Research related to this paper and the development of the fBFP technology in the Zhu lab were supported by NIH grants AI044902, AI077343, AI038282, HL093723, HL091020, GM096187, and TW008753. We thank Evan Evans for inventing this empowering experimental tool, and members of the Evans lab, Andrew Leung, Koji Kinoshita, Wesley Wong, and Ken Halvorsen, for helping us to build the BFP. We also thank other Zhu lab members, Fang Kong, Chenghao Ge and Kaitao Li, for their helps in the instrumentation development.

该协议遵循的指导方针，并已获得由佐治亚理工学院的人类研究伦理委员会。 1.人类红细胞分离，生物素和渗透压调节注：步骤1.1应该由受过训练的医疗专业人员如护士进行，与机构审查委员会批准的协议。 <ol><li>得到的血液8-10微升（一滴）从手指刺，并添加至1ml的碳酸盐/碳酸氢盐缓冲液（ 表1和2）。轻轻涡旋或吸取的混合物离心1分钟，在900×g下。弃上清，洗一次。 </li><li>在小烧杯称量3.5-4毫克生物素- PEG3500-NHS接头（ 表1）。将其溶解在碳酸盐/碳酸氢盐缓冲液，使最终浓度为3毫克/毫升。 </li><li>混合171微升的碳酸盐/碳酸氢盐缓冲液，10微升RBC包和1049微升生物素 - PEG3500-NHS交联剂溶液和在室温下孵育30分钟。用碳酸盐/碳酸氢盐缓冲液洗一次红细胞，然后两次用N2-5％缓冲液（ 见表1和2）。 </li><li>同时，将连接器瓶在玻璃真空干燥器填充在底部和真空5分钟干燥剂瓶盖松动，并填写氩气干燥器。拧紧瓶盖，并采取了瓶子。密封该瓶用塑料石蜡膜（ 表1），将其放入一个容器中填充有在-20℃的底部，并存储干燥剂。 注意：涉及使用生物素 - PEG3500-NHS接头的步骤，包括1.2-1.4（除了保温和洗涤在1.3），需要尽可能快进行越好。 </li><li>稀制霉菌素到N2-5％缓冲液，使40微克/毫升的最终浓度。混合5微升生物素化的红细胞与71.4微升制霉菌素（ 表1）溶液孵育1小时，在0 ＆＃176;℃。与N2-5％缓冲和存储与N2-5％缓冲液+ 0.5％BSA中（ 表1）在冰箱（4℃）洗涤两次。 </li></ol> 2.玻璃珠硅烷化<ol><li>珠面清洗<ol><li>称取50毫克玻璃珠粉和500微升去离子水重新挂起他们。 </li><li>混合0.5毫升30％的H 2 O 2（ 表1）与9.5 DI水毫升，在50毫升烧杯中，然后浓NH 4 OH（ 表1）的加入2 ml和将这一溶液到锅炉的热板上。 </li><li>添加玻璃珠的沸腾溶液，继续煮沸另外的5分钟。轻轻摇动解决方案的每分钟。 </li><li>煮沸后，转移〜5个毫升该热珠悬浮液入15ml微型离心管中并顶与室温去离子水。离心3500 XG 5分钟，捞出弃去上清液。 </li><li>传送另外5毫升热珠悬浮液，并加入到洗过的小珠，再补足更多的去离子水，拌匀，和离心机。重复此过程直至约50毫升去离子水的情况下，这将是一个总的洗涤的4至5倍。 </li><li>转移珠悬浮液进入1毫升的小瓶。重复洗涤用甲醇（ 表1）通过离心珠在17000×g离心5分钟进行3次，最后重新悬浮在1ml的100％甲醇中的珠子。 </li></ol></li><li>珠面硫醇化<ol><li>到50ml离心管中添加45.6毫升甲醇，0.4毫升乙酸（ 表1），1.85毫升去离子水，1.15毫升的三巯基（MPTMS）（ 表1）和1ml珠粒的悬浮液制备2.1，然后在室温下孵育3小时。 </li><li>反应后，除去所有的反应物通过用新鲜甲醇洗涤一次，并重新悬浮珠子入500微升甲醇中。平均分这浓缩的玻璃珠悬挂成一组20干燥和干净的玻璃小瓶螺丝帽。蒸发掉甲醇用干燥的氩气射流，缓慢旋转小瓶，从而使一薄层干珠在每个小瓶的侧面。 </li><li>放置的珠的小瓶进入预加热的干燥炉中在120℃下进行5分钟，然后取出，并迅速将帽（多个）松弛地上。将小瓶中的玻璃真空干燥器填充有干燥剂，在底部和用真空泵真空干燥器中，直到冷却。 </li><li>吹扫真空干燥器用干燥氩气使干燥器正常大气压力。取下盖子干燥器并迅速重新拧紧瓶帽（S）。轴封采用塑料薄膜石蜡的小瓶，在室温下存放在干燥避光贮存箱。 </li><li>在立即使用，采取干珠一小瓶并用磷酸盐缓冲液（ 表1和2），再悬浮成50μl的磷酸盐缓冲液并储存在4℃下洗一次。该浓缩珠制剂将被称为如在下面的步骤“MPTMS珠”。 注意：使用适当的储存，MPTMS珠可以保持工作长达三个月。 </li></ol></li></ol> 3.珠功能化<ol><li>共价蛋白质涂层珠上<ol><li>取该蛋白质的库存的一定体积（ 例如 ，2.5微升）和用碳酸盐/碳酸氢盐缓冲液等体积使溶液1混合。 注意：体积取决于原料浓度和在珠子上表面上的蛋白质的期望的最终位点密度。 </li><li>在小烧杯称量2-3毫克MAL-PEG3500-NHS接头（ 表1）中，用碳酸盐/碳酸氢盐缓冲液溶解，以达到0.231毫克/毫升的最终浓度。 </li><li>混合溶液1与3.1.2中制备的交联剂溶液的等体积。在室温下孵育该混合物30分钟，使溶液2。 </li><li>同时，将连接器瓶瓶盖松动在玻璃真空干燥器˚Filled在底部和真空5分钟干燥剂，并填写氩气干燥器。拧紧瓶盖，并采取了瓶子。密封该瓶用塑料石蜡膜，将其放入一个容器中填充有在-20℃的底部，并存储干燥剂。 注意：这涉及使用MAL-PEG3500-NHS连接的步骤，包括3.1.2-3.1.4（除3.1.3孵化），需尽可能快地完成。 </li><li>混合5微升MPTMS珠溶液2，并添加磷酸盐缓冲液（ 见表1），使250微升的最终体积。 </li><li>孵育珠粒过夜在RT，洗3次，用磷酸盐缓冲液，并重新悬浮在100微升磷酸盐缓冲液并储存在4℃。 </li></ol></li><li>准备蛋白/链亲和素（SA）包被的珠子<ol><li>按照协议3.1.1-3.1.4。 </li><li>混合5微升MPTMS珠溶液2和5微升4毫克/毫升链霉亲和马来酰亚胺（SA-MAL）（ 表1）溶液，然后添加磷酸缓冲液，使250微升的最终体积。 </li><li>孵育珠粒过夜在RT，洗3次，用磷酸盐缓冲液，最后重新悬浮在100微升磷酸盐缓冲液并储存在4℃。 </li></ol></li><li>涂层链霉到玻璃珠<ol><li>混合5微升MPTMS珠5微升4毫克/毫升的SA溶液，并添加140微升磷酸盐缓冲液中。 </li><li>孵育珠粒过夜在RT，洗3次，用磷酸盐缓冲液，并重新悬浮于50微升磷酸盐缓冲液并储存在4℃。 </li></ol></li><li>涂覆的SA包被的珠子用生物素化蛋白<ol><li>混合5微升的SA包被的珠与蛋白质（体积取决于期望的涂层密度），并添加磷酸盐缓冲液，使终体积为100微升。 </li><li>孵育混合物过夜，在4℃或3小时，在室温洗3次，用磷酸盐缓冲液，并重新悬浮于50微升pH值osphate缓冲并存储于4℃。 </li></ol></li></ol> 4.细胞的制备注意：为了纯化的细胞，按照对应于细胞中使用的类型的标准细胞纯化方案，例如T细胞27或某些细胞系21,29。 <ol><li>对于fBFP实验，一旦细胞悬浮液制备中，添加Fura2-AM（ 表1）溶解于DMSO至细胞悬浮液，以达到2μM的终浓度温育30分钟，在RT，然后洗一次。保持这种荧光装细胞悬浮液在黑暗中，直到使用。 </li></ol> 5.准备微量移液器和细胞室<ol><li>准备微量移液器<ol><li>切长毛细玻璃管（ 表1）用玻璃刀成短节约3英寸长。安装一块到微量拉马（ 表1），单击“拉”，但吨，使得毛细管的中间将由机器加热和毛细将被拉上的两端进行两次毛细管具有尖锐尖端（原始微量）。 注意：通过遵循产品准则，原始吸管的所需形态有6-8毫米的锥度和0.1-0.5微米小费。 </li><li>安装原始吸管到微量锻造（ 表1）的吸管持有人。热量融化在伪造的玻璃球。插入玻璃球内的原始吸管尖。冷静下来的玻璃球和拉生吸管从外部打破它，离开它的尖端领域内。直到获得所需的尖端孔重复此过程。 注意：一个微量移液器尖端内径的实例：2.0-2.4微米的红细胞，〜1.5微米的珠，〜2-4微米为T细胞和-7微米为杂交瘤细胞。 </li></ol></li><li>构建一个细胞室 注：电池室建起了家庭狂的基础上，Ë室支架，它由两片金属平方（铜/铝），以及它们链接起来的手柄（ 图1D）。 </li><li>切使用玻璃切割器40毫米×22毫米×0.2毫米盖玻片成两个40毫米×11毫米×0.2毫米件（盖玻片1和2）。胶盖玻片1由润滑脂腔室夹持器，它桥接两个金属平方方式的顶侧，并且类似胶盖玻片2向底侧，这将形成一平行盖玻片池室（ 图1D）。 </li><li>使用移液管注入200微升试验缓冲液在两个盖玻片之间。确保缓冲连接到两个盖玻片。轻轻旋转和摇动所述室让缓冲碰触腔室的两端。 </li><li>仔细矿物油注入室侧翼实验缓冲区从而密封从露天缓冲器的两侧。注入的探针珠悬浮液（例如，的pMHC包被的珠子），RBC和目标在缓冲区的分别的上部，中部和下部区域（例如，T细胞）。 </li></ol> 6. BFP实验<img alt="图1" src="/files/ftp_upload/52975/52975fig1.jpg" /> 图1：fBFP总成 （ 一 ）fBFP硬件系统的总览画面。 （B）中的fBFP硬件系统的示意图。 （C）在其上的高速照相机（蓝色）和一个荧光照相机（白色）被安装的双凸轮系统“DC2”（橙色）。 （D）中的显微镜载物台，能够适应一个实验室和三个微量操纵系统。 （E和F）的BFP设置在试验室中显微照片。 （E） -微量移液器组件示出探针移液器（ 左 ），目标吸管（ 右上 ）和辅助移液器（ 右下飞行）。 （F）探头放置珠。探针珠被操纵的辅助吸管，并连接到一个顶点红细胞形成的力量探头。 <a href="https://www.jove.com/files/ftp_upload/52975/52975fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>打开显微镜（ 表1）和光源。放置室到主显微镜阶段（ 图1A中的D）。 </li><li>安装BFP的所有三个微量（ 图1D左：探头，抢RBC，右：目标，抓住一个细胞或珠，右下：帮手，抢珠）。 <ol><li>使用微注射器（ 见表1）回填与实验缓冲液中的微量。取下吸管持有人（ 表1），并保持在一个较低的位置，让滴水尖。快速插入微管插入支架顶端，并确保无气泡进入微管在插入。拧紧支架螺丝。 </li><li>每个吸管架安装到其相应的微型机器人。推微量朝向腔室，使得它们的尖端进入腔室的缓冲区域。调整微量的位置，并找到他们下视显微镜领域。 </li></ol></li><li>左右移动的腔室保持台找到的三个要素的菌落（红细胞，靶和探针珠）一个接一个。通过转动操纵器的旋钮让微量办法一个小区/珠的前端调整相应微量的位置。通过调整相应的微量内的压力吸出细胞/珠。所有这三个微量将捕获其相应的元素。 </li><li>左右移动的腔室保持台找到一个开放的空间距离，其中该实验将执行的注射元件的菌落。切换显微镜可视化模式，以可视化图像上的补偿uter程序在电脑屏幕上。继续前行枪头所有三个元素融入到节目的视野。 </li><li>对准探针珠和RBC，并小心地操纵探针珠红细胞的顶点，珠简要撞击到红细胞和轻轻缩回。调整辅助微量的压力轻轻吹珠远，因此，它会留下粘到红细胞顶点（ 图1F）。搬开帮手微量并对准目标和探针珠（ 图2A）。 </li></ol><img alt="图2" src="/files/ftp_upload/52975/52975fig2.jpg" /> 图2：BFP方案及其测试周期 （A） 的视频显微照片描绘的力探针（ 左 ）和靶的T细胞（ 右 ）由各自pipettes.The固定力探头抽吸由肿红细胞和一个附加的配体轴承珠。受体轴承T细胞（目标）被安装到对面对准到探针piezotranslator。投资回报率显示在绿色。边缘跟踪显示在一个蓝线。插入描绘了配体（的pMHC， 焊道侧 ）和受体标记在橙色区域的两个相对的表面上（TCR，T细胞侧 ）对。 （B）中的珠缘（A）中的强度分布。在x -方向的ROI区域被绘 ​​制为x轴（在像素数）和光强度（灰度值）由分箱30个像素，沿y -方向平均。 （C）的红细胞的偏转和胎圈的位置，并在力钳测定的试验循环的靶（T细胞）。垂直和水平虚线指示红细胞顶点和时间过程的零力位置，分别。红细胞变形的线边缘跟踪器以蓝色显示在每个面板。粘附频率采用相同但更少的步骤试验（缺少的步骤“钳”和“分离”）和热波动分析（缺少的“游离”的步骤）。 <ol start="6"><li>上节目，在视野窗口中使用工具程序来测量探针微量（R P）的半径分别为，红细胞（R 0），红细胞和探头珠（R C）之间的圆形接触面积，这使得RBC（k）的弹簧常数的估计由下式17,30， <img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/52975/52975equ1.jpg" width="300" /> 其中Δp是在探头枪头抽吸压力。 注意：它遵循从胡克定律的结合力，F可以通过弹簧常数和探针珠（四）的位移， 即，F = D的产物来定量</em&gt;（ 图2C）。 </li><li>输入所需的RBC弹簧常数到程序（请参见协议6.6节的弹簧常数通常设定在0.25或0.3 PN /纳米为动力钳测定和粘附频率测定和0.1 PN /纳米的热波动分析） ，这将返回在水厘米的单元所需的抽吸压力。调节水箱链接到探针的移液管，直到所需的抽吸压力达到的高度。 </li><li>画一条横线穿过红细胞顶点，这将产生一个曲线在相邻的窗口，指示沿着这条线的每个像素的亮度（灰度值）。拖动阈值线是在曲线（ 图2A，B）的大约一半的深度。 注意：下面的阈值线的亮度曲线上的最低点表示胎圈的边界的位置，因此只有一个局部最小值是允许的。如果两个或多个局部最小值是在目前，它表示图像不是最佳（可能是由于图像是离焦，或探针珠和红细胞之间的差的对准）。 </li><li>选择所需的实验方式：热波动分析，附着力测定频率或武力钳测定。根据需要（ 例如 ，冲击力= 15 PN，加载速率= 1000 PN /秒，接触时间= 1秒，锁模力= 20 PN（对于力钳测定）等）设置参数。 <ol><li>单击“开始”，它允许程序移动目标吸管和推动的目标，并用探针接触（见代表结果的细节部分）。数据收集将并联，其中记录实时的探针珠的位置来执行。通过点击按钮“停止试验”，此时会弹出一个窗口出来，以便保存所采集的数据停止程序。 </li></ol></li></ol> 7.荧光BFP（fBFP）实验<ol><li>使用荧光的BFP系统刘哲民功能，打开激励光源（ 表1）和荧光照相机（ 表1），这是由一个独立的程序（ 表1）来控制。上的程序中，选择参数的荧光成像，包括增益，曝光，激发通道（在这种情况下，340纳米和380纳米的光） 等 。按照在BFP实验协议的所有准备工作，包括对准的探针和目标，这将允许通过340纳米或380纳米的激发光激发的靶细胞活荧光图像的可视化。 </li><li>使用切片工具大致部的区域内该单元将在整个记录期间停留。 注意：由于使用的方法接触回缩周期中，细胞将向前和向后重复，因而剖面积小于单元本身大得多。 </li><li>点击“记录”，让340纳米ð380纳米的光交替激发的细胞内荧光染料（Fura2），以及一对相应的荧光图像将被交替地记录大约每秒一次。在节目中同时单击“开始”，开始BFP实验分析的分子相互作用和荧光成像实验监测细胞内钙信号。系统将产生对受体-配体结合的原始数据文件（参见下面的图6A）等一系列的荧光图像中.tiff文件格式的钙信号。 </li></ol> 8.数据分析<ol><li> BFP数据分析<ol><li>附着力测定频率分析数据<ol><li>依次检查每个周期的“力量与时间”信号，并简单地记录其周期含有粘合事件，哪些没有，和总结，产生平均附着力频率。 </li><li>收集各附着力事件的破裂力，这是线性斜坡力的债券破裂前的峰值。收集的范围内的升温速率的破裂力足够量后，求出断裂力分布在从该力依赖离速率的受体-配体的解离使用动态力光谱分析18,31导出每个斜坡率。 </li></ol></li><li>对于热波动化验数据分析<ol><li>依次检查每个周期，这可能包含多个键结合和离解事件的夹紧相位信号。使用在夹紧阶段热波动水平（的珠位置70连续时间点的区间滑动的平均标准偏差）“力量与时间”的信号为导向，以区分债券结合和解离事件，因为债券形成对应于热起伏的降低。 </li><li>从债券分离的瞬间指定的时间间隔（当疗法发作波动恢复到正常水平）到下一个键形成作为等待时间的瞬间，并从其关联到解离为粘合寿命，它是在数据检查期间都采集指定键的持续时间。计算的平均轮候时间及平均寿命的债券，分别反映在零力16,30对利率的偏离率的倒数和。 </li></ol></li><li>对于力钳测定数据分析<ol><li>所有寿命事件，包括平均力和粘结寿命与序列号以及开始时间和结束时间，这将允许一个绘制终生累积曲线的记录参数（例如， 图6C中 ，黄色曲线）。 </li><li>根据收集各种力量的寿命事件有足够量。它们分成不同的力箱，这将产生一个平均寿命在各部队仓，干脆和产生一个“平均寿命VS。力“曲线（ 图4）。 </li></ol></li></ol></li><li>钙荧光成像数据分析<ol><li>调整强度阈值，直到荧光图像显示在两个340纳米和380纳米通道的细胞的清晰轮廓没有背景噪声（ 图5A，B）。然后检查由帧用伪彩色显示的亮度水平（ 图6B），这是基于为340nm / 380nm处的强度比导出的细胞内Ca 2+信号帧，以产生“归一化的Ca 2+强度VS 。时间“曲线（ 图6C）。使用伪彩色荧光图像以产生一个电影，由第二显示荧光水平第二。 </li></ol></li></ol>

