We acknowledge support of this work by the National Multiple Sclerosis Society and Simons Center for the Social Brain. BQ acknowledges support from the U.S. National Defense Science &amp; Engineering Graduate Fellowship program.

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The authors have nothing to disclose.

本文提出的每个技术测量脑组织的机械性能的不同方面。蠕变柔和应力松弛模量随时间变化的机械性能的量度。储存和损耗模数代表率相关的机械性能。冲击压痕还测量速率依赖性机械特性，但在能量耗散的情况下。当表征组织的机械性能，同时启用AFM压痕和流变的常用方法。因为除了提供时间相关的材料性质，不同的实验参数可以用来测量细胞和组织的弹性模量4，甚至频率相关特性24，如先前所述使能的AFM压痕是特别有用的。然而，实验的数据和设计的准确解释是具有挑战性的标准，水合组织。虽然流变措施散装适合组织的联系，使AFM压痕探测相关细胞的微环境微尺度卷。冲击压痕提供的材料以浓缩，动态冲击负荷，这是在像学习造成的焦点冲击创伤性​​脑损伤应用中是有用的上下文中如何变形以具体量化的装置。而从各技术的结果是不能直接比较，通过冲击压痕测量的能量耗散特性遵循相同的趋势，通过流变学测量的剪切损耗弹性模量，如下文所讨论。 在这里说明脑组织的启用AFM压痕，我们通过测量蠕变柔并迫使松弛粘弹性。由于AFM探针的小规模的，这种技术可以测量的脑的解剖学不同的区域，如胼胝体和皮质，分别为（ 图 11的白质和灰质区域的机 ​​械性能。4 </strong&gt;的）。同时也有其他的技术用于测定生物样品的微观机械性能 - 诸如磁加捻术，光学镊子和microrheology - 这些技术都没有被成功地用于对由于组织样品中的一部分的光的相对强散射内半透明组织。 我们发现，以这种方式测得的脑组织的粘弹行为性质上类似于以前报道由埃尔金＆莫里森26的结果。而测得的值的松弛模量的大小不同意，这可能是由于在实验条件的差异。埃尔金 - 莫里森使用直径250微米的平冲头，相对于我们的直径为20微米的球体。此外，埃尔金 - 莫里森脑组织进行测量大鼠，而我们进行了从小鼠的脑组织上的测量。尽管存在这些差异，这两种技术MEAS被保险脑组织内异质的机械性能，或者更具体地，胼胝体的白质具有较低的松弛模量比在冠状面皮层灰质。 要注意的是，虽然我们计算蠕变柔并迫使松弛模量响应于请求步骤负荷或步骤压痕，分别用实验施加载荷和缩进是不理想的（瞬时）步骤的功能是很重要的。载荷和凹槽是施加在短的时间尺度（&lt;1秒），而这些加载历史可以影响测量的蠕变和松弛响应7,25。具体来说，假设低估松弛模量轻微施加的一步缩进效果，同时假设蠕变柔模量略有高估施加阶跃负载的结果。实际和计算出的弹性模量之间的差异将减小，升温速率的施加的负荷和缩进增加。 在进行负荷放松的一个关键步骤是选择保持力的适当幅度（ 即对应，直接关系到所施加的力光电二极管的电压设定值）。用于蠕变柔设定值力，必须选择，使得:（1）该反应是大到足以产生压痕深度容易可测量的变化;和（2）足够小，以保持给定值的力所需的压痕深度不会变得过大，以致该调制AFM悬臂基座的垂直位置的原子力显微镜的压电致动器的范围之外漂移。在所提出的协议中，我们建议5 NN，这对于我们的实验装置运转良好的额定值力。然而，如果AFM压电无法维持该力由于其有限的运动范围（ 参见图4A，插图），该值可以降低。这个实验的问题不是encountERED与力松弛实验即保持通过反馈环路常数，计算出的压痕深度。 与此相反的准静态启用AFM压痕在nanoNewton（NN）比例的力和​​微米尺度深处，冲击压痕应用于MN尺度力的集中动态负载，并测量试样的变形响应于接近毫米级的深度。我们以前使用的影响压痕量化心脏和肝脏9,11,12的行为，并观察到类似的负荷率从这些器官组织耗能响应依赖。 影响压痕可容纳探针半径范围从微米到毫米。此外，冲击压痕实验可以在完全浸入的环境中，其允许水合组织21的机械特性来进行。当测试高度兼容样本的脑组织，如，IMPortant考虑必须加以考虑。第一，最大可测深度进料为约1毫米，限制由仪器本身的长度尺度集;任何进一步的摆位移将由设在摆的顶部和固定磁性板的电磁线圈之间的碰撞被物理停止。对于脑组织，这限制了可成功地应用到约5毫米/秒的最高冲击速度。注意，虽然冲击速度都在顺序毫米/秒，相应的应变能量密度为千焦/米3的顺序，接近弹道的条件下，由于探头半径11的小的尺寸上。第二，它可以潜在地变得困难的仪器来检测探针和组织表面之间的接触。作为样品台朝向探针行进时，当摆锤通过移动样品被推回被检测到的接触。然而，对于高度complian吨样品，钟摆可能无法检测地而探针穿入样品偏转。 为了解决这个问题，我们可以增加在该样品台移动，使得会出现接触时更大的动量，以驱动摆回的速度。样品也应尽可能平坦，以便在检测到适当的接触点进一步减少任何错误。最后，注意，冲击载荷是不是真正的脉冲负载，在于在摆锤顶部的电磁电流继续在第一冲击事件之后提供用于渗透的驱动力。其结果是，可以特别是在更高加载条件下，其复杂的能量耗散特性分析发生蠕变。关于这种技术的进一步工作可涉及解耦从冲击响应蠕变响应，经由显微镜引入温度控制，以使在体温下的研究，并包括组织样品表面的可视化Ë与液体细胞相容。 流变测量在宏观水平的粘弹性固体的频率相关的机械性能。剪切模量部件，存储G&#39;和损耗G"，可以在频率被测量范围通常跨越0.001-0.1弧度/秒至10-100弧度/秒，这取决于仪器，探头的几何形状，和样品13。为了精确的测量，振幅扫应之前的频率扫描被执行，以确定该材料的线性弹性范围，这是对于其中G&#39;和G&#39;&#39;保持不变14,27的应变的范围内选择的频率的剪切应变。扫应的线性粘弹性限度内是尽可能的高（通常为1-2％的剪切应变），使得在测量过程中足够的扭矩来实现的。在测量过程中的扭矩应始终在由生产商以确保所提供的允许范围ufficient信噪比。 此外，流变测定探针使用应尽可能大，以最大限度的转矩，但必须与样品完全13叠加。在制备样品，组织应切片尽可能平坦时接触板之间进行，以尽量减少应力梯度。当接触与样品制作中，组织不应该有它的任何水滴，以尽量减少在该界面滑移。然而，组织也不能之前或在测量过程中干燥出来，因为这将降低组织结构13。该组织应与媒体都板之间接触后充分水化。粘合剂，防水砂纸也可附连到所述板，以减少滑28。此外，轴向压缩已经显示出改变的脑组织29的G&#39;的大小。因为流变样品通常是薄的（约5毫米），高度的小变化（〜500微米）可能会产生较大的压缩应变（ 例如 ，〜10％），并在剪切模量因此显著变化。此外，由于样品是粘弹性，该材料会经受应力缓和由于轴向压缩28，这可能会影响测量结果。因此，重复测量应在相似的操作轴向应变来进行，并且样品应该允许（ 例如，5-10分钟）放松测量之前。这些现象相关的误差是该技术的限制。流变的其它限制包括这样的假设的材料是均匀的和各向同性的，这常常是不组织样本13在真实的。此外，温度应保持在生理条件，因为这将影响G&#39;和G"22，在脑组织中具体地，增加的温度已经显示出减少两个G&#39;不改变幂BEHA和G&#39;&#39;温和vior与频率，从而继时间-温度叠加主要22,30。我们的数据与在猪脑以前的研究22,27，其观察到的G&#39;和G&#39;的类似的量值，以及在这两个G&#39;和G"弱幂频率依赖性一致。 计算出的比率的tanδ= G"/ G&#39;（ 图6）提供流变和冲击压痕之间的比较中的一个基础。在冲击压痕，我们发现，脑组织的能量耗散能力增加负载率增加。使用流变，我们发现作为频率的增加，tanδ的也有所增加。换句话说，该物质是在更高的频率更耗散。另外，虽然冲击压痕测量不直接量化的弹性模量，则穿透深度点¯x 最大值减少直接与increasin克弹性模量。 一起，在本文中所描述的方法使脑组织的力学特性在微观，中观，和宏观尺度，并在不同的负荷率。本文所提出的方法也可以在许多柔性材料，包括生物组织和工程化水凝胶的使用。随着脑组织的多尺度粘弹特性的深入了解，我们可以更好地设计，以模拟大脑的机械响应设计材料。这些组织模拟材料可促进机械损伤和保护战略工程的预测。此外，脑的材料性质可用于设计用于体外 和体内研究的仿生材料，以更好地了解细胞的生长和连接在中枢神经系统中，特别是在神经系统疾病如孤独症和多发性硬化的情况下。

