纽- GFP质粒午山田，美国国立卫生研究院，国立牙科和颅面研究所，马里兰州贝塞斯达，和肌动蛋白- MRFP的质粒的礼物，是一个礼物，佛罗里达州立大学，佛罗里达州塔拉哈西，从迈克尔戴维森。 这项工作是支持由美国国家科学基金会职业奖＃0747334和AHA国家SDG＃0835205N Andreea气管。

1。集成的显微镜系统概述这些研究中使用的显微镜系统，已被描述详细1。简单地说，一个倒置的奥林巴斯IX - 81 TIRF附件显微镜（奥林巴斯，中心谷，PA）结合CSU - 22横河扫描头（横河电机公司，日本）。一个放映机深圳原子力显微镜（威科仪器公司，圣巴巴拉，CA）是安装在光学倒置显微镜上，和整个系统的研究级光学表上方（新港，IRVINE，CA）。 从旋转的磁盘扫描仪，外部滤镜轮和EMCCD相机（罗珀科学Photometrics，图森，亚利桑那州）组成的共聚焦大会是硬性连接铝基板放在硅垫减振必要。共焦扫描仪与显微镜通过法兰与treaded领，可以在原子力显微镜测量显微镜脱钩，没有旋转的磁盘扫描程序的振动会影响的原子力显微镜测量。氩氪激光（光谱物理，加州山景城）是用来作为激发源为TIRF和共聚焦成像模式，并可以加上另外两个光纤提供的激光TIRF附件或扫描头，分别。两台计算机控制系统：Slidebook软件控制的光学成像和奈米级软件控制的原子力显微镜。 2。细胞培养活细胞表达GFP对针对特定的蛋白质的构造应被放置在玻璃底部的细胞培养皿中的机械性刺激实验前24小时。在实验之日起，代替无酚红培养基的细胞培养液中。细胞培养皿中的原子力显微镜的舞台上安装带有磁性的衣领。 3。针尖的制备和显微镜设置 2微米定制针尖biotinilated玻璃珠将与DPBS冲洗5次，然后与抗生物素蛋白（1毫克/毫升）孵育5分钟，再洗净，用DPBS五倍，然后孵育5分钟1毫克/毫升biotinilated纤维连接蛋白。提示将在2月末洗5次以上，装上的原子力显微镜扫描仪，然后将保护硅裙各地的持有人。设置观看视频显微镜针尖在视野的中心对齐。通过放置在悬臂的激光束对准光杆，然后改变目标，高放大倍率的目标，适当的单细胞成像。针尖弹簧常数在实验开始前确定。此参数将采用热调整的方法，在奈米级软件。从摄像头开关EMCCD相机，让花期成像。然后，选择一个单元格，使针尖在接触特定细胞。经过20分钟的等待时间，这是一个强烈的粘着，在原子力显微镜与细胞表面的接触点形成所必需的，机械性刺激过程将开始。 4。活细胞的机械性刺激 TIRF成像3-4将可视化和量化粘着斑的首选方法，将用于可视化整个细胞体内的肌动蛋白细胞骨架和旋转盘共聚焦成像。原子力显微镜6将用于下流体的接触模式成像。 一个细胞TIRF或共聚焦图像将被收购开始前的机械性刺激过程。机械性刺激会诱发功能化的原子力显微镜的探针在离散的步骤，将向上移动过一段时间（1-1.5小时），每3-5分钟。细胞的活性反应将被记录不断一系列高度图像，每一行代表了1秒单位时间内的形象在奈米级软件。虽然收购的AFM数据，操作员显示器针尖Z -位置，在任何时候都确定为往后拉的适当时机。 每个机械性刺激后很可能获得一个细胞Slidebook软件TIRF或共聚焦图像，同时，不断收购的AFM数据。光学图像处理脱线，显示肌动蛋白细胞骨架和粘着斑重组创造电影。此外，每个可以处理图像，定量，以确定细胞的区域，特定的蛋白质面积，并分析一般的细胞形态。 在针尖是从细胞和针尖的灵敏度撤回实验年底将在奈米级软件的测量。 实验是非常敏感的噪音！在接触前的任何谈话或显微镜应避免periment。为了尽量减少在原子力显微镜测量的噪音，旋转磁盘焦应取消加上从显微镜。取决于使用的细胞类型和矩阵涂在刀尖上，形成强有力的细胞表面和针尖之间的粘着的等待时间可能会有所不同。 5。代表性的成果<img src="/files/ftp_upload/2072/2072fig1.jpg" alt="图1" /> 图1。相同的血管平滑肌细胞（VSMC）肌动蛋白- MRFP和纽- GFP转成像是：（一）在根尖细胞表面的原子力显微镜（B）TIRF在基底细胞表面;及（C）纺纱盘共聚焦显微镜作为一个3 - D堆叠的最大投影，整个细胞的身体。共聚焦图像显示。良好的AFM和TIRF或旋转盘共聚焦图像之间的重叠显示在（D）和（E）分别。图像尺寸为64 × 50微米。 <img src="/files/ftp_upload/2072/2072fig2.jpg" alt="图2" /> 图2。机械刺激血管平滑肌细胞与肌动蛋白- MRFP转一个由原子力显微镜和旋转盘共聚焦显微镜成像的箭头指示肌动蛋白纤维：（一）聚合/勾芡，（二）解聚/消失，或（c）捆绑在一起肌动蛋白重组。白色虚线表示的单元格上方的针尖。图像尺寸91 × 91微米。

