This work was supported by the following funding sources: R01-AR063649, AG-020591, F31-AR035931.

所有涉及动物或人类受试者的程序应按照相关准则，法规和监管机构来执行。密歇根委员会对动物的使用和保养密歇根大学医学中心机构审查委员会的大学大学（UCUCA），并批准了所有的动物在本文中描述的人类手续。 1.解剖和存储解决方案股票注:在下面的指令中的最后一个卷可以放大或缩小的期望。 <ol><li>在一个1000毫升烧杯中，加800毫升去离子水（ASTM类型1）。保持温和搅拌，都在表1中列出的化合物添加到去离子水中，并允许他们溶解。 <table><tbody><tr><td> 复合 </td><td> 期望中的浓。 （M）的 </td><td> 式重量（g / mol）的 </td><td> 加至1升（克） </td> &lt;/ TR&gt; <tr><td> K-丙酸</td><td> 0.250 </td><td> 112.17 </td><td> 28.040 </td></tr><tr><td>咪唑</td><td> 0.040 </td><td> 68.08 </td><td> 2.720 </td></tr><tr><td> EGTA </td><td> 0.010 </td><td> 380.40 </td><td> 3.800 </td></tr><tr><td>氯化镁2•6H 2 O </td><td> 0.004 </td><td> 203.31 </td><td> 0.813 </td></tr></tbody></table></li></ol> 表1:解剖和存储原液组件。 <ol start="2"><li>带来至1000毫升去离子水的最终体积。注意，这是不必要至pH的溶液在这个时候。储存原液于4℃。 </li></ol> 2.存储解决方案<ol><li>为了使将200ml的存储解决方案，开始用100毫升解剖和储存原液在250毫升烧杯中。添加足够的Na 2 H 2的ATP带来最终腺苷三磷酸（ATP）的浓度为2毫摩尔。带来至200毫升与甘油的最终体积。调节至pH 7.00与氢氧化钾（KOH）。储存在-20℃下贮存溶液。 注意:由于甘油的粘性性质，它可能难以准确地按体积分配。由于这个原因，我们一般按重量计添加甘油（100毫升的丙三醇的重量大约为126克）。 </li></ol> 3.解剖解决方案<ol><li>使200毫升解剖溶液，开始用100毫升的解剖和储存原液在250毫升烧杯中。 </li><li>添加足够的Na 2 H 2的ATP使最终ATP浓度为2毫摩尔。调节pH值至7.00用KOH。使终体积为200毫升去离子水。等分试样中以2.5ml体积并储存在-80℃。 </li></ol> 4.进行剖析与解决方案58的Brij 注意:的Brij 58是一种非离子型洗涤剂是破坏（permeabilizes）脂质双层。 &lt;OL&gt; <li>使200毫升解剖用的Brij 58的溶液，开始用200毫升中的250毫升烧杯中的解剖溶液。保持温和搅拌，添加1克的Brij 58（0.5％重量/体积）的解剖溶液，并允许它以溶解。等分试样中以2.5ml体积并储存在-80℃。 </li></ol> 5.测试解决方案注:以下是改编自Moisescu和Thieleczek 1978年（6）。见讨论有关准备测试解决方案的补充意见。 <ol><li>制备三个独立千毫升烧杯标记为"放松"，"预活化"和"激活"。加入400毫升去离子水到各烧杯中。 </li><li>在表2所示的化合物添加到适当的烧杯中，然后加热该溶液至之间70℃和80℃。保持70-80℃的溶液温度下进行30分钟，同时连续搅拌。 注:70-80℃助攻的ELIMI温度碳酸的国家由碳酸钙用EGTA在活化溶液中的反应而形成的。放松溶液和预活化溶液处理以相同的方式作为活化溶液，以保持一致性。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2" style="border:0px #fff solid"></td><td colspan="2"> RELAXING解决方案 </td><td colspan="2"> PRE-活性溶液 </td><td colspan="2"> 活化溶液 </td></tr><tr><td>复合</td><td>式重量（g / mol）的</td><td>所需浓度（mM） </td><td>必需的海量（G） </td><td>所需浓度（mM） </td><td>必需的海量（G） </td><td>所需浓度（mM） </td><td>必需的海量（G） </td></tr><tr><td> HEPES（酸） </td><td> 238.30 </td><td> 90.0 </td><td> 107.24 </td><td> 90.0 </td><td> 107.24 </td><td> 90.00 </td><td> 107.24 </td></tr><tr><td> 氧化镁 </td><td> 40.31 </td><td> 10.3 </td><td> 0.208 </td><td> 8.5 </td><td> 0.171 </td><td> 8.12 </td><td> 0.164 </td></tr><tr><td> EGTA（酸） </td><td> 380.40 </td><td> 52.0 </td><td> 9.890 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td> 52.00 </td><td> 9.890 </td></tr><tr><td> HDTA（酸） </td><td> 348.36 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td> 50.0 </td><td> 8.709 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td></tr><tr><td> 碳酸钙 </td><td> 100.10 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td> 50.00 </td><td> 2.503 </td></tr></tbody></table> 表2:重新laxing，预活化和活化溶液组成。 <ol start="3"><li>冷却该溶液至室温，并添加足够的NaN 3 / KOH，使其终NaN 3的浓度为1mM。 注意:NaN 3（和叠氮化钠）是有毒的。请参阅之前处理这种化学物质的化学MSDS。 <ol><li>至100ml 100mM的叠氮化钠溶液中，添加0.65克的NaN 3至10ml的1N KOH。调整至100ml去离子水的最终体积。 </li></ol></li><li>将pH调节至约7.10使用的KOH。 </li><li>以下步骤5.4添加足够的Na 2 H 2的ATP，使最终ATP浓度至8毫和足够的Na 2 CRP以使终肌酸phosophate（CRP）浓度至10mM。 </li><li>带来每种溶液的使用去离子水500毫升最终体积。寒意或加热溶液到在该实验将执行的温度，然后用氢氧化钾使pH至7.10，同时保持该温度。 </li><li>添加放松溶液至包含预活化溶液的烧杯，使得最终的预活化溶液为1份放松在500预活化溶液溶液。等分试样中以2.5ml体积并储存在-80℃。 </li></ol> 6.进行缝合循环<ol><li>开始的非无菌的USP 10-0单线尼龙缝合线料。 </li><li>使用镊子来创建与使用双上手结技术的链环。减少大小的结约750微米的直径。环直径可在显微镜下用目镜刻度标记进行评估。 </li><li>使用microdissecting剪刀以除去多余的缝线只留下在任一侧的环和小的（500微米）的尾巴。的成品环的例子示于图1。 </li><li>直到6.2-6.3可用4回路已重复步骤。商店循环在硅氧烷弹性体镀彼得我菜供将来使用。 注意:四缝合环所需要的每根纤维进行测试。 </li></ol> 7.束样本注意:下面的步骤描述的过程解剖原始样本成更小的实验&#39;捆&#39;从其中单纤维将最终被提取和测试。在任何时候都应该样品小心对待。对于这个描述的目的，说明将给出如果研究者是右撇子。 <ol><li>获得的感兴趣的样品，并将其转移到设备，在夹层将发生。 注:组织活检的方法将根据实验模型和研究设计有所不同。在可能的情况，肌肉灌注应保持，直到活检时。 </li><li>如果样品是收获站点和解剖部位之间传送，传送它在含有冷冻解剖溶液中，同时保持在冰上的小瓶。</li><li>准备5cm的硅弹性体镀培养皿冷冻解剖溶液和两到三个昆虫安装管脚（直径100微米，不锈钢）。 </li><li>样品转移到菜。确保样品仍浸没通过增加更多的解剖解决方案，如果有必要的。 </li><li>检查在显微镜下的样品和操纵它对齐的纤维的纵向轴线朝向所述研究者（ 图2）的右肩。然后，通过钉扎在角锚定样品的菜。 注意:请使用任何剩余的结缔组织如在这个时候，因为这将最大限度地提高样品的使用和保护纤维的完整性锚固点。束可以固定在小的张力，以帮助限定纤维间边距。 </li><li>与在左手镊子和在右边的microdissecting剪刀，开始沿纤维之间的纵向边缘轻轻解剖束 注:根据其总长度，进一步剖析捆绑成无数个较小的包。 </li><li>确保束尺寸测量约0.5-1毫米宽和≥3毫米长。 ，或者将一个统治者名下的菜评估使用在目镜刻度标记显微镜的尺寸。 </li><li>取下并丢弃任何由镊子或销作为解剖过程的结果受损组织。 </li><li>重复该过程，直到束的足够数量的被解剖或直到样品已经用完。 注意:可以得到将取决于很多因素，包括大小和初始样品的情况下，肌肉的形态，和研究者的技能束的数量。 </li></ol> 8.纤维透<ol><li>转移从解剖溶液束到含有2.5新鲜，冷冻，用非离子型洗涤剂"的Brij 58&#39;解剖溶液的小瓶中加入（0.5％，重量/体积）。在冰上孵育30分钟，偶尔，轻轻摇动。确保束保持整个孵化淹没。 </li><li>在30分钟温育结束时，捆转移到含有新鲜解剖溶液（无的Brij 58）的小瓶中，并轻轻搅动，并简要以除去任何残留的洗涤剂。 </li></ol> 9.准备捆绑的存储<ol><li>转移捆含冷藏存储解决方案小瓶和孵化一夜之间在4℃。 </li></ol> 10.商店捆绑<ol><li>第二天，准备一个收纳盒，能够承受-80℃，有足够的个人0.5毫升旋盖锥形管，以适应在解剖过程中（每管一个捆绑）获得的所有包。每个锥形管应充满200-400微升新鲜的存储解决方案。 </li><li>转移捆绑成单独标记锥形管。确保束不坚持锥形管或浮在溶液表面上的一侧。盖住锥形管和储存样品在-80℃直至测试日。 </li></ol> 11.准备实验装置注:自定义装置由容纳长度控制器和力传感器，运动室系统和10X解剖显微镜的一个阶段。千分尺驱动装置允许精准操控的纤维附着表面。激光衍射图案被用于估计小节的长度。实验过程中所产生的数据被记录在个人计算机上。参考图3，对本实验的建立注释的图像。 <ol><li>每个解冻放松，预激活和激活解决方案之一小瓶和冰上维护。需要注意的是ATP和CRP是应保持在低温下不稳定的化合物。 </li><li>准备显微镜，测试设备和相关联的计算机使用。 </li&gt; <li>填写第一个试验室放松的解决方案。在我们的装置中，第一腔室含有棱镜允许研究者拍摄从侧光纤。填第二试验室具有预活化溶液和第三与活化溶液。 </li><li>调节温度，以使在腔温度计显示读数15℃。螺纹两人准备缝合环上同时从力传感器和长度控制器（ 图5A）延伸的不锈钢连接表面。 </li></ol> 12.提取透单纤<ol><li>解冻感兴趣的纤维束，并转移到有机硅弹性体镀培养皿中的新鲜，冰鲜轻松的解决方案。固定捆绑销在任一端，并确保它被浸没。 </li><li>使用镊子，把握光纤在一端和顺利开始沿其纵轴提取它。 注:为压缩破坏引起的镊子纤维到底是不是在此时，因为在这方面的收缩特性不会测试一个关注的问题。