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D., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+dynamic+catch+bonds+between+TCR+and+agonist+peptide-MHC+triggers+T+cell+signaling.">Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.</a> Cell. 157, 357-368 (2014).</li><li>Lou, J., 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=Flow-enhanced+adhesion+regulated+by+a+selectin+interdomain+hinge.">Flow-enhanced adhesion regulated by a selectin interdomain hinge.</a> The Journal of cell biology. 174, 1107-1117 (2006).</li><li>Fiore, V. F., Ju, L., Chen, Y., Zhu, C., Barker, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+catch+of+a+Thy-1-alpha5beta1+syndecan-4+trimolecular+complex.">Dynamic catch of a Thy-1-alpha5beta1+syndecan-4 trimolecular complex.</a> Nature communications. 5, 4886 (2014).</li><li>Chen, W., Zarnitsyna, V. I., Sarangapani, K. K., Huang, J., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Receptor-Ligand+Binding+Kinetics+on+Cell+Surfaces:+From+Adhesion+Frequency+to+Thermal+Fluctuation+Methods.">Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods.</a> Cellular and molecular bioengineering. 1, 276-288 (2008).</li><li>Marshall, B. T., Sarangapani, K. K., Lou, J., McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+history+dependence+of+receptor-ligand+dissociation.">Force history dependence of receptor-ligand dissociation.</a> Biophysical. 88, 1458-1466 (2005).</li><li>Xiang, X., 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=Structural+basis+and+kinetics+of+force-induced+conformational+changes+of+an+alphaA+domain-containing+integrin.">Structural basis and kinetics of force-induced conformational changes of an alphaA domain-containing integrin.</a> PloS one. 6, e27946 (2011).</li></ol>

