive microcantilever force sensor and sensor module

In the present work, we report fabrication and characterization of a low-cost MEMS based piezoresistive micro-force sensor with SU-8 tip using laboratory made silicon-on-insulator (SOI) substrate. To prepare SOI wafer, silicon film (0.8 µm thick) was deposited on an oxidized silicon wafer using RF magnetron sputtering technique. The films were deposited in argon (Ar) ambient without external substrate heating. The material characteristics of the sputtered deposited silicon film and silicon film annealed at different temperatures (400–1050 °C) were studied using atomic force microscopy (AFM) and X-ray diffraction (XRD) techniques. The residual stress of the films was measured as a function of annealing temperature. The stress of the as-deposited films was observed to be compressive and annealing the film above 1050 °C resulted in a tensile stress. The stress of the film decreased gradually with increase in annealing temperature. The fabricated cantilevers were 130 μm in length, 40 μm wide and 1.0 μm thick. A series of force–displacement curves were obtained using fabricated microcantilever with commercial AFM setup and the data were analyzed to get the spring constant and the sensitivity of the fabricated microcantilever. The measured spring constant and sensitivity of the sensor was 0.1488 N/m and 2.7 mV/N. The microcantilever force sensor was integrated with an electronic module that detects the change in resistance of the sensor with respect to the applied force and displays it on the computer screen.     

High-density data storage based on the atomic force microscope

The atomic force microscope (AFM), with its ability to image and modify surfaces on the nanometer scale, offers the potential for simple, compact, high-density data storage devices. At the heart of the technique is a microfabricated cantilever with a sharp tip on the end. Using modern micromachining techniques, it is possible to batch fabricate cantilevers with tips that are sharp on the scale of 100 Å. We have pursued a particular AFM storage scheme based on mechanical readback of topographic data using high-frequency piezoresistive silicon cantilevers. Areal densities of 65 Gbit/in² have been demonstrated, with readback rates greater than 10 Mbit/s. Nanoreplication techniques have been used to produce read-only disks. In addition, a write-once scheme was developed that uses integrated heating elements on the cantilevers in order to perform thermomechanical writing on a polymer substrate. Considerable progress has been made in addressing critical issues such as data rate, reliability, and practical implementation, but significant challenges still remain, both in the technology and in finding the most suitable applications     

Mechanical properties and applications of custom-built gold AFM cantilevers

Silicon and silicon nitride metal-coated cantilevers have been in use for a long time in several scanning probe microscopy applications that require electrically conductive probes. However, conventional metalized cantilevers present several drawbacks such as limited life-time of the metal layer due to wear, and increased tip radius. This work focuses on monolithic metallic cantilevers developed in order to overcome the limitations of conventional metalized atomic force microscopy (AFM) probes. These custom-made cantilevers are designed for several applications such as in current-sensing AFM (CSAFM), Kelvin probe force microscopy (KPFM), and tip-enhanced Raman spectroscopy (TERS). Determination of the dynamic and static mechanical properties of these cantilevers in a non-destructive way is reported here. Key parameters under investigation are the cantilever spring constant and the frequency response using finite element method (FEM). Gold cantilevers are selected for this study, which allow optimizing the design and the process of developing these metallic cantilevers with parameters engineered for the applications mentioned above. This work contributes to the establishment and applicability of custom-made probes in advanced scanning probe microscopy methods and their performance understanding using computer simulations.     

CMOS monolithic mechatronic microsystem for surface imaging and force response studies

We report on a standalone single-chip (7 mm /spl times/10mm) atomic force microscopy (AFM) unit including a fully integrated array of cantilevers, each of which has individual actuation, detection, and control units so that standard AFM operations can be performed only by means of the chip without any external controller. The system offers drastically reduced overall size and costs and can be fabricated in standard CMOS technology with some post-CMOS micromachining steps to form the cantilevers. Full integration of microelectronic and micromechanical components on the same chip allows for controlling and monitoring all system functions. The on-chip circuitry notably improves the overall system performance. Circuitry includes analog signal amplification and filtering stages with offset compensation, analog-to-digital converters, digital proportional-integral-derivative (PID) deflection controllers, sensor-actuator compensation (SAC) filters, and an on-chip digital interface for data transmission. The microsystem characterization evidenced a vertical resolution of better than 1 nm and a force resolution of better than 1 nN as shown in the measurement results. This CMOS monolithic AFM microsystem allows for precise and fully controlled mechanical manipulation in the nanoworld.     

亚微米级硅尖端传感器的研制

提出了一种全新的亚微米级温度传感器的构想。作者在借鉴AFM悬臂测头的基础上，采用了微机械加工技术中的各向同性和各向异性腐蚀技术，在硅材料上完成了悬臂梁与硅尖制作。制成的梁的厚度在6μm左右、尖端曲径半径远小于1μm。随后对悬臂梁前端的硅尖进行尖端放电，隧穿其顶端的Si_3N_4层形成温敏器件──一个微型的热电偶。最后进行了这一温敏传感器的引线和封装。由于制作所形成的硅尖曲径半径在0．1μm左右，从而可以在其尖顶上形成亚微米级的温度传感器，在集成电路的探伤与修补以及生物技术领域，有着广阔的应用前景。     

两种基于MEMS谐振器的原子力显微镜探针

目前商用原子力显微镜(AFM)大多使用的微悬臂式探针,力灵敏度可达到pN级别。然而受到工艺水平及检测方法限制,微悬臂谐振频率难以超过3.5 MHz,且Q值较低,制约了AFM的成像速度以及在液体中的成像效果。另外,光杠杆的检测方法无法与探针本身进行片上集成,较小的悬臂也给激光束的聚焦带来困难。基于以上考虑,本文提出两种基于MEMS谐振器的探针,振频率可达11 MHz,Q值为4 000,并集成了执行与传感功能以及批量加工纳米针尖的工艺。目前两种探针都已经实现对树脂图案的成像功能,力灵敏度最高可达5pN/√Hz。