美国国家科学基金会，MRI检查程序（CHE:1229103）被确认为购买新的电子控制装置，软件，液体电池，并装配了双AFM / STM所需的其他设备提供资金。我们承认在芝加哥NSF-MRSEC计划大学（DMR-0820054）与原子力显微镜仪器，培训和成像援助共用设施和仪器时提供的材料研究设施网络（DMR-0820054）。我们特别感谢泣涕郭医生，贾斯汀Jureller博士和嘉怡Lee教授为美国国家科学基金会，核磁共振建议，带来了必要的仪器，我们的校园资助之前，欢迎我们的学生。 （:P031C110157号）颁发给东北伊利诺伊大学所提供的暑期研究奖学金的学生和教师，以及支持消耗品我们承认，从第三章干补助资金。最后，我们感谢布鲁克纳公司的持续性支持和许可于reproduce剧情呈现PeakForce QNM的机制，用这个词ScanAsyst来形容自动化软件。

1，电脑和显微镜设置<ol><li>打开氮气气缸的阀和调节旋钮，以确保空气表是浮动电平。 </li><li>打开计算机，控制器和光纤在该顺序的光。 </li><li>将扫描仪的AFM / LFM模式和中心相机在原子力显微镜头。 </li><li>打开软件。选择实验类"纳米力学性能"，"定量纳米机械制图"下的实验组，与"PeakForce QNM流体力学"下的实验。点击载试验，然后安装。 </li><li>使用Z客观看舞台的表面对焦。使用粗上/下压电体移动大约1mm的原子力显微镜头孔的表面之下的阶段。 </li></ol> 2，准备云母表面的<ol><li>按粘合剂贴到一个干净的样品支撑盘（直径15毫米，厚0.8毫米）的脸。推云母V-4级盘（25×25×26毫米）上的粘合剂，直到牢固地附着。 </li><li>擦了一块透明胶带的到云母确保其完全遵守，没有气泡。从一个侧面拉出带了云母均匀地切割它。如果有必要，以确保该云母是平坦重复。 </li><li>抓地力的支持下盘，清洁云母面朝上，有一对圆形的镊子（磁盘夹子），并放置到扫描仪。云母应低于AFM头孔的水平。 </li></ol> 3，流体细胞大会和云母表面成像<ol><li>冲洗液细胞，O形环，注射器接头，软管接头，并在100％乙醇中的喷射软管。在冲洗去离子水的液体电池。让所有部件的空气完全干燥。 </li><li>将软管适配器插入干净的流体单元的端口。轻轻按下O型圈进用平头镊子流体单元的中心压痕。 </li><li>按春风等闲探头固定夹，并把它远离groovE对于小费。 </li><li>用平头镊子抓住探头上的长端离一角。把探头放入朝向小区的中心的末端的槽。推动在春季和调整固定夹在探头固定（ 图1B）。 </li><li>签上的原子力显微镜的夹紧，以确保它完全缩回（ 图1C）。 </li><li>将流体细胞，O型​​环和探针朝下，在云母样品的顶部。允许流体细胞休息到原子力显微镜的螺栓。放下夹具，以稳定在舞台上的流体单元。按下显微镜下开关，直到视力检查显示O型圈支撑盘和流体细胞之间紧紧压缩。 </li><li>一边看着监视器，聚焦粗调旋钮来可视化屏幕上的流体池的顶部。 </li><li>移动显微镜，直到有尖端的使用X / Y轴调节旋钮以及Z客观清晰的图像。该提示将APP耳朵像金属，金三角。移动的前端更接近使用在显微镜的下压电杆的样品表面。专注于尖端的反映。保持关系，它的反射轨迹的实际悬臂。在此阶段，应紧密配合，但不完全重叠。 </li><li>附上注射器接头到无菌1ml注射器。附加的喷射软管的注射器适配器的一端，并放置在缓冲溶液的容器，软管的另一端。充分延伸注射器的柱塞。 </li><li>连接软管上的流体单元的软管接头。轻轻按压活塞，直到液体完全充满中心室。检查细胞以确保没有气泡。检查，看看有没有流体从流体细胞溅出到显微镜。放置在一个凸起的固相支持物的注射器。 </li><li>使用相机饲料和X / Y方向平移旋钮来定位屏幕上的激光（漫红点）。使用激光MOVEM耳鼻喉科控制旋钮，将激光上一角。 </li><li>精细地调整激光及反射镜控制，直到在显微镜下显示的和信号被最大化（值= 4 - 6）。 </li><li>调整用的左后侧上的旋钮和扫描头的左上侧，以降低在显微镜接近零越好（±0.1）所显示的垂直和水平偏转的象限二极管的角度。 </li><li>在软件中，选择收购方案。至少，选择身高（跟踪和回扫），峰值​​力误差和变形。其他选项是依赖于用户的，并且可以根据期望的实验者的特定信息进行定制。 </li><li>选择"行"的RT飞机适合在每个采集窗口。选择"无"旧约飞机适合在高窗。设置的X和Y偏移量和扫描角为零时，如果尚未完成。 </li><li>设置扫描速率为1赫兹，每行的样品256中，ScanAsyst自动控制个别和ScanAsyst Auto Z技术限制为关闭。改变Z轴限制到500纳米。设定扫描尺寸为1微米。 </li><li>点击参与。在这一点上，末端接近的表面上。而尖端临近，观察屏幕的左下角Ž压电电压变化。如果针尖进入的范围之外，撤回尖和重新的做法。如果这是不成功的，尖端可能需要为了有一个成功的方法要改变。一旦该尖端部接合，该线将开始出现在每一个采集窗口，这将构成一个图像。 </li><li>取云母的图像以1×1微米和放大慢慢以确保该云母表面是干净的和平坦的。点击连续捕捉图像保存，因为它们出现。放大和周围的表面根据需要移动。 </li></ol> 4，蛋白沉积和样本进行成像的制备<ol><li>得到纯化的蛋白样品38并保持在冰上。如果存储在冷冻箱中，在解冻的样品冰浴5分钟。 </li><li>稀释蛋白质到0.0010毫克/毫升用冷藏成像缓冲液。这里所使用的成像缓冲器是15毫摩尔Tris-盐酸（pH值= 8.0），150mM的NaCl和15mM的MgCl 2的。 </li><li>补4室（100×15毫米）培养皿冷藏影像缓冲溶液。填充每个腔室具​​有至少5毫升溶液。轻轻混匀与连接到微量移液器尖端稀释的蛋白溶液。 </li><li>申请加入200μl稀释的蛋白溶液，以云母的已制备的表面。 30秒后，拿起蛋白/云母表面与圆形镊子（磁盘夹持器）。保持平行实验台上的样品。 </li><li>同时保持样品，立即浸入冷水中含有的培养皿中（ 步骤4.2）的第一象限中的缓冲溶液中的样品，同时确保该样品是平行于培养皿的盆地。经过1秒，取出样品。 </li><li>继续保持平行的试样的盆在培养皿中，重复步骤4.5其余三个象限。 </li><li>放置在干燥的无尘布的盘，以吸收多余的水分和盘转移到显微镜的阶段。请勿触摸实际表面用布。不使样品干燥。 </li><li>重复步骤3.6 - 3.16。 </li></ol>在云母5成像蛋白质<ol><li>点击检查参数框在屏幕的左侧。弹簧大小和针尖半径应根据所使用的尖端被设置。 </li><li>点击参与。类似于云母成像过程（ 步骤3.17），观察到的z压电窗口的前端的方法。 </li><li>允许扫描仪图像的相同区域的至少两个连续的通行证。点击连续拍摄，保存收集的每个图像。 </li><li>将扫描仪到一个新的区域和/或根据需要改变图像的大小。 </li><li>之后，自动化软件已经完成调整扫描参数为特定的图像大小，把软件关闭。 </li><li>为了手动调节扫描速率和z限制，行数，和电压微调的图像的焦点。如果图像变得太远失焦，打开软件，重新回到原来的起始位置。以增加，而对蛋白质放大的图像的分辨率，降低扫描速率和增加的行数成比例。 </li><li>在实验结束后，清洗液细胞用100％乙醇和冲洗用DI H 2 O。使细胞液充分干燥。 </li><li>打开软件的数据处理原始数据图像。首先，通过点击平铺图标拼合图像。选择零阶 ，然后点击执行。根据图像的不同，平面拟合可能是必要的，以获得适当的平面图像。在这种情况下，选择合适的飞机图标和借鉴使用光标图像的平坦区域的平面。点击执行。根据需要重复</li><li> SEL等截面标签来测量蛋白质的尺寸。绘制光标线移动在分子或区域进行分析。重复用于多个领域，该软件会产生叠加。导出照片或​​用于生成图像兼容其他软件和绘图软件中的格式的数据。 </li></ol>

