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，四象限光电二极管的（QPD，S5980与C5460SPL 6041板 - 滨松）和一个光电二极管（PD，PDA100A-EC - Thorlabs公司）的基础上。 激光剥离源是次纳秒脉冲UV Nd:YAG激光波长为355 nm，力晶（PNV-001525-040纳米紫外激光脉冲 - 天河城光电）。一个声光调制器（MQ110-A3-UV，355nm处的熔融石英-AA-光电），控制传递到样品的紫外激光的功率。 集成的全息光镊和激光微解剖上的变形的正立显微镜（BX51 - 奥林巴斯）配有60X，0.9 NA水浸渍显微镜objective.The阶段是由一个3轴直线直流电机微定位系统（M-126.CG1的，物理仪器）背着一个独立的3轴压电纳米定位的阶段（P-733.3DD的，物理仪器）相结合的样品粗动亚纳米分辨率的压电阶段。的显微镜载物台系统配备有两个控制循环协同作用捕集焦点维持在合适的位置，这取决于所选择的工作模式（位置或力钳，静态或动态的）16。特别是，一个内部反馈回路作用于压电体的阶段，保持在胎圈，在一个专ED从陷阱中心的距离。其它外部回路控制利用跨越的区域更大范围内的面积的比其行程17由压电致动器的电动载物台的位置。当压电阶段达到在一个方向上的行程的限制，外部环路移动在相反的方向，从而在压电微阶段恢复朝向其中心位置，因为它是跟踪被困珠粘附到样品。当压电阶段达到其场范围的中心位置，微阶段停止。 16,17 Guiggiani 等报道了该系统的进一步的细节。 珀耳帖元件（的QE1电阻加热TC-344B双通道热水器控制器 - 华纳仪器）控制在显微镜下的细胞培养的温度（37°C）。在文化，pH值和湿度保持在生理条件下，通过曝气定制设计的聚二甲基硅氧烷（PDMS）的套筒（集成显微镜物镜）与加湿卡波金（95％O 2，5％CO 2）。 2。细胞培养制备所有实验的协议是由意大利卫生部批准。原代培养小鼠海马（C57BL6J，查尔斯河）获得在胚胎18天（E18）。 <ol><li>镀在玻璃底培养皿中的25000个细胞/ ml的浓度为（P35G-0-14-C - 公司MaTek）神经元。低浓度的培养的细胞是必要的，以避免形成致密的网络已经在第一天的体外 。上面的提到的细胞浓度，发现没有连接到其他细胞具有较长的神经突起的分离的细胞的可能性高得多。 </li></ol> 3。珠涂层<ol><li>涂有聚聚合物微球（直径4微米，COOH终止刘海实验室的，产品代码PC05N/6700）-D-赖氨酸的polyLink蛋白偶联试剂盒（Polysciences的，产品代码19539）中描述的程序之后。珠子都涂有用于弥补文化的支持和青睐细胞粘附分子。 </li></ol> 4。选择隔离神经元。分离珠文化基板，陷阱和移动下一步的神经<ol><li>文化培养皿放在显微镜舞台上。重点对样品阶段运动后，纠正设置科勒照明的照明目标冷凝器的位置。对齐的照明光学系统，设置Kolher照明条件是必要的，以改善亮场成像质量。利用这样的对应条件下，它也需要最大限度的红外激光的收集效率（通过客观冷凝器），从散射被困探针，来执行其干涉跟踪的QPD和PD。 </li><li>用移液管在几微升的包衣的微球原液培养皿。微将存款和坚持文化衬底。注入培养皿微升的数量依赖于微球浓度的储存溶液。理想的状况是有一至两微每成像视野。当过多的微球加入到培养皿中，它们已经可以随机附加的培养的神经元。 </li><li>在培养皿中四处移动，寻找一个孤立的神经元具有较长的神经轴突（轴突），在显微镜阶段保存的位置。 </li><li>走动，搜索珠坚持的文化支撑。 </li><li>打开IR俘获光上。在这个阶段，没有全息图投影（SLM）和红外激光光斑的位置与紫外激光的光点位置一致。设置红外现货2-3微米以上的文化支撑表面上，通过显微镜载物台运动的轴向位置。 </li><li>置紫外激光功率小于1μW传递到样品。该先前已校准的紫外激光剥离器14: 5-6μW被烧蚀玻璃支撑 4μW神经元的连接完全解剖 2.5μW部分病变神经突 &lt;1μW冲击波在培养皿中产生。光冲击波强大到足以分离珠玻璃支撑。 </li><li>开启紫外激光，而留下的红外激光，移动UV点以上坚持珠显微镜阶段运动。在胎圈从表面分离，并且被困住的红外激光。