包含生物器官最软组织是在机械上和结构复杂，低刚度相比矿化骨或工程材料，并显示出非线性的和取决于时间的变形。相比，在身体的其他组织，脑组织是非常柔顺的，以帕1的100秒的数量级上的弹性模量例如脑组织呈现出具有鲜明和相互交叉的灰质和白质区域也有所不同功能结构的异质性。理解脑组织力学会在材料和计算模型的设计有助于向模拟大脑损伤时的反应，促进机械损伤的预测，并启用的保护策略工程。此外，这样的信息可以用来考虑的组织再生的设计目标，以更好地理解脑组织是与疾病，如多发性硬化症和孤独症有关的结构变化。 HERE，我们描述和证明了可用来表征机械柔性组织中，包括脑组织的粘弹性能几个实验方法，在微观，中观和宏观尺度。 在微尺度，我们进行了蠕变遵守和用原子力显微镜（AFM） - 启用压痕力松弛实验。通常情况下，使能AFM压痕用于估计样品2-4的弹性模量（或瞬时刚度）。然而，同样的仪器也可用于测量微尺度粘弹性（时间或速率依赖性）性质:5-10。这些实验中，在图1所示的原理，是缩进悬臂探针进入脑组织的AFM，保持力或压痕深度指定大小，并测量在分别压痕深度和力，相应的变化，随着时间的推移。通过这些数据，我们可以计算出蠕变补偿分别lianceĴC和松弛模量G R，。 在尺度，我们在维持组织结构和水化水平液体浸泡条件下进行冲击试验压痕，使用的是基于钟摆仪器化纳米压痕。实验装置如图2所示，随着钟摆成与组织接触，探针直到振荡摆锤涉及到组织内休息位移记录为时间的函数。从探头所得阻尼谐振荡运动，我们可以计算出最大穿透深×max时 ，耗能能力K和耗散品质因数Q（其涉及能量耗散率）的组织11,12。 在宏观尺度，我们使用了平行板流变仪来量化该频率依赖剪切弹性模量，称为储能模量G&#39;和损耗模量G"时，组织的。在这种类型的流变的，我们应用一个谐波角株（和对应的剪应变）在已知振幅和频率，并测量reactional扭矩（和相应的剪切应力） ， 如图3，从所测量的转矩的产生幅度和相位滞后和系统的几何变量，我们可以在感兴趣13,14施加的频率计算G&#39;和G"。