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There are no competing financial interests or conflicts of interest.

原子力显微镜是一种扫描探针显微镜方法完全能够成像的蛋白质和其他生物大分子的生理相关条件。相比于X射线晶体学和核磁共振，原子力显微镜中的一个限制是它不能达到相同的分辨率，特别是横向分辨率。当使用原子力显微镜来分析在任何表面上的分子，该表面的分子的图象上造成的影响和探针也必须当数据进行分析考虑。所获取的图像上的探测​​器的影响去卷积可以用适当的软件来完成。显影的理论模型进行比较，以实验的结果也可以被证明是非常有用的。 在本文中，细菌红光感光，RpBphP3（P3）的AFM图像，呈现（ 图3）。而在显微镜组件和软件操作的描述是针对特定设备模型使用的，云母和蛋白样品制备程序是通用的，可以应用于任何的AFM实验。 必须遵循，以获得在云母表面所需的蛋白质覆盖该协议的几个关键步骤。首先，将稀释的蛋白溶液必须慢慢混合之前它被施加到云母表面（ 步骤4.3）。由于P3的分子量高，沉积溶液是相当的胶体;因此，P3沉降到试管底部随着时间的推移使该溶液的非均相。一个温柔的轰动补救这个问题。第二，一旦蛋白被施加时，表面必须被清洗以去除任何松散结合的分子（ 步骤4.5 - 4.6）。如果松散结合P3保持在表面上，所述分子将被任一释放到溶液中时，液体池充满缓冲溶液和/或在实验过程中拾取的原子力显微镜探针。这两个事件的阻碍成功的成像实验。如果液细胞变成与P3的污染，必须将其拆开并彻底清洗。如果AFM探针拾取的分子时，它通常必须更换或牺牲图像质量。最后，冲洗该蛋白质样品的方法是获得优异的图像的关键。云母是一种非选择性表面到P3;因此，该分子应当发现是随机取向的。在协议中漂洗描述的方法是，以避免重新排列在表面上的分子在一个单一方向的一种方式。关键是要始终保持样品湿和避免保持表面在角和字面上表面喷涂用缓冲液。如果样品是通过浸渍，同时保持其平行于培养皿的盆冲洗，P3的表面由缓冲溶液的力电流在失真最小化。 所有部件的液晶单元，探针，扫描仪和光学头是非常微妙的，必须小心处理。该sprin持有的扫描仪和光学头GS一起是非常强的。当务之急是组装或拆解任何件时，一只手放在扫描仪上。为PF-QNM与ScanAsyst软件，如在协议中所述，有必要关闭的Z极限的自动化软件控制，该值设定为至少500纳米，如果原来的扫描尺寸为1μm（ 步骤3.16） 。这保护，如果表面已经成为意外粗糙或已被污染的扫描仪。一旦单个图像被收集并可以确认，在成像的区域是光滑的，在Z极限可以向下手动调节，以实现更高分辨率的图像。至关重要的是背面调整的Z限制到至少为500纳米时，扫描仪被移动到表面的新区域，或者如果前端缩回任何原因。 虽然本文已经集中在使用PF-QNM，得到高品质的地形影像，该方法本身可以提供一个库的有关的机械强度和表面结构的灵活性的附加信息。这个功能可以通过简单地在实验过程中选择合适的信道导致大约功能进一步的见解。16,18通过适当的改变沉积溶液和/或该表面的浓度，这种方法可以是通常使用的PF-QNM调查生物大分子通过液 - 细胞的AFM。随着软件的自动帮助，学生在上层科学课程与原子力显微镜没有现成的经验可以更快速地完成液体细胞原子力显微镜实验比传统的敲击模式原子力显微镜。学生可以体验到原子力显微镜的基本知识，并获得在一个典型的3成为可能，这种强大的技术的跨学科研究的味道 - 4小时的实验室部分时间限制。