然后，关掉紫外激光。 </li><li>移动被困胎圈几微米以上的玻璃支撑（20-30微米）。起重珠到这个位置，防止它与其他细胞接触，而它正在走向先前选定的神经元。使珠先前保存的舞台上的地位。阶段速度设置为较低的值（10微米/秒），以便拖动流体的力不超过光阱平力。 </li></ol> 5。移动陷阱的位置，就由计算机生成的全息激光剥离器现货和轴突位置，并校正光学镊子刚度<ol><li>将胎圈朝向玻璃支撑，以可视化的轴突。以上的细胞被保持在胎圈（约5μm以上的玻璃支承），以避免接触。 </li><li>移动显微镜载物台移动的UV轴突的宽度的中心的焦点。保存当前舞台上的地位。 </li><li>将IR的焦点位置由计算机生成的全息图。在此步骤中，在先前步骤中所选择的位置是停止的阶段。当投射在SLM的计算机生成的全息图，被困珠移动相对于轴突。移动IR激光光斑有被困珠神经突起的中心对齐，与UV激光点为中心的相同的神经突的太多。沿神经轴突的红外点，从而定位被困珠5-10微米远离UV点。我们的位置移动的红外光相对于突起的几何形状取决于UV点。的位置的情况下，光镊SLM的系统都没有配备，可以是多种多样的，通过倾斜的反射镜，或声光偏转器上的IR光路。 5-10微米的远红外激光光斑可能被定位从解剖梁与被困的探头，它可能会影响它的布朗运动和改变力显微镜的痕迹，以避免光学作用的UV点。 </li><li>在定义新的IR现货头寸UV点和轴突的位置，移动定位被困珠远离轴突和避免碰撞的舞台。被困胎圈的轴向位置移动到约2μm以上的玻璃盖。 </li><li> QPD对准中心的干涉条纹:x和y QPD差分信号是零QPD中心时。收购5秒的布朗运动被困珠干涉米，在50 KHz采样率。获取刚度（PN /纳米）和灵敏度（V /纳米）的光陷阱功率谱方法18。 </li></ol> 6。将珠轴突。执行干切断同时测力<ol><li>提高被困珠约4微米以上的玻璃盖的位置，并将其移动到之前保存的舞台上的地位（见第3.2步）。 </li><li>将被困珠轴突向下延伸，直到它接触的神经轴突。被困的胎圈和轴突之间的碰撞情况是由在z QPD被困胎圈在轴向方向上的位移信号检测。 </li><li>等待10秒被困珠推反对的轴突有利于它的突起膜的附着力。 </li><li>移动微阶段位移轴突被困珠，从而验证其在细胞膜的粘附性。如果珠坚持，从光学陷阱逃脱。 </li><li>回迁激光陷阱粘附珠切换与受力情况等于零力钳循环。在这样的方式中，压电阶段重新定位的胎圈，连接到电池上的光陷阱中心。如果系统没有一个反馈环路控制激光陷阱的位置可以移动显微镜阶段中心上坚持的探头。陷阱的中心时达到QPD胎圈会被捕获，未粘附的细胞（x和y QPD信号等于零，则在z QPD信号给出了相同的电压的和四个象限的信号是相同的当被困住的胎圈从任何表面）。 </li><li>设置捕集激光定位在z轴上，稍为超过所述胎圈的中心（约100nm），以产生预张力附着的捕获探针上的力钳条件。光阱的位置的情况下，系统没有配备的力钳控制，可以在显微镜载物台的步长为25nm上升。在z QPD信号允许监控。因此，使用氏S信号来设置相对于附着的探针的位置的激光陷阱。这样的预张力所需的感测轴突损伤后的膜的应变，以及因此而减少附着的探针的布朗运动。已经提出了一种类似的方法测量的张力释放，在由视频成像19的膜系绳。 </li><li>开关关闭力钳反馈测量附着的探针上的力的位置钳条件（压电阶段被阻塞）。 </li><li>开始被困探头位置的同时记录通过干涉仪（20 KHz采样频率），细胞在激光切断后的时间推移明场成像（20赫兹帧速率）。 </li><li>开启紫外激光提供激光​​脉冲，直到病灶变成可见图像的轴突（一般需要200-400光脉冲，每个脉冲的能量:25新泽西），然后关掉紫外激光。 </li><li>约3英里干涉继续录制N，直到x，y和z QPD痕迹达到一个高原。 </li></ol> 7。量化总张力释放<ol><li>转换录制的QPD的痕迹（以伏特为单位），由校准的灵敏度的光阱，在纳米取得各自的痕迹。 </li><li>过滤器的x，y和z位移的痕迹通过一个高通滤波器，带切断为10赫兹。 </li><li>计算的总方差占被困珠的布朗运动的过滤后的痕迹。方差计算步长在25毫秒，500毫秒（10,000个数据点）20重叠的时间窗口。