护理应，但是，可以提取束的纤维时，由于纤维与细胞外基质之间的粘连会导致过度的张力，最终导致拉伸诱导的损伤采取。需要注意的是相当大的可变性存在于程度肌肉其中纤维粘附到周围的细胞外基质中。被怀疑已损坏由这样的拉伸的纤维应被丢弃。 </li><li>用剃刀刀片或手术刀来修改一个吸管尖所示（ 图4）。光纤引入连同少量放松溶液的尖端。从硅氧烷弹性体镀培养皿的单纤维转移到试验室中，其中包含了轻松的溶液。 </li></ol> 13.安装单纤维注:一步一步depicti上可以在图5中看到。 <ol><li>用镊子轻轻引导，从吸管尖取出​​纤维，并将其固定的长度控制器（左）使用第一缝合线环。 <ol><li>紧缩与镊子循环时使用一个单一的，平滑的运动。确保相等且相反的张力被施加到缝线的任一端。 </li><li>第一环路应从长度控制器安装面的端部捆绑约1至2mm。 </li></ol></li><li>操纵朝向的力换能器的光纤的另一端（右），并使用相同的方法固定在纤维上。使用microdissecting剪刀（ 图5C）删除多余的缝线。 </li><li>通过增加使用的x坐标微米驱动器（ 图3A）的长度控制器和力传感器臂之间的距离放置下少量张力的纤维。 </li><li>螺纹的第二环在第一和锚定纤维在在0.2毫米的力换能器安装面（ 图5D）的端部的一个点。 <ol><li>使用microdissecting剪刀去除多余的缝线。 </li></ol></li><li>纤维附着过程可导致从腔室的溶液的损失。如果必要的话，加入更多的放松溶液以确保溶液的表面是平坦的（既不凹也不凸）。评估采用激光衍射小节的长度，当一个平坦的表面是非常重要的。 </li><li>通过调整在y轴方向上的长度控制器的位置对齐平行于试验室的侧壁上的纤维。 </li><li>使用棱镜侧视图勘测纤维和调整长度控制器在z轴的方向上的位置，直到该纤维是平行于腔室的地板上。 注:定位平行于腔室的地板上的纤维可以不室棱镜由第一聚焦在纤维和叔一端来实现母鸡，不调整显微镜聚焦，利用其z轴千分尺驱动使光纤的另一端成为焦点。 </li><li>如果纤维是以任何方式扭曲，扭曲或损坏作为安装过程的结果，该纤维应当被丢弃，一个新的光纤连接的。 </li></ol> 14.设置最佳小节的长度<ol><li>当纤维已在腔室中被正确地对准，插入校准目标屏幕到显微镜的前方面和对齐在第一实验室。 注意:目标屏是采用标准的光栅方程校准，λ= SLSINθ，其中SL是小节的长度，θ为0°和1°衍射光束和λ之间的衍射角是激光的波长。 </li><li>打开激光器和调整，使得激光通过光纤的中心的舞台的位置。 注意:浓缩的激光光可以破坏牛逼Ø视力。不要试图通过显微镜以可视化的光纤时，激光器上。 </li><li>相对于光纤定位到激光束衍射激光和观察的干涉图案的校准目标屏幕上（ 图6）。关灯更清晰地显现了这种模式。 注意:如果，用激光束的正确定位，没有干涉图案可以看到的，这表明，在纤维的肌原纤维组件是异常/损坏的，而且纤维应更换用新鲜。 </li><li>要设置小节的长度，增加或减少有关使用长度控制器x轴千分尺驱动器，直至衍射光的期望间距目标屏幕上观察到的纤维的张力。 注:纤维的最佳小节的长度将依赖于动物的从其获得样品的物种。 2.7微米的肌节长度一般认为驴的时候是最佳唱从人体组织7,8的纤维。 </li><li>后最佳小节的长度已定，测量两个最内缝线之间的距离。这是通过使用其控制腔室的x轴运动的微米驱动器上的数字读出最容易完成。定位腔，使得所述目镜的垂直横毛发上最里面的缝合线的最内边界对齐和置零微米驱动器上的数字读出。 </li><li>平移沿x轴相对于所述显微镜的阶段，直到到达另一最内缝合。数字显示将指示所述光纤的长度。此值应记录为纤维长度L F。 注意:应该理解的是，这两个最内缝线之间的距离将决定收缩组织的功能长度被评估。研究者应当争取在一个在这方面的一致性（ 即 L F）一系列的实验。 </li></ol> 15.估计截面面积（CSA）的<ol><li>保持光纤为 L f和使用显微镜载相机来同时从顶视图和侧视图捕获纤维的中央部分的高放大倍率的图像。侧视图的图片可使用嵌入在腔室的一侧的棱镜被捕获。 注意:当顶视图和侧视图之间移动来移动所述显微镜仅在y方向上，以确保这两个图像是"寄存器"，因此显示了纤维的同一部分的两个不同的观点是重要的。 </li><li>获得的测量此时在研究后正常化的绝对力。该技术用于获得这些测量后述和在图7A和7B所示。 </li></ol> 16.引导学生回答等长收缩注意:虽然这些实验过程中所产生的数据之间可以收集和解释，而无需使用一台计算机，软件，允许对力的响应的获取，显示，存储和分析是有利的。由我们实验室建立的定制LabVIEW软件允许这些功能，以及有能力设计&#39;运动列车"支配的实验期间的长度控制器的动作。 <ol><li>确认放松，预活化的温度和激活溶液是稳定在15℃。 </li><li>使用腔室控制软件对纤维移动到包含预活化溶液的腔室，并培育有3分钟。 注意:预活化溶液是弱缓冲以供的Ca 2+，导致引入光纤到活化溶液中时非常迅速激活和力的发展。 </li><li>用10秒中剩余的预活化溶液，并保持为L f显示纤维长度，建立零FO在实验记录RCE水平。 注:长度控制器的"查找强制零&#39;运动表明对应于零力的力换能器的水平（ 即纤维简要地变得松弛作为移动的结果）。被动力是零，并且力水平之前到换能器调零运动之间的差异。 </li><li>在3分钟的末尾，将纤维以含有所述活化溶液的腔室，并允许最大等距力发展就证明了在力的高原是前面有一个快速上升。 </li><li>达到最大等长收缩力后，使用长度控制器，以识别对应于零力在含有活化溶液腔室中的力换能器的输出。 注意:这是必要的，因为这对应于零力的力传感器的输出是，在一般情况下，对于每个溶液填充腔的不同。 </li><li>继实现第二个力PLateau，返回该纤维含有该放松溶液的腔室。测试完成。任何一个会话期间吸测试多种纤维所有的解决方案，并添加新的，冷冻的解决方案。 注:以上的长时间引发宫缩最大布伦纳时的循环程序应予以考虑。此协议已被证明在最大限度活化纤维9保存结构和机械性能。 </li></ol>