原子力显微镜（AFM）已成为自1986年发明（ 图1），调查表面，薄膜，和单个分子的结构和机械性能的非常重要的工具。1-3使用的液体芯，该方法具有成为在生物大分子的研究，即使活细胞是特别有用的生理学相关的环境。4-10轻敲模式原子力显微镜在传统上被用于成像软材料或松散结合的分子的表面，由于接触模式AFM通常是不合适的，由于由悬臂引起的横向力的损害作用在样品11攻牙模式AFM基本上由具有前端间歇触摸表面，而不是在恒定接触而减少了这些力。在这种模式下，悬臂摆动达到或接近其共振频率与表面垂直。同样地，以非接触式原子力显微镜模式，地势肛门通过绘制Z-压电的运动为XY（距离）的函数yzed。 悬臂动力学可以是相当不稳定的或接近共振;因此，他们是非常具有挑战性的自动化的"稳定状态"的情况外。具体地，这些动力学依赖于两个样品的性质和扫描环境。对于软分子吸附到硬盘（ER）的表面，经过良好调优的反馈回路的分子可能会导致反馈振荡的表面。操作在流体进一步复杂悬臂的调谐。温度变化或流体水平要求设定点，收益和其他成像参数的不断调整。这些调整往往是非常耗时和对用户的挑战性。 峰力定量纳米机械性能映射（PF-QNM），如轻敲模式原子力显微镜，通过样品（ 图2）间歇性地接触，避免了横向的互动。12-15 &lt;/ SUP&gt;然而，PF-QNM工作在非谐振模式和频率比轻敲模式原子力显微镜低得多。这消除了轻敲模式原子力显微镜，特别是那些由流体的存在而加剧的调谐挑战。随着PF-QNM，图像通过采取武力响应曲线在每一个接触点收集。通过加入ScanAsyst软件，15调整扫描参数可被自动和高分辨率的图像由没有经验的用户获得在几分钟之内。一旦用户变得更加熟悉的原子力显微镜，任何自动或所有参数可以在任何时间，其允许实验者微调图像质量手动禁用。公司自成立以来，PF-QNM已经应用到地图细菌视紫红质，膜蛋白，以及其他天然蛋白在亚分子水平。16-18对细菌视紫红质，有弹性的蛋白质和X-射线晶体结构之间有直接的关系。12犯规-qnM的被利用来研究具有高分辨率的活细胞。19,20此外，PF-QNM数据已经阐明的红血球膜，这对细胞的完整性和功能是至关重要的内结构和力学之间的重要连接。21 我们已经采用扫描探针显微镜（SPM）的方法，22，包括原子力显微镜，23学习的红色光的光感受器的结构称为bacteriophytochromes（BphPs）。24,25它们由共价连接到信令执行器模块这样的光感测模块的因为组氨酸激酶（香港）26的光感测模组通常包含一个胆色素生色团，其在吸收光子发生结构转变，一系列深远的信号效应器模块，并导致整个蛋白质的全球转变的结构性变化，24,27-29基于这种转变，有两种截然不同的光吸收šBphPs，红色和远红外光吸收状态的美帝，表示为Pr和PFR。 Pr为热稳定的，暗适应状态下的最BphPs 28的PR / PFR光转化的分子基础是不完全是由于这些蛋白质的有限结构的知识的理解。随着从D除了一个结构球菌 ，这些蛋白质的30所有发表的X射线晶体结构是在暗适应状态下，缺乏效应器结构域。完整BphPs是太大而不能有效地通过核磁共振（NMR）研究，并且非常难以在其完整形式结晶（特别是在光适应状态下）的X射线晶体结构。 BphPs最近被改造为红外荧光蛋白标记（IFP的），这些蛋白31的结构特征可以进一步帮助有效IFP设计。32-36 本文的重点是介绍一个程序，影像通过PF-QNM使用液体电池的AFM BphPs的。该方法是从光合细菌R.表现在bacteriophytochrome RpBphP3（P3）的光适应状态下的学习沼泽 。这里介绍的AFM程序是用于蛋白质的成像方便和简单的方法，以及其他的生物大分子。用这种方法，单个分子的结构细节可以被收集的时间，类似于上位科学实验室课程会话短时间。通过测量断面，完成进一步的维分析，实验数据可以比较有效的计算模型。37-42