The authors have nothing to disclose.

一个成功的实验fBFP限嗣继承了一些关键的考虑因素。首先，对于力计算是可靠的，微量，红细胞，和探针珠应该对齐接近同轴越好。红细胞的吸移管内的投影应约一个探针移液器直径，使得红细胞和吸管之间的摩擦是微不足道的。对于一个典型的人RBC，最优吸管直径为2.0-2.4微米，这将产生等式1 17,30的最佳拟合。第二，以确保在力钳测定及热波动分析测量大多为单键，也可以保持在20％以...

细胞-细胞和细胞-细胞外基质（ECM）的粘附是由细胞表面受体，细胞外基质蛋白和/或脂 ​​质1之间的结合介导的。结合允许细胞以形成功能结构1，以及认识，沟通，并做出反应的环境1-3。不像可溶性蛋白（ 例如 ，细胞因子和生长因子）从一个三维（3D），其结合流体相到细胞表面受体，细胞粘附受体形成具有跨越窄交界间隙及其配体桥接两个相对的表面，限制分子键扩散在二维（2D）接口4-7。与此相反，以三维动力学是由传统的结合分析（ 例如 ，表面等离子体共振或SPR）常用的测量，2D动力学已与诸如原子力显微镜（AFM）8-10专门技术来量化，流室11,12，微管13,14，光镊子15和生物膜力探针（BFP）16-21。 以上仅仅提供物理连锁蜂窝内聚力，粘附分子是信令机械为细胞与其周围环境进行通信的主要组成部分。出现了在理解越来越大的兴趣如何粘附分子的配体接合启动细胞内信号传导，以及如何在初始信号被转导的细胞内。直观地，受体 - 配体的性质的结合可影响它诱导的信号。然而，很难使用，因为它们的许多限制， 例如 ，一个差时间分辨率和完全缺乏空间分辨率的生物化学分析的传统合奏解剖外相互作用和细胞内信号事件之间的机械关系。现有的方法，使生物物理（2D受体 - 配体结合动力学）和生化（信令）观察活细胞包括可调刚性22的荧光能力24-26掺入底物，弹性体柱阵列23和流动室/微流体装置。然而，信令和受体 - 配体结合的读数必须（通过不同的方法最多）分别求出，从而难以解剖的粘结性能与信号事件的时间和空间关系。 传统的BFP是一个超灵敏光谱力高时空分辨率17。它使用一个挠性红细胞（RBC），为力传感器，可实现单分子2D动力学，机械性能和构象变化14,16,19-21,27-29的测量。荧光成像基于BFP（fBFP）相关与结合触发的细胞信号在单分子尺度的受体 - 配体的结合动力学。采用这种设置， 原位细胞信号活动表面mechani的上下文卡尔刺激中观察到的T细胞27。该fBFP是通用的，可以用于细胞粘附和信号传导由在其他小区的其他分子介导的研究。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Table 1: Reagents/Equipment</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic Monohydrate (NaH2PO4&#8226;H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>MAL-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5002-1</td>
			<td>Bead functionalization</td>
		</tr>
		<tr>
			<td>Biotin-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5026-1</td>
			<td>RBC biotinylation</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N6261</td>
			<td>RBC osmolarity adjustment</td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide (NH4OH)</td>
			<td>Sigma-Aldrich</td>
			<td>A-6899</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>BDH</td>
			<td>67-56-1</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide (H2O2)</td>
			<td>J. T. Barker</td>
			<td>Jan-86</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Acetic Acid (Glacial)</td>
			<td>Sigma-Aldrich</td>
			<td>ARK2183</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>3-MERCAPTOPROPYLTRIMETHOXYSILANE(MPTMS)</td>
			<td>Uct Specialties, llc</td>
			<td>4420-74-0</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Borosilicate Glass beads</td>
			<td>Distrilab Particle Technology</td>
			<td>9002</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Streptavidin&#8722;Maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>S9415</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A0336</td>
			<td>Ligand functionalizing</td>
		</tr>
		<tr>
			<td>Fura2-AM</td>
			<td>Life Technologies</td>
			<td>F-1201</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Quantibrite PE Beads</td>
			<td>BD Biosciences</td>
			<td>340495</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>BD LSR II</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Capillary Tube 0.7-1.0mm x 30&#34;</td>
			<td>Kimble Chase</td>
			<td>46485-1</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>sutter instrument</td>
			<td>P-97</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Pipette microforce</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Fisher Scientific</td>
			<td>BP2629-1</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-G</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micro-injector</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>BD&#160;</td>
			<td>309602</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Narishige</td>
			<td>HI-7</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Home-designed mechanical parts and adaptors fabrications using CNC machining.&#160;</td>
			<td>Biophysics Instrument</td>
			<td>All parts are customized according to the CAD designs.</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Microscope (TiE inverted)</td>
			<td>Nikon</td>
			<td>MEA53100</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Objective CFI Plan Fluor 40x (NA 0.75, WD 0.72mm, Spg)</td>
			<td>Nikon</td>
			<td>MRH00401</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Camera, GE680, 640x480, GigE, 1/3&#34; CCD, mono</td>
			<td>Graftek Imaging</td>
			<td>02-2020C</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Prosilica GC1290 - ICX445, 1/3&#34;, C-Mount, 1280x960, Mono., CCD, 12 Bit ADC</td>
			<td>Graftek Imaging</td>
			<td>02-2185A</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Manual submicron probehead with high resolution remote control</td>
			<td>Karl Suss</td>
			<td>PH400</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Anti-vibration table (5&#8217; x 3&#8217;)</td>
			<td>TMC</td>
			<td>77049089</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>3D manual translational stage</td>
			<td>Newport</td>
			<td>462-XYZ-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SolidWorks 3D CAD software</td>
			<td>SOLIDWORKS Corp.</td>
			<td>Version 2012 SP5</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>LabVIEW software</td>
			<td>National Instruments</td>
			<td>Version 2009</td>
			<td>BFP system, BFP program</td>
		</tr>
		<tr>
			<td>3D piezo translational stage</td>
			<td>Physik Instrumente</td>
			<td>M-105.3P</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Linear piezo accuator</td>
			<td>Physik Instrumente</td>
			<td>P-753.1CD</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Micromanager software</td>
			<td></td>
			<td>Version 1.4</td>
			<td>fBFP system, fluorescence imaging program</td>
		</tr>
		<tr>
			<td>Dual Cam (DC-2)</td>
			<td>Photometrics</td>
			<td>77054724</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Dual Cam emission filter (T565LPXR)</td>
			<td>Photometrics</td>
			<td>77054725</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Fluorescence Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA-R2 C10600-10B</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Plastic paraffin film (Parafilm)</td>
			<td>Bemis Company, Inc</td>
			<td>PM996</td>
			<td>bottle sealing</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Table 2: Buffer solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate/bicarbonate buffer (pH 8.5)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>8.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>10.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer (pH 6.5-6.8)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaPhosphate monobasic&#160;&#160;&#160;NaH2PO4&#8226;H2O</td>
			<td>27.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. NaPhosphate dibasic&#160;&#160; Na2HPO4</td>
			<td>28.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2-5% buffer (pH 7.2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>20.77g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>2.38g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>0.13g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>0.71g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>9.70g/L</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence biomembrane force probe: concurrent quantitation of receptor-ligand kinetics and binding-induced intracellular signaling on a single cell