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的透单骨骼肌纤维的收缩特性评估用在各种各样的环境中，调查肌肉功能。例子包括已老化评估12的影响的研究，锻炼10,13,14，航天15，伤害2,3,16，药物治疗17,18，疾病和19对纤维的结构和功能基因操纵20,21。由于直接评估其天然构肌原纤维的收缩性能的能力，这种技术提供了从中形成潜在的混杂影响是存在的肌原纤维功能缺失的理解，一个有吸引力的平台时，神经肌肉信号传输和激励引起的钙释放被包括在所研究的系统。另外，单纤维的功能性测试可用于补充收缩蛋白鉴定结果如通过免疫组织化学或凝胶电泳+免疫印迹获得22。 之一的骨骼肌的主要功能是产生力。因此的sF O，一个收缩系统的内在动力发电能力的措施，是非常感兴趣的肌肉生理学家。的sF 邻可靠的估计需要两个纤维CSA和FØ准确的措施。因为纤维是，在一般情况下，在横截面中在沿其长度的CSA既不圆形，也不均匀，非常小心，应推定的CSA时服用。为此，测量是在沿光纤的长度的几个位置制成，并在每个位置，从两个角度90°分开。 ˚F 邻可靠的措施，需要注意几个细节，包括占被动力，调整肌节的长度，最大限度地厚，薄丝重叠，采用与钙浓度的测定活化液帽子结果在最大活化，保持所需实验温度，并保持现有的实验当天纤维的最佳储存条件（温度和时间）。 而此处列出的步骤描述了用于评估最大等长收缩力的方法，它是经常需要评估的骨骼肌纤维其它重要的功能特性。这可以通过延伸实验方案以包括纤维的额外的机械操作来实现。例如，在其中纤维缩短针对一系列不同负载的速度的测量允许确定的力-速度关系，从中力功率和速度-功率关系可以计算10,23,24。此外，卸载的缩短的速度可以通过采用"松弛测试"25，其由应用一系列松弛诱导缩短的步骤和measuri来确定纳克由光纤所需要的时间，以消除松弛。即经常报告另一个动力学参数k 潮流 ，以下的机械扰动暂时分离所有的速率常数力重建crossbridges 26。最后，钙离子浓度和活性力产生（"强制-PCA关系"）之间的关系是感兴趣18常，并且可以通过使纤维的一系列与钙浓度范围从低于所述阈值用于激活收缩溶液来确定系统对那些足以引起最大活化，因此最大的力（F O）。 虽然大部分提到设备的需要，以评估单纤维的收缩，其他设备不是绝对必要的。长度控制器，例如，为任何实验方案，需要快速或准确加长纤维或缩短必要，但不是评估最大等长收缩力（虽然在力创纪录零水平力仍然必须通过某种手段来识别）绝对必要的。允许观察从侧面的纤维，而评估横截面积有用的棱镜，实验室中定位的纤维时并不是绝对必要的。此外，另外的装置，用于暴露纤维的各种实验方案可以采用，其中包括制定一个手工操作的腔室或单腔室，其允许快速填充系统和排空的解决方案。最后，虽然子生理实验的温度如15℃下通常用于改善机械测量1,2,3,5,8,12,17,27的再现性，就可以产生在其他温度23有效数据，28只要温度对溶液性质（钙浓度，pH值等 ）的影响考虑在内。 </P&gt; 测试溶液的组合物中的这里所描述的通透纤维技术的最关键的方面。关于解决方案组成的考虑很复杂，超出了本文的范围。在协议部分的步骤5中描述的解决方案的设计重点放在了透化纤维在从预激活到激活解决方案，同时保持恒定的离子强度，阳离子组合物，和摩尔渗透压浓度6,29其传输快速活化。其他的方法来解决成分已受聘具有显着的成功由其他研究小组，并且通常利用出版结合常数和计算工具27,30,31的。钙离子在各种活化溶液中的浓度是在涉及次最大活化如力-PCA评价研究尤为重要。对于实验，其中纤维被完全激活，例如那些描述ð这里，在活化溶液中的钙浓度通常超过由一个舒适的余量，需要以达到最大的力，使得它的精确知识不那么重要。另外磷酸肌酸是用于缓冲，否则将与收缩活动相关联的intramyofibrillar的ATP和ADP的波动很重要的。肌酸激酶是需要从催化的磷酸肌酸磷酸转移到ADP。根据该导致高的ATP周转率，包括在高温下工作或测量高速缩短在快纤维32的实验条件下，肌酸激酶必须加入到该溶液中，以补充其保持结合到纤维中的内源性肌酸激酶。对于要求较低的实验条件下，ATP的再生系统是不太关键27。 的渗透性作用单纤维技术的局限性包括以下内容。通过这些试验产生的数据定义附于实验装置，该特定的肌原纤维单元的收缩性能。因此，该捕获来自该段得到，这反过来，代表了肌肉内纤维的总数的一小部分，整个多核光纤的仅一小部分。研究者因此应该仔细考虑支持从实验中得出的任何结论所需要的采样。此外，评估对纤维功能的运动训练干预的影响假定评估纤维在培训期间确实招募。虽然该协议试图模仿纤维的天然细胞内环境，肌膜透过程是非特异性的，必然允许可溶性胞内组分，以自由地扩散到洗澡的解决方案。细胞膜的通透性的另一后果是渗透平衡由肿胀纤维体积33证据发生变化。该纤维肿胀增加导致肌丝系统34,35的减少钙敏感性的肌动蛋白和肌球蛋白纤丝之间的距离，但可以通过引进大，渗透活性化合物34逆转。最后限制要考虑的是用于将纤维附着到实验装置的技术的结果。这总是需要在附近的附着点歪曲丝系统内部的空间关系，与主治功能缺损。具体而言，纤维在和相邻的连接点的区域在功能上受到影响，从而有助于人为的系列弹性的测量系统。 总之，我们已经描述了通过该评估体外化学透骨骼肌纤维的力产生能力的装置。尽管本文的重点是最大等长收缩力generatin评估人骨骼肌纤维的克容量，实验方法可以修改和扩展，以确定在一系列物种，哺乳动物或其他的各种动力学参数和关系。

