Name: measurement of tension release during laser induced axon lesion to evaluate axonal adhesion to the substrate at piconewton and millisecond resolution
Uploaded: 2013-05-27T12:00:00
Description: Watch this Scientific Journal Video about Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution at JoVE.

阿尔贝托Guiggiani开发实时控制的系统，埃弗利纳Chieregatti和花子对马岛见地的讨论，普鲁佐和亚历山德罗·贾科莫Parodi的定制的电子产品和软件的发展，克劳迪娅Chiabrera和滨海南尼他们在细胞培养制备的专家建议和援助。

1。光学设置整个光学系统的前面描述的15。简单地说，是根据光镊系统镱连续波（CW）光纤激光器1064 nm处（IPG激光GmbH公司）。空间光调制器（SLM）（LCOS-SLM模型X10468-07 - 滨松）传入的红外激光束来控制培养皿上由计算机生成的全息图捕获焦点的位置不同阶段。蓝镊子软件免费提供的（网页连结设备表）产生全息图投射在空间光调制器。武力干涉仪光谱测量，四象限光?...

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我们报告了这项工作的定量方法比较突起文化基板的附着力，同时执行力时的光谱测量激光诱导的细胞病变。测得的张力释放基板的细胞粘附的程度有关：细胞具有较高的粘着斑的数目应释放张力小。测量张力的释放在piconewtons方面提供的物理量评价轴突粘着培养支持在不同的实验条件下14。 几个实验室与光镊系统集成的激光剥离，采用独特的光学设计23。我?...

光学显微镜是一种较少侵入性的成像系统，可观察活细胞。随着开采的辐射压力（如在光学镊子1），或高能量光子通量（在激光剥离器2）的影响，如，这种技术被扩展到纳米操纵。的光学成像系统，可视化和操纵子的细胞的目标3。提供精确的控制。与此同时，得益于精确标定交付的激光功率，光工具完成软或侵入性的样品操作，以前所未有的重现性。 几个实验室的集成，在相同的实验装置，光学镊子和激光剥离器，以消融细胞器4，不同的细胞融合在一起5星 ，或光学驱动的货物6,7刺激细胞。虽然光镊后校准的光刚度，以便的作用力细胞一个piconewton规模的控制，激光剥离系统可以调节光学操纵，单细胞器或亚细胞结构的解剖消融范围从照片蒸发膜。然而，激光剥离校准依赖于光学操纵实体与定性评估的能量传递到样品，主要是基于图像分析说明形态的变化而引起的标本8。在该方法中，我们将演示如何执行力在一个发展中的神经元轴突解剖激光光谱测量，量化，上piconewton规模，产生的力平衡，改变细胞骨架结构的亚细胞室9。培养神经元坚持为基材，并在开发过程中两极化。极化阶段发生在第一天的体外 。在第二阶段的偏振，的EXTRUD之一的神经突起变长，它会分化成为轴突10。轴突生长锥伸长响应牵引力先前已为蓝本由Dennerl的模型11。最近，这种模式被扩展12包括突起粘附到细胞外基质底物的作用。这种生物物理模型，实验观察13后提出，表明拉力沿神经轴突生长锥，传播，调制焦点粘连基板。同样，轴突病变向细胞体中传播产生了一个局部的张力释放。因此，我们建议，测量等发布张力之间的病变沿轴突和胞体的位置，提供阻尼结果不受影响的焦点粘连的可能性进行评估。 我们校准所需的能量的光子通量的激光剥离器来控制的程度，造成了轴突damagE，完全横断局部病变。校准之后，我们重复部分病变几个分化的神经元的轴突和发展的协议的张力解除量化，从而得到一个量化参数估计的轴突到基板14的粘附。 在目前的工作中，我们详细描述了所开发的协议，即代表了精确的实验程序评估和比较与piconewton灵敏度的轴突对基材的附着力，在不同的实验条件下，如化学处理14，或不同类型的细胞培养支持。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Polymer microspheres, &Oslash; 4 &mu;m, COOH coated</td>
			<td>Bangs laboratories</td>
			<td>PC05N/6700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PolyLink Protein Coupling Kit</td>
			<td>Polyscience</td>
			<td>19539</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>IR laser</td>
			<td>IPG Laser GmbH</td>
			<td>YLM-5-SC-LP</td>
			<td>ytterbium continuous wave (CW) fiber laser operating at 1064 nm, with linear polarization</td>
		</tr>
		<tr>
			<td>Spatial light modulator</td>
			<td>Hamamatsu</td>
			<td>LCOS-SLM 10468-07</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue-tweezers software</td>
			<td>Optics group, University of Glasgow</td>
			<td>Free downloadable software</td>
			<td><a href="http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/" target="_blank">http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/</a></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>Free downloadable software</td>
			<td><a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/ </a></td>
		</tr>
		<tr>
			<td>QPD</td>
			<td>Thorlabs</td>
			<td>S5980 with C5460SPL 6041 board</td>
			<td>Four quadrant photo-diode to measure x, y trapped probe displacement</td>
		</tr>
		<tr>
			<td>PD</td>
			<td>Teem Photonics</td>
			<td>PDA100A-EC</td>
			<td>Photodiode to measure z trapped probe displacement</td>
		</tr>
		<tr>
			<td>nano-Pulse UV laser</td>
			<td>AA-optoelctronics</td>
			<td>PNV-001525-040</td>
			<td>Pulsed UVA laser, pulse length 400 ps</td>
		</tr>
		<tr>
			<td>Acoustic Optic Modulator</td>
			<td>Olympus</td>
			<td>MQ110-A3-UV, 355 nm fused silica</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Andor</td>
			<td>BX51</td>
			<td>Equipped with a 60X, 0.9 NA, water dipping objective</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>Warner Instruments</td>
			<td>V887ECSUVB EMCCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peltier device</td>
			<td>Physic Instruments</td>
			<td>QE1 resistive heating with TC-344B dual channel heater controller</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope stage: micro+piezo stage</td>
			<td>National Instruments</td>
			<td>Three linear stages M-126.CG1 carrying a separate 3-axis piezoelectric nano-positioning stage P-733.3DD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Daq</td>
			<td>&nbsp;</td>
			<td>NI PCI-6229</td>
			<td>Acquiring the x, y, z position of the trapped probe, and sending feedback loop signals to microscope stage</td>
		</tr>
		<tr>
			<td>Linux Real Time Application Interface (RTAI) machine</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Real time feedback loop system, to control stage position, developed on a dedicated PC desktop</td>
		</tr>
	</tbody>
</table>