膜受体 - 配体相互作用介导许多细胞功能。结合动力学和由这些分子之间的相互作用引起的下游信号很可能受到机械环境中结合和信号发生。最近的一项研究表明，机械力可以调节抗原识别由与触发T细胞受体（TCR）的。这成为可能，我们开发了一种新技术，荧光地称为生物膜力探针（fBFP），它结合了单分子力谱荧光显微镜。使用超软人类红细胞作为敏感的力传感器，一个高速摄像机和实时成像跟踪技术，该fBFP是〜1 PN（10 -12 N）〜3纳米和〜0.5毫秒力，空间和时间分辨率。随着fBFP，可以精确地测量力下单调节受体 - 配体结合动力学，同时结合图像触发细胞内的CALcium信令在单个活细胞。这种新技术可以用来研究其他膜受体 - 配体相互作用和信号下机械调节的其它细胞。

的BFP技术是在1995年17率先由埃文斯实验室，这picoforce工具已被广泛用来测量固定化在表面上的蛋白质的相互作用，从而分析二维粘附分子动力学与它们的配体16,19,20相互作用， 30，测量分子的弹性21,29，并确定蛋白的构象变化21。对于fBFP，一组额外的与相应的软件系统（ 表1）加落射荧光关联装置（ 图1A - C）。 <p cla...

Watch this Scientific Journal Video about Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell at JoVE

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力：受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

fluorescence-biomembrane-force-probe-concurrent-quantitation-receptor

Research

JoVE Journal

Bioengineering

12.3K 次观看.  Georgia Institute of Technology. We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

视频: Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

科研

生物工程

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

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. ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...