高通滤波的痕迹，排除了布朗运动的变化，由于膜的波动或皮层肌动蛋白的运动。在胎圈的布朗运动，在细胞膜上的的光学力和粘附力减少。当膜紧张由于张力的释放，增加其粘度，从而在胎圈的布朗运动，立即检测到采用干切断后，开始减少。 </li><li>定义T0:的布朗运动的方差减少的开始，在交货后的紫外激光能量。时刻t0表示膜应变的开始。 </li><li>定义T1:年底减少了布朗运动的方差（当它到达高原）。的时间瞬间t1表示结束膜应变。 </li><li>进行视频跟踪的碎片或刮盖玻片上的支持，来衡量任何样品漂移的测力。要跟踪的颗粒在玻璃上的支持，我们首先应用阈值的明视场图像，获取一个栈二进制图像的黑色背景上的白色颗粒。然后，我们跟踪的粒子的质量中心的亚像素精度（使用软件ImageJ软件）。 </li><li>中减去测得的漂移来自位移QPD的痕迹。由各 ​​自的光学校准刚度（K X，K乘以漂移校正QPD痕迹<suB&gt; Y，K Z）获得FX，FY，FZ piconewtons的痕迹。 </li><li>计算总兵力跟踪F TOT =√（FX 2 +风云2 + FZ 2）。 </li><li>计算总发行T0和T1之间的力F = F 发布TOT（T1） - F TOT（T0）。要减去在t0测量的力，因为它是由于探头从陷阱中心的位移，在胎圈的神经突的附着。 </li><li>计算被困珠和紫外激光光斑位置突起之间的接触面积亮场图像测量长（L）和短（D）轴之间的神经轴突被困珠和紫外激光光斑位置。轴突接触面积A 轴突 =长x D. </li><li>规范化公布总释放的力F 轴突接触面积A 轴突 。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decrease+in+membrane+tension+precedes+successful+cell-membrane+repair.">A decrease in membrane tension precedes successful cell-membrane repair.</a> Mol. Biol. Cell. 11, 4339-4346 (2000).</li><li>Kress, H., Stelzer, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+act+as+phagocytic+tentacles+and+pull+with+discrete+steps+and+a+load-dependent+velocity.">Filopodia act as phagocytic tentacles and pull with discrete steps and a load-dependent velocity.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 11633-11638 (2007).</li><li>Togo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+plasma+membrane+stimulates+rearrangement+of+microtubules+and+lipid+traffic+toward+the+wound+site.">Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site.</a> J. Cell Sci. 119, 2780-2786 (2006).</li><li>Huang, H., Kamm, R. D., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics+and+mechanotransduction:+pathways,+probes,+and+physiology.">Cell mechanics and mechanotransduction: pathways, probes, and physiology.</a> Am. J. Physiol. 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作者宣称，他们有没有竞争经济利益。