measurement of tension release during laser induced axon lesion to evaluate axonal adhesion to the substrate at piconewton and millisecond resolution

一个发展中的神经元网络的功能连接的形成是受外部线索。神经突起生长发育中的神经元受到化学和机械信号的机制，感知和响应机械信号却知之甚少。阐明细胞的成熟过程中的力的作用，使支架的设计，可以促进细胞粘附和细胞骨架耦合到衬底，因此提高损伤后再生的能力，不同的神经元类型。 在这里，我们描述了一种方法，同时力光谱测量应用在激光诱导的细胞病变。测量张力释放部分病变轴突附着膜的轴突的光学捕获探针通过同时干涉跟踪。我们的实验协议检测的张力释放与piconewton灵敏度和动态的张力释放毫秒级的时间分辨率。因此，它提供了一个高分辨率的方法来研究细胞和基片之间的机械耦合，可以调制的药物治疗和/或通过在基板的不同的力学性能。

细胞产生牵引力在基板上，其焦点粘连。细胞骨架元素产生的力处于平衡状态的培养基材的反作用力。激光诱导后的神经突的病变，有些紧张的细胞骨架电缆被破坏平衡的张力被释放，因为基板的附着力的反作用力消除。张力释放部分地分布于未受影响的粘着斑，胎圈附着在细胞膜上，保持在一个光阱，测量等释放的部分未抵消基板锚定细胞的细胞骨架元素（见示意图图1） 。 <p c...

Watch this Scientific Journal Video about Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution at JoVE.

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

我们测量了张力释放部分病变进行附着膜的轴突的光学捕获探针上的的同时力显微镜测量用激光剥离器通过在一个轴突。所开发的实验协议评估轴突的培养基材的粘附性。

在激光诱导轴突病变张力释放测量评估轴突在Piconewton和毫秒级的分辨率对基材的附着力

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Research

JoVE Journal

Bioengineering

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

轴突伸展生长：神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

构建一个低预算的激光干切断系统研究轴突再生 C。线虫</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

通过电场诱导释放和测量（EFIRM）检测外体生物标志物

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

基于数字图像相关的骨折检测中股骨皮质应变的估算方法

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

Intraductal Delivery to the Rabbit Mammary Gland

导管内送货到兔乳腺

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

预测神经植入物软化行为的环境动力力学分析

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

集成张力传感器在亚微米分辨率下的成像整合和细胞力

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...