骨骼肌的主要功能是产生力。肌肉力通过事件的复序列，其中包括运动神经动作电位，神经肌肉传递，肌肉纤维的动作电位，细胞内钙的释放，活化的监管和收缩蛋白的系统引起体内 。因为力产生是该序列的最终结果是，在力的赤字可以由一个单独的步骤的一个或多个的故障引起的。透化纤维制备的关键属性是它消除了大部分的体内所需力产生的步骤，以仅与肌原纤维装置其余相关的法规和收缩功能。研究者假定控制激活钙和能量（ATP）的交付，并奖励了简化系统，使孤立的监管和收缩的结构评估其天然的合作nfiguration。评估体内观察到的肌肉功能的改变，当使用透骨骼肌纤维的力测量因此价值。例如，我们已经使用此技术来纤维的力发电量从氯化筒箭毒碱缺陷小鼠1表征并评估持续肌无力表现出以下慢性肩袖撕裂2,3的原因。 现代透纤维的方法，可以追溯到早期有影响力的研究和4,5是当前正在使用由多个研究小组。虽然该技术已在文献中进行了描述，它们尚未被呈现在视频格式。本文的目的是要说明一个更新，有效和可靠的技术，用于测量单纤维从化学透化骨骼肌样品的最大的力的发电量。为了做到这一点，一个单个纤维段（在此被称为一个̶0;纤维"）是从纤维的预通透束抽出并置于含有一个轻松溶液的实验腔室中，限定功能，它是一种钙离子浓度即&lt;10纳米。然后使光纤在一端连接到一个力传感器，并在另一端连接到一个长度控制器。与在最佳小节的长度保持的纤维，将其转移到活化溶液，其具有钙浓度足以引起最大活化，从而最大等长收缩力。力数据采集，储存和使用个人计算机进行分析。