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细胞骨架的 7-10和粘着斑 11-16是动态的结构组合，分散和细胞迁移或回应机械力 17 。他们有重要的功能，调节细胞生长，生存，和基因表达。知道他们的具体分配和动态，提供了一个更好地了解细胞如何与基体和它们之间的沟通。 这种新的显微镜系统是大致跨越任何附着的活细胞的分子动力学研究的广泛适用。集成的显微镜，使亚细胞快速变化的定?...

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<td>AFM tip</td>
<td>&nbsp;</td>
<td>Novascan Technologies</td>
<td>&nbsp;</td>
<td>2 micron Borosilicate bead, 0.01N/m, Biotin Surface</td>
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<td>avidin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A9275</td>
<td>1mg/mL in DPBS</td>
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<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>F4759</td>
<td>1 mg/mL</td>
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<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21600-051</td>
<td>&nbsp;</td>
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<td>Amaxa transfection kit for Smooth Muscle Cells</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPI-1004</td>
<td>&nbsp;</td>
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<td>Mattek glass bottom dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P60G-1.5-30-F</td>
<td>Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5</td>
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live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy

要理解这一机制的活细胞检测的机械力，以及他们如何应对和适应他们的环境，新的技术能够在亚细胞水平研究细胞的行为，具有高时空分辨率的开发。集成与全内反射荧光（TIRF）显微镜和快速旋转的磁盘（FSD）的共聚焦显微镜，原子力显微镜（AFM）。大致横跨在任何附着的活细胞的分子动力学研究的一个广泛适用的集成系统，可以直接实时的机械刺激细胞反应的光学成像。微丝和粘着斑的重大重排的结果表明，由于地方根尖细胞表面的机械刺激，诱导到整个细胞体细胞结构的变化。这些创新技术将提供新的信息，了解机械力活细胞的结构调整和动态。一个详细的协议和有代表性的数据显示活细胞机械性刺激的反应是。

Watch this Scientific Journal Video about Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy at JoVE.com

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

本文旨在指导读者在原子操作的综合力量光学成像显微镜文化的活细胞的机械性刺激。一个一步一步的协议。一个有代表性的数据集显示活细胞的机械性刺激的反应。

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

live-cell-response-to-mechanical-stimulation-studied-integrated

Research

JoVE Journal

Biology

11.2K 次观看.  Texas A&M Health Science Center. 本文旨在指导读者在原子操作的综合力量光学成像显微镜文化的活细胞的机械性刺激。一个一步一步的协议。一个有代表性的数据集显示活细胞的机械性刺激的反应。

视频: Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

科研

生物学

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

光镜研究沉默突触的突触前

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

微型机械的肺组织利用原子力显微镜表征

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

分析细胞表面粘附重塑回应机械张力使用磁珠

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

凸出力显微镜: 一种量化细胞突起力的方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

使用原子力显微镜表征活植物器官中原代细胞壁的机械性能

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...