我们感谢Gijs Wuite，欧文JG彼得曼，彼得总的帮助，微流体和阿莱西娅Tempestini的帮助，样品制备。这项研究是根据资助协议资金由欧盟第七框架计划（FP7 / 2007-2013）N°284464和意大利外交部，教育，大学和研究外国投资审查委员会2011 RBAP11X42L006，清新汽油在Ricerca 2013 RBFR13V4M2，并在框架该旗舰项目NANOMAX。

1，光镊设置与纳米稳定性实验装置必须提供两个光镊在低于1％的捕集激光器的纳米级和强度波动指向稳定性。这些条件结合，将确保在典型的拉哑铃纳米稳定性（1 PN -几十PN的），陷阱刚度（0.1 PN /纳米）和测量带宽（图像采集速度为20秒-1）。该实验装置的方案， 如图1所示。 <ol><li>光镊设计和施工15,17： <ol><li>安装与激活隔离器的光学平台，实验设备，以减少机械振动。安装在弹性体隔振器的显微镜的结构来吸收噪声和显微镜结构的机械共振。 </li><li>插入一个光隔离器，在俘获激光的路径附近的激光源，于r得出的随机波动的幅度，由于光反馈。 </li><li>降低捕集激光尽可能的路径长度和包围在一个密封的盒子的整个路径，以减少由于空气流和湍流激光指点波动。 </li><li> （：YAG激光器，1064纳米波长的Nd）分为两束正交的偏振通过分割一个单一的近红外激光源产生双光镊。不要使用分时陷阱，因为它们所造成的哑铃振荡张力下12。 </li><li>使用由直接数字频率合成器（DDS中）驱动声光偏转器（1AOD及第2AOD）允许（至少）一个陷阱和DNA张力精确调节精细动作。 DDS中可以更好的陷阱稳定性比压控振荡器（VCO）。 </li><li>插入两个象限检测器光电二极管（QDPs）在冷凝器中的后焦面检测的捕获珠亚纳米精度的位置。 </li></ol></li><li>检查强度F.在显微镜入口俘获激光的luctuations是在1％以下通过使用光电二极管带带宽比图像采集的速率较大。 </li><li>检查两个捕获激光指向稳定性： <ol><li>制备硅珠在磷酸盐缓冲液的稀释：20微升的硅石珠粒（1.54微米，10％的固体）在1ml丙酮中，超声处理30秒，旋涡简单地说，离心2分钟，在19000×g下。悬浮在1毫升丙酮和重复洗。重悬在1ml的50mM磷酸缓冲液，洗2次，最后悬浮于100μl的50mM磷酸盐缓冲液中。 </li><li>校准光镊与硅珠：通过将盖玻片用双胶带（约60微米厚）载玻片制备流通池。流程1毫克/毫升BSA，并等待3分钟。流动硅珠稀释1 / 1,000磷酸盐缓冲液和密封流室与硅脂。在每个陷阱陷阱一胎圈和校准使用的功率谱法18陷阱。 </lI&gt; <li>制备硅珠在乙酸异戊酯及硝化纤维素：稀释20微升的硅石珠粒（1.54微米，10％的固体）在1ml丙酮中，超声处理30秒，旋涡简单地说，离心2分钟，在19000×g下。悬浮在1毫升丙酮和重复洗。重悬在1ml乙酸戊酯和洗涤2次。最后重悬他们在乙酸异戊酯的0.1％硝化纤维素溶解在乙酸异戊酯。 </li><li>差价2微升的使用第二盖玻片（24×60毫米）溶解在乙酸戊酯+ 0.1％硝酸纤维素到盖玻片（24 x 24毫米）的表面1.54微米直径的硅珠。通过使用两片双面胶带来创建一个流动池附着在盖玻片到显微镜载玻片上。填充流动池用50mM磷酸盐缓冲液和用硅酮油脂封住它。 </li><li>图片1硅珠在明显微镜在2000倍的放大倍率。视野应该是7×5微米，在768×576像素的收购，从而使图像珠的直径为190，第ixels。补偿热漂移与反馈软件，如在参考文献17中 ，计算出坐标位置的图像的质心和z的衍射环和校正漂移运动的压电阶段以纳米精度或更好。 </li><li>重叠左阱与胎圈中心的中心（从QDP在xy信号电平必须是相同的校准过程中测量的）和测量位置的噪声和其从QDP标准偏差。重复测量的正确陷阱。 </li></ol></li></ol> 2，单分子定位在纳米精度<ol><li>荧光显微镜的设计和施工15,19 <ol><li>使用高量子效率和电子倍增CCD来达到所需的纳米定位精度高的信号 - 噪声比。 </li><li>选择光学器件来获得的图像的像素尺寸〜80纳米（例如，图像放大率〜200X为16微米的照相机的像素大小）。这是sufficiently小到忽略了定位误差，由于像素化8，但足够大，收集了相当数量的每个像素的光子。 </li><li>作为激发源，可使用激光光是非偏振的（通过穿过非偏振保持光纤）或圆偏振（通过穿过λ/ 4波片），以最大限度地提高单一发色团的激发，而不管其方向如何。 </li><li>选择发射带通滤波器，可有效切断两者激发和捕捉激光灯。注：在我们的实验中，我们标记了蛋白质Atto532染料和使用集中在600nm与100nm的带宽的带通滤波器的排放。 </li></ol></li></ol> 3，微流体为了实现缓冲的交流与&#39;哑铃&#39;组装， 即单个DNA分子的两个光学微被困之间的固定，一个定制的层流多通道的精确控制系统必须开发。这是由液流腔，一个压力容器和压力控制系统（ 图2）组成。 <ol><li>流动池的准备 请注意：用于单分子实验的蛋白-DNA相互作用的流动腔室具有优选4的入口和1个出口，以允许多达4个并行缓冲流，每一个都含有实验所需的不同部件中的一个：球，DNA荧光标记的蛋白，并且成像缓冲器。在流动通道的组件的分离允许哑铃的高效装配。此外，成像通道是有用的，以避免背景荧光扩散的蛋白质，并获得所需的DNA结合蛋白具有几个纳米的数量级的精确定位的高的信号 - 噪声比。 <ol><li>钻头直径为1mm的孔，在显微镜载玻片（76×76×1毫米）用金刚石尖，以创建4个入口和一个出口。 </li><li>清洁日Ë显微镜载玻片，用乙醇和居中端口与O形环插入并在包围通道孔滑动的粘合环。它是基本在此步骤中要戴手套避免指纹，这会扰乱胶合处理。 </li><li>夹紧NanoPorts是在滑动，并将其放置在预热至180℃下进行1小时，以允许完全附着的烘箱。 </li><li>绘制流动池的轮廓图案在一张纸上。渠道应该是〜3mm宽和匹配的显微镜载片上的孔的位置。 </li><li>广场上的两片封口膜上方的绘图，并用手术刀做锐利和连续切削沿绘制轮廓。去除形成的任何突起。弃下封口膜层，它只是用来保证一个干净的支持。 </li><li>放置含所附NanoPorts是在金属支撑倒置显微镜载玻片。 </li><li>小心地将封口膜腔轮廓与孔，确保日在石蜡膜不管闲事腔室的入口和出口。 </li><li>盖上清洁显微镜的盖玻片（62 X 56 X0.15毫米），小心地取出围绕室内多余的封口膜。 </li><li>将两个热块，预先在120℃下加热，在流动池的顶部施加在其上恒定的压力。让封口膜融化约25分钟。 </li></ol></li><li>蓄压器和缓冲容器 蓄压应有机玻璃（或类似），并允许六个缓冲容器住宿。具有至少两个额外的缓冲容器的可能性提高了仪器的灵活性。注被困气泡的透明容器和管道大大有助于流动系统操作和故障排除。无菌且一次性鲁尔锁定尖端注射器（2.5ml）中，从该柱塞之前已经去除，是良好的缓冲容器中，并允许与所述入口管的连接方便。确保与蓄压器，必需以获得平滑且稳定的流动条件内的空气体积相比较的总流体体积是小的。 <ol><li>连接切断阀的缓冲容器，在实验过程中打开或关闭的缓冲流的出口。 </li><li>安装在显微镜舞台上的流动池。连接的截止阀的流动腔室进气口与聚醚管（内径〜150微米）和凸缘接头。加入〜3厘米氟化乙烯丙烯管（内径〜500微米）的管和流动池的NanoPort组件之间的连杆。这一步是必要的，以减少在腔室入口的缓冲液流，以避免在玻璃的流动池或NanoPort脱离的破损。使用大内径的管子单独，另一方面，将需要的生物样品的庞大体积。 </li><li>流室的出口连接到在大气压下的开放Falcon管中。这管用作处置流出的流动室的缓冲液中。 </li></ol></li><li>压力控制系统<ol><li>构建一个能够精细地调节压力储层内的空气压力的压力控制系统。这可以通过使用两个计算机控制的电磁阀，另一个连接到0.5个大气压的压力源（压缩机），第二个大气压力下进行。电磁阀反复打开约7毫秒，以增加或减少压力的线，直到所期望的值为止。注意：这个方法是改编自卡洛斯·布斯塔曼特20，并产生更平滑的流比使用步进电机注射泵21。 </li><li>通过添加自定义编写的软件控制的压力传感器来监控有效压力施加在蓄压器。典型的工作压力为20毫巴的哑铃组件和200毫巴用于清洗流动系统部件的数量级。 </li></ol></LI&gt; </ol> 4，尾矿的DNA与生物素标记的脱氧胞苷三磷酸（生物素的dCTP） 该DNA分子应长于1​​微米，以促进下流动和锚定到被困珠及其扩展。此外，长的DNA可以防止捕获红外光照射的DNA结合蛋白。这是一个特殊的意义，因为红外光的吸收，从激发生色结果增加了漂白。请注意：在本研究中，该DNA分子是3.7微米长。该分子包含的主要运营商O1一式三份，每两个辅助运营商，O2和O3的一个副本。这些序列被插入到该分子的中间，从而导致从捕获珠子和红外激光束大致等距离。 <ol><li>混合1皮摩尔线性DNA 3&#39;-末端与60皮摩尔生物素14的dCTP的，4微升的5X末端转移酶（TDT）缓冲液（1M氢CAcodylate，125毫摩尔Tris，0.05％（体积/体积）的Triton X-100，5mM的氯化钴（pH为7.2，在25℃））和1.5微升末端转移酶和DDH 2 O的以20微升的总体积。