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			<td>Polystyrene culture test tube with cap</td>
			<td>Fisher Scientific&#160;</td>
			<td>14-956-3D</td>
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			<td>Fisher Scientific&#160;</td>
			<td>02-681-334</td>
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			<td>0.5 mL microcentrifuge caps with o-ring</td>
			<td>Fisher Scientific&#160;</td>
			<td>02-681-358</td>
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			<td>Fisher Scientific&#160;</td>
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			<td>Fine Science Tools</td>
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			<td>Microdissecting scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
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			<td>Leica Microsystems</td>
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			<td>Parker Hannifin</td>
			<td>3936M</td>
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			<td>Physitemp</td>
			<td>BAT-12</td>
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			<td>Aplha Omega Instruments</td>
			<td>Series 800</td>
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			<td>National Instruments</td>
			<td>-</td>
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			<td>Varied</td>
			<td>-</td>
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			<td>Aurora Scientific</td>
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			<td>Aurora Scientific</td>
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			<td>Aurora Scientific</td>
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			<td>TCI America</td>
			<td>P0510</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
			<td>A7699</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
			<td>529699</td>
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			<td>TCI America</td>
			<td>D2019</td>
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			<td>Sigma-Aldrich</td>
			<td>C4830</td>
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			<td>Sigma-Aldrich</td>
			<td>S8032</td>
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			<td>Sigma-Aldrich</td>
			<td>35113</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
			<td>P7936</td>
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			<td>Fisher Scientific</td>
			<td>SB115</td>
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			<td>pH 7 standard</td>
			<td>Fisher Scientific</td>
			<td>SB107</td>
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measurement of maximum isometric force generated by permeabilized skeletal muscle fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

