Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages

Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented.
AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip–sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

刚性的球头铣刀切削力模型

基于切削力与切屑负载之间的经验关系,通过对球头铣刀的微分化方法,建立了球头铣刀基本切削力模型,并着重研究刀具的径向跳动对径向未变形切屑厚度的影响,提出了刚性的球刀铣刀切削力模型,最后用切槽实验对模型进行了验证。     

振动攻丝动态切削力研究

分析攻丝时静态攻丝扭矩和动态攻丝扭矩的变化规律,研究动态切削力分析方法,探讨影响动态切削力变化的因素,通过试验研究动态切削力功率谱特点以及不同频率段信号能量的分布状况,结果动态切削力功率谱图可以反映攻丝过程,可以用信号功率谱峰变化和信号能量变化对攻丝过程进行监控。     

A stochastic finite element dynamic analysis of structures with uncertain parameters

The real life structural systems are characterized by the inherent uncertainty in the definition of their parameters in the context of both space and time. In the present study a stochastic finite element method has been proposed in the frequency domain for analysis of structural dynamic problems involving uncertain parameters. The harmonic forces as well as earthquake-induced ground motion are treated as random process defined by respective power spectral density function. The uncertain structural parameters are modelled as homogeneous Gaussian stochastic field and discretized by the local averaging method. The discretized stochastic field is simulated by the Cholesky decomposition of respective covariance matrix. By expanding the uncertain dynamic stiffness matrix about its reference value the Neumann expansion method is introduced in the finite element procedure within the framework of Monte Carlo simulation. This approach involves only single decomposition of the dynamic stiffness matrix for entire simulated structure. Thus a considerable saving of computing time and the facility that several stochastic fields can be simultaneously handled are the basic advantages of the proposed formulation. Numerical examples are presented to elucidate the accuracy and efficiency of the proposed method with the direct Monte Carlo simulation.     

Dynamics of a rotating Rayleigh beam subject to a repetitively travelling force

The dynamics of a rotating Rayleigh beam subject to a force travelling at a constant speed along the axial direction is studied. The beam is chosen as a simple model of the workpiece in the lathing process. A technique is developed for modeling the repetitive cutting force on the workpiece. The amplitude of the cutting force is chosen to be either constant or dependent on the motion of the beam. The discretized equations of motion of the rotating beam are obtained by Galerkin’s method. The time response of the rotating beam subject to the external force is discussed. The possible resonant conditions resulting in divergent solutions are studied. The stability of the response, due to a travelling motion-dependent force, is determined by the method of multiple scales. The effects of varying the rotating frequency, the travelling speed of the external force, and the movement of the force are also investigated.     

Observations of Contact Measurements Using a Resonance-Based Touch Sensor

A study of the contact mechanisms between a resonator probe sensor and a broad range of engineering surfaces is presented. The resonator-based touch sensor used in these studies consists of a prismatic beam clamped at one end with a spherical probe attached at the other. Pairs of piezoelectric (PZT) elements cemented at either side of the beam along its axis are employed; one to actuate and the other to pick up the strain signals. Over a band of frequencies near to a resonance, the sensor behaves like second-order system. When oscillated near to its resonant frequency, interactions between the probe tip and specimen are detected by monitoring phase or frequency shifts using phase-locking techniques. As the probe approaches and contacts a surface, a range of phenomena are observed. Approximate theoretical models have been developed to predict the effects characteristic of added mass, stiffness, and damping (i.e., kinetic, potential and dissipative energy transfer) for contacts between clean solids and when contaminant films are present. These models predict that phase and frequency shifts can either increase or decrease depending upon the dominant phenomena in the contact region. For example, when a solid surface is contacted by a clean probe, the resonant frequency can either increase or decrease depending upon the ratio of elastic modulus to density, and this is demonstrated with contact measurements made on metals and rubbers. A systematic method for the identification of the dominant effect (if there is one) based on observations of frequency or phase shifts using either constant phase or constant frequency monitoring is presented.     

Mechanical properties of L929 cells measured by atomic force microscopy: Effects of anticytoskeletal drugs and membrane crosslinking

To shed light on the architecture of the cytoskeleton, we used the atomic force microscope (AFM) to measure the elasticity, viscoelasticity, and plasticity of L929 cells. The initial elastic response (Young's modulus ～ 4,000 Pa) of the cells to an applied force was followed by a slow compression of the cytoskeleton (τ_1/2 ≈ 10 s). When force application was terminated, the cytoskeleton underwent a sudden partial decompression and a subsequent slow, incomplete recovery. The role of the cytoskeletal elements in cell mechanics was accessed in AFM measurements carried out on cells treated with cytochalasin D, nocodazole, or col-cemid. Cytochalasin D treatment reduced both elasticity (～45%) and cytoplasmic viscosity (～65%), whereas cells treated with nocodazole or colcemid exhibited a marked increase in elasticity (～100%) and a slight increase in viscosity (～15%). The AFM force measurements also provided evidence that the cell membrane and the cytoskeleton are mechanically coupled. Tightly adherent cells were stiffer than cells that were loosely attached. Moreover, cells crosslinked with either glutaraldehyde, 3,3 ‘-dithiobis’sul-fosuccinimidylpropionate] (DTSSP), or Concanavalin A were more rigid than untreated cells. It is of interest that cells crosslinked with Concanavalin A, but not DTSSP, displayed plastic behaviors that may reflect the induction of cytoskeletal reorganization by Concanavalin A.     

EFFECT OF ALLOY ELEMENTS ON QUASIEQUILIBRIUM MICROSTRUCTURE IN Fe-Mn-Ni-Cr STEELS

The microstructures of Fe-Mn-Ni-Cr steels with medium carbon and the effect of alloy ele-ments on them have been investigated by means of X-ray diffraction quantitative phase ana-lyses,metallography and hardness tests.The volume fraction of martensite in steels quenchedat 1100℃ can be expressed as:f_M~(1100)=162.643-15.482Mn-7.36Ni-4.286(Cr+Mo+V)Based on this expression,the quasi-equilibrium microstructure diagram has been obtained.Itis shown that the effect of Mn on the austenite stability is greater than that of Ni.The equiva-lent [Ni]=2.104Mn+Ni and equivalent [Cr]=Cr+Mo+V.The driving force for martensite transformation at M_s point has been calculated to hedrastically decreased by element Mn.     

温度对Ni—P化学镀层结合力影响的研究

用弯曲法、划痕法研究了镀后加热温度对Ni-P化学镀层结合力的影响,结果表明:镀后退火温度越高,镀层结合力越好,但温度过高时,镀层硬度将下降。用金相观察及电子探针分析,探讨了镀后退火温度影响结合力的机理。结果指出:退火时将在镀层和基体界面上发生Ni与Fe的互扩散,形成Ni-Fe合金扩散层,从而提高了结合力。为了能在保证镀层硬度的前提下提高结合力,本文提出了二次镀覆的设想,获得较好试验结果。