<table><tbody><tr><td>Tris-HCl</td><td>Fisher Scientific</td><td>O4997-100</td><td></td></tr><tr><td>NaCl</td><td>Acros Organics</td><td>7647-14-5</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Acros Organics</td><td>7791-18-6</td><td></td></tr><tr><td>Multimode 8 AFM</td><td>Bruker-Nano</td><td>492-008-011</td><td>equipped with Nanoscope V controller and J scanner</td></tr><tr><td>Probe</td><td>Bruker-Nano</td><td>SNL-10, ScanAsyst-Fluid+</td><td></td></tr><tr><td>Tapping Mode Fluid Cell</td><td>Bruker-Nano</td><td>MTFML</td><td></td></tr><tr><td>Mica V-4 Grade</td><td>SPI supplies</td><td>1150503</td><td>25 x 25 x .26 mm</td></tr><tr><td>Sample support disk</td><td>nanoSurf</td><td>BT02236</td><td></td></tr><tr><td>Petri dish</td><td>Plasta-Medic, Inc.</td><td></td><td>100 mm x 15 mm&amp;nbsp;</td></tr><tr><td>micropipettors</td><td>Denville Scientific</td><td>XL 3000i</td><td></td></tr><tr><td>RpBphP3</td><td></td><td></td><td>Prepared according to cited references</td></tr><tr><td>Nanoscope software</td><td>Bruker-Nano</td><td></td><td></td></tr><tr><td>Fiber Optic Light</td><td>Digital Instruments Inc.</td><td>F0-50</td><td></td></tr><tr><td>Pelco AFM Disc Gripper</td><td>Ted Pella Inc</td><td>1668</td><td>12 mm</td></tr><tr><td>1 ml syringe</td><td>McKesson</td><td>102-ST1C</td><td></td></tr><tr><td>Eppendorf tubes</td><td>Denville Scientific</td><td>C2171</td><td></td></tr><tr><td>The Pymol Molecular Graphic System v.1.5.0.1</td><td>Schrodinger, LLC</td><td></td><td></td></tr></tbody></table>

atomic force microscopy of red-light photoreceptors using peakforce quantitative nanomechanical property mapping

原子力显微镜（AFM）使用连接到悬臂探测表面的力响应金字塔尖。尖端的偏转可以被测量到〜10 PN通过激光和扇区检测器，它可以转换为图像的形貌。振幅调制或"轻敲模式"AFM涉及制备与表面间歇性接触，在其谐振频率而振荡，以产生图像的探测器。用于与流体细胞结合，轻敲模式原子力显微镜使生物大分子的影像，如在生理条件有关的蛋白质。轻敲模式原子力显微镜需要的探头和多种可挑战经验的用户扫描参数的频繁调整的手动调整。为了获得高质量的图像，这些调整是最耗时的。 PeakForce定量纳米机械性能映射（PF-QNM）通过测量力respon产生图像本身曲线的每个点与样品接触的。与ScanAsyst软件，PF-QNM可以自动化。这个软件调整设定点，驱动频率，扫描速度，增益和其他重要的扫描参数自动给定的样本。这不仅处理同时保护脆弱的探针和样品，它显著降低，得到高清晰度的图像所需的时间。 PF-QNM是原子力显微镜成像流体兼容;因此，它具有用于成像的生物相关物质的广泛应用。 本文提出的方法描述PF-QNM的应用程序来获取细菌红光感光，RpBphP3（P3）的图像，从光合河沼泽红假单胞菌在其光适应状态。使用这种方法，P3的个别蛋白二聚体和二聚体的聚集体已经观察到在云母表面在成像缓冲器的存在。以适当调整表面和/或溶液浓度，这种方法通常可应用于其它生物学相关的大分子和软质材料。