健康，透化学纤维单丝应该出现均匀的形状，并具有一致的条纹间距在高倍率下观察时。当纤维用钳子操纵是不灵活或有明显的结构性损坏应丢弃。 在步骤15采取高倍率数字图像进行5成对直径测量沿光纤的中部进行分析。估计纤维CSA假设椭圆形横截面，平均5个独立的CSA测量值作为图7A的所示 。 图7B还用于说明如何在一个视图纤维尺寸与在其它视图（即，交叉配对尺寸相比可以显著不同部分都没有，在一般情况下，圆形）。 从人类慢和快纤维代表力迹线示于图8A和8B分别。 Voltag在测试过程中被采集和转换用数据采集和分析软件（LabVIEW中），以强制（MN）的力换能器电子输出。 图9示出了用于评估最大活性力（F o）就方法，它是通过减去计算出的以保持纤维在最佳小节的长度所需要的力，而在松弛状态（无源力F P），从在最大纤维活化开发的最大等长收缩力（总力F T）。由于力传感器，对应于零力的输出，在一般情况下，对于每一个不同的沐浴室不同，我们简要放松光纤在两个预活化和活化的解决方案来捕获在零力水平实验记录。由纤维CSA最大的主动力正常化是用来生成比力（SF O）的更多的信息价值。因为它考虑到光纤的CSA，顺丰Ø </s的ub&gt;提供了纤维的收缩装置的固有的力发电能力的量度，从而允许不同尺寸的纤维之间的功能比较。它应该然而，应当注意，CSA测量不能够区分由收缩长丝与由其他亚细胞结构所占的比例占据的纤维的比例。 的健康典型特征，克拉夫林等2011 10为人类，Mendias 等2011 1从成年纤维鼠标和Gumucio 等人 2012 2大鼠都在表3中详细描述。在表3中的所有数据使用生成在这篇文章中描述的技术。 <table border="0" cellpadding="0" cellspacing="0" style="text-align:center"><tbody><tr><td rowspan="3" style="border:0px #fff solid"></td><td colspan="4"> 人的 （股外侧） </strong&gt;的</td><td> 鼠标 （EDL） </td><td nowrap="nowrap"> 鼠 （冈） </td></tr><tr><td colspan="2">男</td><td colspan="2">女</td><td>男</td><td>男</td></tr><tr><td>类型1 </td><td> 2A型</td><td>类型1 </td><td> 2A型</td><td> （没有输入） </td><td> （没有输入） </td></tr><tr><td> CSA（微米2） </td><td> 4880 - 6900 </td><td> 5270 - 8380 </td><td> 3870 - 5470 </td><td> 4010 - 5610 </td><td> 1850 - 3080 </td><td> 5290 - 8010 </td></tr><tr><td> ˚FO（MN） </td><td> 0.79 - 1.17 </td><td> 1.02 - 1.54 </td><td> 0.64 - 0.97 </td><td> 0.71 - 1.07 </td><td> 0.14 - 0.25 </td><td> 0.55 - 0.97 </td></tr><tr><td> 的sF O（千帕） </td><td> 142 - 182 </td><td> 165 - 210 </td><td> 156 - 193 </td><td> 172 - 214 </td><td> 67 - 94 </td><td> 102 - 131 </td></tr><tr><td> ñ </td><td> 129 </td><td> 160 </td><td> 149 </td><td> 207 </td><td> 37 </td><td> 94 </td></tr></tbody></table> 表3.典型的人类股健康成年纤维的特性外侧10，鼠标趾长伸肌1和大鼠冈2肌肉。肌节的最佳长度分别设定为2.7微米的纤维人类7,8,9 2.5微米的两个鼠标（36， 37）和大鼠纤维（38）。实验L˚F范围（25 次和第 75 个四分位数）为1.39-1.73毫米，1.17-1.53 ​​毫米，1.32-1.59毫米，用于分别人类，小鼠和大鼠。所示范围表示第 25和第 75位数和n是纤维测试的次数。 在测试过程中遇到的最常见的问题包括一个缝合线套环滑动，这导致了力response为一个"抓"，如在图10A中示出，并且该纤维，这导致力响应返回突然朝向或到（中断）的部分或全部厚度的撕裂零而纤维仍浸在活化溶液（ 图10B）。如果打滑，撕裂或断裂的实验过程中发生，该纤维应当被丢弃，数据排除在外，虽然保持了光纤故障的记录也可以是信息11。另一个负面结果可能遇到是纤维的过早活化，而 ​​在预活化的溶液（ 图10C）。部分活化在预活化溶液表明显著交叉以及污染（ 即，活化的预孔的无意增加钙离子浓度的）。在这种情况下，所有的缸应吸出并用离子交换水充分漂洗。干燥室之间的分隔表面也recommenDED湿气或冷凝在这些领域可能会导致排汗浴场之间的解决方案。该决定包括或排除数据最终将取决于实验的重点和设计研究时，因此应该予以考虑。 <img alt="图1" src="/files/ftp_upload/52695/52695fig1.jpg" /> 图1:缝合环（10-0单线尼龙缝线）。 <img alt="图2" src="/files/ftp_upload/52695/52695fig2.jpg" /> 图2:捆绑解剖钳是在左手，显微切割剪刀在右边。红线表示手腕和剪刀与纤维的纵向轴的有利方向。 <img alt="图3" src="/files/ftp_upload/52695/52695fig3.jpg" /> 图3: </strong&gt;的（A）测试设备与部件标记。 （a）在透明底的试验室中。 （二）长度控制器。 （三）强制换能器。 （四）光源。 （五 ）长控制器XYZ微米驱动器，数字显示。 （ 六）舞台千分尺驱动器，数字显示。 （G）强制换能器XYZ微米的车程。 （H）平台校准激光衍射目标屏幕。 （ 一）隔振表。 （B）的实验室特写图。 （j）条不锈钢安装面从长度控制器延伸。 （k）的不锈钢连接表面的力换能器延伸。 （L）侧视棱镜。 （M）为住房热电冷却模块。 （N）热电偶报告室TEmperature。 <img alt="图4" src="/files/ftp_upload/52695/52695fig4.jpg" /> 图4:修改用于传输从解剖菜光纤到实验室100微升枪头。 <img alt="图5" src="/files/ftp_upload/52695/52695fig5.jpg" /> 图5:安装单纤维到实验装置（A）中准备的缝合线套环拧到不锈钢附连表面。 （B）的纤维转移至实验室。由第一对缝合线套环用过量的缝合除去（C）的纤维锚定到不锈钢附连表面。 （D）的第二对缝合环拧在第一缝合线环的顶部和并列代替。 <img alt="图6"SRC ="/文件/ ftp_upload / 52695 / 52695fig6.jpg"/&gt; 图6:肌节长度由激光干涉图案的投影分摊到校准目标屏幕 （ 一 ）激光源。 （二）镜像。 （三）目标屏幕。 （四）激光干涉图案。 <img alt="图7A" src="/files/ftp_upload/52695/52695fig7A.jpg" /><img alt="图7B" src="/files/ftp_upload/52695/52695fig7B.jpg" /> 图7:（A）测定，纤维的横截面面积的最佳小节的长度（人= 2.7微米）。假定一个椭圆形横截面，CSA计算每个的沿着光纤中部五个地点和五个单独测量的平均值报告为纤维的CSA。 2a表示俯视直径为<html>椭圆的一个轴，图2b表示侧视图直径和是椭圆的另一个轴。 （B）的代表性的纤维的图像示出各在顶部和侧面视图采取的五个相应直径测量。 <img alt="图8" src="/files/ftp_upload/52695/52695fig8.jpg" /> 图8:从健康人股代表力的痕迹外侧肌纤维（A）1型纤维（CSA:5710微米2，F O型:0.89万和SF○:156千帕）。 （ 二）2A型纤维（CSA:9510微米2，F O型:1.66万和SF○:174千帕）。纤维肌球蛋白重链的类型是通过利用电泳分离和银染色技术22确定。 OAD / 52695 / 52695fig9.jpg"/&gt; 图9:最大活性力（F O）计算的纤维力响应在预活化溶液引发长度控制器的松弛诱导运动期间（ 一 ）扩大图。 在 fp是保持2.7微米的小节的长度与纤维在休息所需的力。 （二）长度控制器松弛引起的运动展开视图。注意了f O = F T - 在 fp。 <img alt="图10" src="/files/ftp_upload/52695/52695fig10.jpg" /> 图10:（A）缝合循环支路，掘起力过程中证明有效跟踪一个"抓"。检查，以确保循环是激活光纤之前安全。激活过程中（B）光纤断裂。在缝线环的地方可能是由于不良的纤维完整性或腐蚀性纤维处理换货。 （ 三）部分过早激活纤维由于预活化室与钙污染2+。

Watch this Scientific Journal Video about Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers at JoVE.com

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

化学剥皮，或透化，骨骼肌纤维的收缩特性的分析提供了强有力的手段，其中在单个肌细胞的水平来评估肌肉功能。在这篇文章中，我们勾勒出一个有效和可靠的技术准备和测试透骨骼肌纤维体外 。

最大等长收缩力的测量通过透骨骼肌纤维生成

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

measurement-maximum-isometric-force-generated-permeabilized-skeletal

Research

JoVE Journal

Bioengineering

25.3K 次观看.  University of Michigan Medical School. 化学剥皮，或透化，骨骼肌纤维的收缩特性的分析提供了强有力的手段，其中在单个肌细胞的水平来评估肌肉功能。在这篇文章中，我们勾勒出一个有效和可靠的技术准备和测试透骨骼肌纤维体外 。 

视频: Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

总结凝聚力测量组织表面Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

科研

生物工程

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

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 ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...