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。我们的选择是设置固定光路和显微镜载物台运动。要做到这一点，我们引入一个空间光调制器中的红外激光束路径移动的捕集点，相对于紫外激光的位置。尽管galvanometrIC镜子使激光焦点位置，而不转向移动样品，他们可以引进机械噪音影响力光谱测量系统。此外，我们决定重新定位红外激光聚焦在样品中，由于布朗运动分析允许快速重新校准的光学刚度。移动到样品上的紫外激光的光点位置可以产生影响质量的UV焦点本身，这是可以忽略的小位移的光束从其中心位置的球面像差。因此，激光剥离器的光学性能的修改可能需要重新校准交付的激光功率与实体损坏。 由力显微镜测量，我们可以观察到张力解除由几秒钟的快相，和一个缓慢的阶段，可能会持续一些几十秒钟。据报道在以前的工作14，有时慢阶段可能会持续几分钟。出于这个原因，我们不得不纠正视频跟踪一个从无到有的文化支撑力的测量。在未来，一个更好的办法将取消阶段漂移一个反馈回路，自动校正显微镜舞台上的地位，通过视频或干涉跟踪珠附着到玻璃盖。这种类型的配置中，在文献中已经报道，需要连接的胎圈中心的第二激光束，并确保稳定阶段与亚纳米级精密24,25。 在未来，我们要应用开发协议，比较在不同分化阶段的文化支持轴突的附着力，并在不同病理条件下提供了定量分析药物治疗。此外，相同的协议可以适用于植入支架的设计，用来比较的神经元的不同类型的材料的粘附性，具有鲜明的机​​甲anical或化学性质26。