在37℃下将混合物15分钟。这将导致增加每脱氧核糖核酸末端可达60个核苷酸。请注意，DNA的3&#39;-突出端尾和更高的效率比凹或平端。 </li><li>纯化用离心柱或酚/氯仿抽提和随后的乙醇沉淀尾的DNA，从末端转移酶反应缓冲液除去氯化钴。 </li><li>纯化后，可将DNA贮存在4℃几周或在-20℃下的时间较长，但避免反复冻融和再冻结。 </li></ol> 5，标记蛋白质的巯基与ATTO532 通过半胱氨酸残基的修饰标签必须不改变蛋白质的活性。为了克服这个问题，我们使用了了LacI突变，LacIQ231C，whicħ携带只是每单体一个半胱氨酸的位置231。该突变体保留了野生型和化学修饰，在位置231的相同特征已被证明不与蛋白质稳定性22干涉。注：我们与ATTO532马来酰亚胺标记的蛋白质。 <ol><li>确保该蛋白溶解在缓冲液中的pH值范围从7.0至7.5，并用浓度为2〜10毫克/毫升。注意：在这些实验中，了LacI溶于磷酸钾0.3米，5％葡萄糖，pH为7.5（缓冲液A）。 </li><li>溶解三（2 -羧乙基）膦盐酸盐（TCEP）至100毫克/毫升的最终浓度在H 2 O。 TCEP的解决方案必须在使用前新鲜制作。 </li><li>混合100微升3μLTCEP和涡仔细的蛋白质。 </li><li>于N，N二甲基甲酰胺（DMF）中的染料溶解到10毫克/毫升的最终浓度在低光条件下工作。涡街至完全溶解。 </li><li>反应英里X：添加33微升染料与蛋白质+ TCEP和涡流小心。快速旋转，以收集尽可能多的解决方案成为可能。 </li><li>在20℃下孵育在摇床（转速8000rpm）的反应混合物中进行2小时（或在4℃CO / N），避光。 </li><li>溶解L-谷胱甘肽还原（GSH）至100毫克/毫升的最终浓度在H 2 O。加入3微升至反应混合物中，并孵育在摇床上以8000 rpm离心15分钟。 GSH将停止反应阻断的巯基的反应性。 </li><li>使用标准的凝胶过滤柱，透析或旋转浓缩纯化标记蛋白。请注意：在这些实验中，标记了LacI使用10kDa的截止旋转浓缩器进行纯化。 <ol><li>具有最多12个1ml缓冲液A的稀释该反应混合物，将其应用到集中器装置，和离心机它在8000 XG，1小时，4℃。因此，标记了LacI浓缩至约500微升的最终体积和过量的染料传递膜中的FLOW-通过。丢弃的流量通过，并重复步骤5.8.1至少三次。 </li></ol></li><li>划分标记蛋白成小分装并储存于-80℃。避免反复冻融。 </li></ol> 6，组合式光学捕获和单分子荧光成像实验<ol><li>加入1 ml缓冲液A到缓冲容器和应用200毫巴压力清洗连接管和流动池。最大化的结合蛋白对DNA，在这里和在以下步骤中扩散，缓冲液A可以被替换为低离子强度缓冲液中。注意：在这些实验中，我们使用TBE 0.5倍。 </li><li>检查是否有空气气泡的存在，这可能会扰乱流动的光滑度。在出口管轻轻一捺，将有助于消除任何残留的气泡。 </li><li>降低压力到20毫巴，并确认所有的通道和管是从气泡清楚，在所有信道相似的速度在缓冲流。 </li><li>准备样品300微升的最终体积：链亲和素包被聚苯乙烯珠1.87微米; 20日下午线性DNA，生物素化的根据第4两个四肢;根据第5条300分了LacI四聚体标记ATTO532。 </li><li>每个样品加入到注射器的容器中以下列顺序之一：在所述第一信道，DNA与成像缓冲器只（缓冲液A +氧清除剂系统），并在第四个信道的标记蛋白，第二，第三信道的珠子。 </li><li>转流上，在200毫巴下进行3分钟，让样品到达的流路内。然后将压力降低到20毫巴。 </li><li>而成像在明视场照明的第一信道，开关上的两个光学镊子和捕捉在每个阱的聚苯乙烯珠粒。 </li><li>快速移动到所述第二信道，以避免其他珠落入陷阱。注意，在到达该DNA信道是由珠流动的末端容易引人注目。 </li><li>使用邻NE AOD，移动一个陷阱（珠）来回于另一胎圈的附近，以允许流动延伸的DNA中的两个光学捕获的珠之间的连接。当形成的DNA哑铃，静态珠开始以下的积极来回移动的陷阱的胎圈的运动。 </li><li>快速移动到缓冲通道，并关闭缓冲液流。执行力-延伸曲线分析4,23-25 ​​验证单个DNA分子的存在。如果多于一个的分子存在（ 即焓，提高力-延伸曲线的相位发生在长度大于该DNA轮廓长度短），则丢弃该哑铃并从步骤6.7重新开始。 </li><li>一旦获得单个DNA哑铃，打开流量再次与移动哑铃的蛋白通道。切换到宽视场荧光显微镜监测标记-了LacI与DNA的相互作用。关闭流并开始采集。 </li></ol>