感光体蛋白，P3，在其光适应状态下的有代表性的AFM图像示于图3和图4。一个刚切割的云母衬底（ 图3A）是合适的，平坦的表面蛋白质的吸附。收集干净云母的图像作为阴性对照是非常重要的有以下几个原因。首先，它确保了液体电池的清洁，并从先前的实验无残留物质会污染地面。第二，它测试探针的质量。如果探头脏了或变形，这样会出现图像条纹或显示自己的噪音。最后，一​​个干净的云母图像确认探头可适当关注这方面的实验。 单一感光体蛋白二聚体可使用PeakForce QNM如果表面覆盖为宜（ 图3B，C）被观察到。箭头信号单独二聚体;星号表示蛋白质聚集体。该蛋白质样品，沉积时间，并且成像缓冲液的离子强度的浓度影响的表面覆盖。23对于单个分子的高分布，非常稀的溶液具有短的沉积时间是必需的。例如，加倍的沉积时间得到了更大的聚集体高分布。如果蛋白质浓度提高到0.100毫克/毫升，观察感光体的薄膜，而无需修改沉积时间或缓冲液的离子强度。 利用图像分析软件，在图像被压扁（ 图4）的蛋白质的横截面可以被测量。的横截面为XY-数据与相应的高度的测量。这些测量值可以被用来提供结构细节，蛋白质/表面相互作用，机械强度等的XY数据可能由AFM探针进行卷积;然而，这种效果可以通过使用尖锐的探针被最小化。事实上，町探针的冰是卷积的可接受量和成像材料被锋利的探针的耐损伤之间的微妙平衡。尖端卷积还可以从图像中减去，如有必要，通过使用适当的软件。 这些照片可以直接相比先前公布的图像收集的用轻敲模式原子力显微镜。P3的云母使用PeakForce QNM 20所测得的尺寸范围内用轻敲模式原子力显微镜获得的值的5％。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/52164/52164fig1highres.jpg" width="600" /> 图1液细胞的原子力显微镜。 （一）激光，悬臂式，探头和表面校准。探针和悬臂贴在液晶单元内部的（B）液体的细胞。探针时，悬臂和样品完全浸没在流体中（C）器Comp1ETE组装。液晶单元被连接到一个测微计。图为光学头和扫描仪的关系，以液态电池。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52164/52164fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="3in" src="/files/ftp_upload/52164/52164fig2highres.jpg" width="300" /> 图2:PeakForce定量纳米机械性能映射这是一个小的峰值力的图（A）方法（B）跳转到联系（C）峰值力（D）附着力（五）退刀。这个数字是复制和改编许可。15 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/52164/52164fig3highres.jpg" width="600"/&gt; 图3原子力对云母P3的显微镜。干净的云母（1×1微米）的（B）图像（1微米×1微米）P3的（A）图片。适用于云母。箭头表示的单一蛋白二聚体的例子。星号表示蛋白二聚体的聚集体（C）图像的两个蛋白二聚体（170×170纳米）与单个二聚体的立体插页。插入符号表示该蛋白二聚体存在于插图。所有图像拍摄的影像缓冲区的存在（蛋白浓度= 0.0010毫克/毫升的沉积）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52164/52164fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="3in" src="/files/ftp_upload/52164/52164fig4highres.jpg" width="300" /> 图4横截面P3的对云母分析。 </stronP3的云母（170×170纳米）与截面高度/距离测量g&gt;的AFM图像指出了两个二聚体。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52164/52164fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping at JoVE.com

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

atomic-force-microscopy-red-light-photoreceptors-using-peakforce

Nanyang Technological University

Research

JoVE Journal

Engineering

11.7K 次观看.  Northeastern Illinois University. A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface. 

视频: Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

科研

生物化学

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

生物工程

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

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

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 ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

使用光学镊子测量禁闭中的亚细胞力学的直接力

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

发育生物学

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

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 ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

原子力显微镜罐体功能化,具有单T细胞或单粒子免疫单细胞力光谱

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

免疫与感染

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

凸出力显微镜: 一种量化细胞突起力的方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...