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） 。 我们在此报告，在图2中，具有代表性的结果的上述实验的协议。神经元，在左边的面板图2a，提出了一个较长的神经轴突，标识区分Ñ轴突EURON。在图2a中右面板中，示出相同的神经元诱导轴索损伤（由白色箭头表示）后， 图2b示出记录的胎圈中的x，y和z方向上的位移的痕迹。 图2c报告划痕的视频目标跟踪在玻璃上的支持，以考虑到并消除在测量过程中的阶段漂移。 在图3a中，在左侧面板上，我们展示了损伤的神经轴突，而白线上游的病灶部位，我们计算kymograph的轴突的直径测量的张力释放。右侧面板kymograph的，显示如何轴突的直径略有增加后立即病变，然后变得更薄，由于张力的胞体释放。在图3b中中，我们量化的kymograph，结果。神经轴突出现明亮的背景，因为我们定位阿塔录取通知书珠的焦点中心的目标，因此轴突稍微失焦。的确，通过沿的kymograph线的时间推移视频记录在每帧的报告的像素强度的总和，我们取得的估计突起的直径在释放过程中的张力。面板上3c中 ，我们报告的过程中的张力释放的连接的胎圈的总方差，计算根据记录，由干涉仪（ 图2b），在10 Hz的截止频率的高通滤波后的痕迹。我们可以观察到的方差材料上游的病灶部位的积累期间与轴突直径增加。然后，方差开始下降（ 图3c T0）当膜紧张，太轴突的直径减小。的方差减少达到稳定（在图3c中的t1），当张力释放停止。 T1之后，布朗运动开始增加，因为的膜松弛可能是由于胞吐21（参见图3c）。 在图4a中，白色的方块表示的估计突起的接触面积A 轴突的文化支撑。甲轴突之间的紫外激光的光点位置的捕获探针的中心计算。 图4b报告公布的总释放的力F的幅度跟踪由各自的校准漂移校正的的QPD位移的痕迹（在图2b中）相乘后获得的光阱刚度（K Y，X，K KŽ）。我们计算力在时间t1和T0（ 图4b中ΔpN）的测量之间的差异，然后除以一个轴突 ，得到PN / 2微米的张力释放。 通过不同的神经元在样品上重复相同的协议，我们就可以开始用化学因素在一些文化中的实验会话或镀在不同的载体上，在不同的实验条件下得到的平均值，最后比较。在图5中 ，我们显示张力释放后轴突病变沾在基板上的焦点粘连，因此，较高的张力释放值意味着少一个轴突附着在基材上。因为我们诱导的部分，和不完整的，病变部位的轴突，我们研究了张力测量值的依赖性释放的总能量传递，上突起的病变部位和被困的胎圈之间的接触面积。我们试图摧毁更多的骨架元素，通过提供更高的光脉冲，从而导致较高的张力释放。否则，神经轴突接触面积增加，我们预期来衡量一个较低的张力释放。在图5中，我们证明了上述两个参数的依赖尚不清楚。因此，我们推断，诱导局部病变（与先前校准的每脉冲能量低，参照协议步骤4.5）的消融点8，其中不使用相同的显微镜的物镜和相同的能量时，改变的尺寸，是关系到每脉冲在采样。此外，张力释放的接触面积是不相关的事实是令人惊讶的，因为它是众所周知的，这样的参数，并不代表的细胞粘附到基板的一个很好的估计，粘着斑占据只有1％的基底面22。 <img alt="图1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50477/50477fig1highres.jpg" src="/files/ftp_upload/50477/50477fig1.jpg" /> 图1。激光干切断中断期间的骨架平衡的图形表示。细胞局灶性粘连基板坚持。蓝箭头Specify细胞骨架元素（通过弹簧）产生的牵引力。淡蓝色的箭头表示的反作用力所产生的刚性的培养基材。在一个简化的情况下，病变前，配对的骨架和基材部队和平衡。激光诱导的神经突的病变后，一些弹簧和基片之间的连接被破坏。因此，基片已不再是抵消牵引力的cytoskletal元素。被困的胎圈，附着膜，跟踪释放的细胞骨架力的方向。 <img alt="图2" fo:content-width="4in" fo:src="/files/ftp_upload/50477/50477fig2highres.jpg" src="/files/ftp_upload/50477/50477fig2.jpg" /> 图2。被困珠，激光诱导病变过程中的海马神经元的轴突膜。（一）明场干涉跟踪<html>采集的图像在消融过程中的轴突。前病变在左侧面板上。在右侧面板上的病变。甲聚-D-赖氨酸涂覆的胎圈连接到膜（polystirene珠，直径4微米），并保持在一个光阱。的样品的红外激光的平均功率为14 mW。白色箭头指示病变部位。酒吧是5微米（B）记录痕迹焦平面干涉珠在光学陷阱的位置。蓝色，绿色和红色的痕迹代表的胎圈的位置，分别沿x，y和z轴。取样频率为20千赫。（三）一个从无到有的文化支持视频跟踪测量的位移监测阶段漂移。蓝色和绿色的痕迹是x和y位置通过以下方式获得视频目标跟踪。帧速率为5.5赫兹。 <html> 77fig3.jpg"/&gt; 图3。 （一）损伤的轴突，在左侧面板上的亮场图像分析的张力释放过程中，膜应变损伤轴突轴突直径 。轴突垂直白线表示的位置在一个kymograph计算。在右侧面板中，kymograph的轴突直径，病灶部位的上游。的kymograph的每一行对应于0.18秒（二）较的张力释放期间估计的轴突直径kymograph每一行的像素强度的总和，（c）总方差的胎圈的布朗运动，附着在轴突（T0和T1分别表示在解剖后轴突释放紧张的开头和结尾）。 0477/50477fig4.jpg"/&gt; 图4。定量的张力释放后干切断（一）损伤轴突的明场图像。白色方块表示估计病变部位和被困珠的中心突起之间的接触面积（B）的总幅度微量干切断后测得的力（F TOT =√（FX 2 +风云2 + FZ 2））。外汇， Fy的，和Fz分别计算由Hooke定律（F = KX），其中的位移的痕迹是图2b中所示的那些。的刚度，在三个正交方向中，所计算出的功率谱的方法（K X，为y = 7.9 PN个/μm，K Z = PN 2.3个/μm）的光阱。 ΔpN表示时刻T0和T1的测力之间公布。 <img alt="图5" fo:content-width="4.5in" fo:sRC ="/ files/ftp_upload/50477/50477fig5highres.jpg"SRC ="/ files/ftp_upload/50477/50477fig5.jpg"/&gt; 图5。测得的张力释放的能量传递到样品量的依赖，并在的突起接触面积。3 DIV鼠标hippocamapal神经元的轴突从26个实验数据。（一）散点图张力释放与神经轴突接触面积（二）病变和被困珠位置。散点图张力释放的能量传递给样品轴突的接触面积与总金额。

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 ...

measurement-tension-release-during-laser-induced-axon-lesion-to

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K 次观看.  National Research Council of Italy. 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。

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

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

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

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

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

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 ...

科研

生物工程

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

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

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

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

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

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

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

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

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)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

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

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

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 ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

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

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

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

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10&#8211;60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

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 ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...