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The authors have nothing to disclose.

在过去的十年中，单分子操纵和成像技术已经看到在空间和时间分辨率方面很大的进步。的操纵和成像技术的组合是在的强大工具的基础，现在允许一个单一的生物聚合物的机械条件，如DNA，RNA或细胞骨架细丝的控制，和单一的蛋白质同时定位与同一聚合物相互作用。控制困聚合物的机械条件是特别感兴趣的。事实上，在活细胞中，核酸被连续经历引起相互作用的酶和蛋白质的机械应力;同样的，...

单分子（SM）的技术有了很大的发展，在过去三十年来克服一些传统的，本体溶液测量1-3的限制，需要作出回应。单一生物分子的操纵创造了机会，以测量生物聚合物4的机械性能和控制的蛋白-蛋白5和蛋白质-DNA相互作用6,7的机械参数。 SM荧光检测，另一方面，代表一个非常灵活的工具用于研究蛋白质的活性在体外和体内 ，导致定位和跟踪单分子与纳米精度的可能性。通过仪器的点扩散函数与SM图像的拟合，实际上，可以实现定位与精度主要取决于信号噪声比（SNR）和达到约一纳米8,9的限制。这些方法找到强大应用在马达蛋白的动力学的研究，以及对底层中的DNA结合蛋白的目标搜索的扩散过程。确定扩散常数为DNA序列上的目标函数，停留时间和精确地测量在一维扩散事件探索了DNA的长度的能力，代表一个功能强大的工具，蛋白质-DNA相互作用动力学的研究和调查的特定目标的搜索机制。 最近，这两种技术的结合，产生了新一代实验装置10-14实现同时操控的生物基质（如肌动蛋白丝或DNA分子）的相互作用伙伴酶检测/定位（例如肌凝蛋白或DNA结合蛋白）。这些技术的优点主要停留在施加机械控制被困聚合物的可能性，从而ENA金光闪闪的相互作用动力学与力或力矩的研究。此外，该方法允许从表面为止测量的生化反应，避免了经典的SM的方法， 即，需要对所研究的分子固定在表面上（玻璃载玻片或微球体）的主要限制之一。 两个单分子技术的结合需要克服几个技术难题，所产生的主要是从机械稳定性和足够的信噪比（SNR）（特别是当需要定位以纳米精度）15的要求。特别地，耦合SM荧光检测用光学镊子，噪声的降低时，并从捕获红外激光器16和生化缓冲液的生物复合物的装配和实验测量11的性能的控制漂白是最重要的。在这里，我们描述了方法，用于执行成功测量双套色/ SM荧光定位设置。该方法被示出与含有特定了LacI结合序列（ 例如，运营商）乳糖阻遏蛋白（了LacI）的荧光标记（用Atto532）和检测到的，因为它结合到DNA分子（两个光学镊子之间的残留）的例子。我们证明在检测了LacI的DNA和扩散约束力沿其在目标搜索过程中轮廓的方法的有效性。该方法适用于DNA序列和DNA结合蛋白中的任意组合，以及与其他系统（微管或肌动蛋白丝和马达蛋白与它们进行交互）。

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:203px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td style="width:151px;">Comments/Description</td>
			<td style="width:203px;">Web Address</td>
		</tr>
		<tr height="114">
			<td height="114" style="height:115px;">elastomeric isolators</td>
			<td style="width:141px;">Newport</td>
			<td style="width:167px;">Newdamp</td>
			<td style="width:151px;">Choose the appropriate Newdamp elastomer depending on the microscope weight and resonance frequencies</td>
			<td><a href="http://www.newport.com/">http://www.newport.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical isolator</td>
			<td style="width:141px;">Optics for Research</td>
			<td style="width:167px;">IO-3-YAG-VHP</td>
			<td></td>
			<td><a href="http://www.ofr.com/">http://www.ofr.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nd:YAG laser, 1064 nm wavelength</td>
			<td style="width:141px;">Spectra-Physics</td>
			<td style="width:167px;">Millennia IR</td>
			<td></td>
			<td><a href="http://www.newport.com/">http://www.newport.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acousto-optic deflectors (AODs)</td>
			<td style="width:141px;">A&#38;A optoelectronic</td>
			<td style="width:167px;">DTS-XY 250</td>
			<td></td>
			<td><a href="http://www.aaoptoelectronic.com/">http://www.aaoptoelectronic.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Direct Digital Synthesizers</td>
			<td style="width:141px;">Analog Devices</td>
			<td style="width:167px;"></td>
			<td></td>
			<td><a href="http://www.analog.com/">http://www.analog.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">quadrant detector photodiodes</td>
			<td style="width:141px;">OSI optoelectronics</td>
			<td style="width:167px;">SPOT-15-YAG</td>
			<td></td>
			<td><a href="http://www.osioptoelectronics.com/">http://www.osioptoelectronics.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">DIO and FPGA board</td>
			<td style="width:141px;">National Instruments</td>
			<td style="width:167px;">NI-PCI-7830R</td>
			<td></td>
			<td><a href="http://www.ni.com/">http://www.ni.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Halogen lamp</td>
			<td style="width:141px;">Schott</td>
			<td style="width:167px;">KL 1500 LCD</td>
			<td></td>
			<td><a href="http://www.schott.com/">http://www.schott.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Condenser</td>
			<td style="width:141px;">Olympus</td>
			<td style="width:167px;">U-AAC 1.4NA Aplanat Apchromat</td>
			<td></td>
			<td><a href="http://www.olympus-global.com/en/">http://www.olympus-global.com/en/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">Objective</td>
			<td style="width:141px;">Nikon</td>
			<td style="width:167px;">CFI Plan Apochromat 60x 1.2NA water immersion</td>
			<td></td>
			<td><a href="http://www.nikoninstruments.com/">http://www.nikoninstruments.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">532 nm laser</td>
			<td style="width:141px;">Coherent</td>
			<td style="width:167px;">Sapphire</td>
			<td></td>
			<td><a href="http://www.coherent.com/">http://www.coherent.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CCD 200X and 2000X</td>
			<td style="width:141px;">Hamamatsu&#160;</td>
			<td style="width:167px;">XC-ST70 CE</td>
			<td></td>
			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">electron-multiplied CCD</td>
			<td style="width:141px;">Hamamatsu&#160;</td>
			<td style="width:167px;">C9100-13</td>
			<td></td>
			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">piezo stage with nm-accuracy</td>
			<td style="width:141px;">Physik Instrumente</td>
			<td style="width:167px;">P-527.2CL&#160;</td>
			<td></td>
			<td><a href="http://www.physikinstrumente.com/">http://www.physikinstrumente.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Emission Filter</td>
			<td style="width:141px;">Chroma Technologies</td>
			<td style="width:167px;">600/100m</td>
			<td></td>
			<td><a href="http://www.chroma.com/">http://www.chroma.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silica beads (1.54 mm)</td>
			<td style="width:141px;">Bangs Laboratories</td>
			<td style="width:167px;">SS04N/5303</td>
			<td></td>
			<td><a href="http://www.bangslabs.com/">http://www.bangslabs.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum (BSA)</td>
			<td style="width:141px;">Sigma Aldrich</td>
			<td style="width:167px;">B4287</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">pentyl acetate&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">46022</td>
			<td style="width:151px;">Flammable liquid and vapour (No 1272/2008)</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">nitrocellulose</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">N8267-5EA</td>
			<td style="width:151px;">Flammable solid&#160; (No 1272/2008)</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">heat block</td>
			<td style="width:141px;">MPM Instruments Srl</td>
			<td style="width:167px;">M502-HBD</td>
			<td style="width:151px;">with 2 removable blocks; preheated at 120&#176; C</td>
			<td><a href="http://www.mpminstruments.com/">http://www.mpminstruments.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoPort assemblies</td>
			<td style="width:141px;">Upchurch Scientific Inc.</td>
			<td style="width:167px;">N-333</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">polyetheretherketone tubing&#160;</td>
			<td style="width:141px;">Upchurch Scientific Inc.</td>
			<td style="width:167px;">1535</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:203px;">home-made metallic holder for the assembly of the flow-chamber pressure reservoir made of Plexiglass</td>
			<td style="width:141px;"></td>
			<td style="width:167px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luer lock-tip syringes 2.5 mL</td>
			<td style="width:141px;">Terumo</td>
			<td style="width:167px;">SS 02LZ1</td>
			<td></td>
			<td><a href="http://www.terumomedical.com/">http://www.terumomedical.com</a></td>
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			<td height="42" style="height:42px;">shut-off valves&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">P-732</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
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			<td height="42" style="height:42px;">flangeless fittings&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">LT-111</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:203px;">fluorinated ethylene propylene tubing&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">1549</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:203px;">two computer-controlled solenoid valves</td>
			<td style="width:141px;">Clippard, Cincinnati, USA</td>
			<td style="width:167px;">ET-2-H-M5</td>
			<td></td>
			<td><a href="http://www.clippard.com/">http://www.clippard.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pressure transducer</td>
			<td style="width:141px;">Druck LTD</td>
			<td style="width:167px;">PTX 1400</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">biotin-14-dCTP&#160;</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:167px;">19518-018</td>
			<td></td>
			<td><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com/</a></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Terminal deoxynucleotidyl Transferase (TdT)</td>
			<td style="width:141px;">Thermoscientific</td>
			<td style="width:167px;">EP0161</td>
			<td></td>
			<td><a href="http://www.thermoscientificbio.com/">http://www.thermoscientificbio.com/</a></td>
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			<td height="21" style="height:21px;">ATTO532 maleimide</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">68499</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
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			<td height="157" style="height:157px;width:203px;">N,N-dimethylformamide (DMF)&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">227056</td>
			<td style="width:151px;">Combustible Liquid, Harmful by skin absorption., Irritant, Teratogen. H226; H303; H312; H316; H319; H331; H360; P201; P261; P280;P305; P351; P338; P311</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP)&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">C4706</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
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		<tr height="45">
			<td height="45" style="height:45px;width:203px;">L-Glutathione reduced (GSH)</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">G4251</td>
			<td style="width:151px;">Acute toxicity, Oral (Category 5), H303</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:203px;">Amicon Ultra-15, PLQK Ultracel-PL Membrane, 10&#160;kDa cutoff spin concentrators</td>
			<td style="width:141px;">Merck Millipore</td>
			<td style="width:167px;">UFC901024</td>
			<td></td>
			<td><a href="http://www.merckmillipore.it/">http://www.merckmillipore.it/</a></td>
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			<td height="42" style="height:42px;width:203px;">streptavidin-coated polystyrene beads 1,87 &#181;m</td>
			<td style="width:141px;">Spherotech, Inc.</td>
			<td style="width:167px;">SVP-15-5</td>
			<td></td>
			<td><a href="http://www.spherotech.com/">http://www.spherotech.com/</a></td>
		</tr>
	</tbody>
</table>

combining single-molecule manipulation and imaging for the study of protein-dna interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

在一个成功的实验中，一个（或多个）标记的蛋白质经受装订沿着DNA分子（ 图3A）/解除绑定和/或单维扩散。沿所述DNA分子的蛋白质的定位允许的动力学参数的量化的DNA序列的功能。当缓冲液条件引起的一维扩散被施加，所以能够追随蛋白的轨迹，并确定，例如，扩散系数D 1D。 单点的精确位置，在每一帧，可通过用二维高斯函数拟合的点扩散函数（PSF）?...

Watch this Scientific Journal Video about Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions at JoVE.com

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

在这里，我们描述了仪器和方法，用于检测单荧光标记的蛋白质分子与悬浮两层光学俘获微球之间的单个DNA分子的相互作用。

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

combining-single-molecule-manipulation-imaging-for-study-protein-dna

Research

JoVE Journal

Biology

11.5K 次观看.  University of Florence. 在这里，我们描述了仪器和方法，用于检测单荧光标记的蛋白质分子与悬浮两层光学俘获微球之间的单个DNA分子的相互作用。 

视频: Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

该芯片外型方法：确定蛋白质-DNA相互作用与近基部精密配对

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

自噬研究中蛋白质-蛋白质相互作用的研究

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

结合3D磁力致动器和多功能荧光成像来研究细胞核力学生物学

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...