<table><tbody><tr><td>Xylazine</td><td>Lloyd Laboratoried</td><td></td><td>perscription drug</td></tr><tr><td>Ketamine</td><td>AnaSed Injections</td><td></td><td>perscription drug</td></tr><tr><td>Vibratome (Vibrating blade microtome)</td><td>Leica</td><td>VT1200</td><td></td></tr><tr><td>Hibernate-A Medium</td><td>Gibco</td><td>A1247501</td><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent neural medium for adult tissue</td></tr><tr><td>Atomic Force Microscope, MFP-3D-BIO</td><td>Asylum Research</td><td>-</td><td></td></tr><tr><td>Petri Dish Heater</td><td>Asylum Research</td><td>-</td><td></td></tr><tr><td>AFM Probe, 0.03 N/m, 10&amp;nbsp;&amp;micro;m radius borosilicate sphere</td><td>Novascan</td><td>PT.GS</td><td></td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td>mussel-derived bioadhesive</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>alternate suppliers can be used</td></tr><tr><td>Sodium Hydroxide, 1 N</td><td>Sigma-Aldrich</td><td>59223C</td><td>alternate suppliers can be used</td></tr><tr><td>Instrumented Indenter, NanoTest Vantage</td><td>Micro Materials Ltd.</td><td>-</td><td>probe tip needs to be machined (steel flat punch, 1&amp;nbsp;mm diameter, 4-5&amp;nbsp;mm length)</td></tr><tr><td>NanoTest Liquid Cell</td><td>Micro Materials Ltd.</td><td>-</td><td></td></tr><tr><td>Parallel Plate Rheometer MCR501</td><td>Anton-Parr</td><td>-</td><td></td></tr><tr><td>PP25&amp;nbsp;</td><td>Anton-Parr</td><td>-</td><td>25 mm diameter flat measurement plate</td></tr><tr><td>Adhesive Sandpaper</td><td>McMaster-Carr</td><td>4184A48</td><td>alternate suppliers can be used</td></tr><tr><td>Loctite 4013 Instant Adhesive</td><td>Henkel</td><td>20268</td><td>alternate suppliers can be used</td></tr></tbody></table>

characterizing multiscale mechanical properties of brain tissue using atomic force microscopy, impact indentation, and rheometry

设计和工程师由大脑的属性激发的材料，无论是机械模拟物或组织再生的研究中，脑组织本身必须很好地表征在不同的长度和时间尺度。像许多生物组织，脑组织呈现出一个复杂的，分层结构。然而，与大多数其它组织，脑是非常低的机械刚度，以Pa的100s的顺序上杨氏弹性模量即这种低刚度可以呈现挑战关键的机械性能的实验表征。在这里，我们表明，已适应测量水分，符合生物材料的脑组织等，在不同的尺度和负荷率弹性和粘弹性能几种机械表征技术。在微尺度，我们进行使用原子力显微镜启用压痕蠕变遵守和力量松弛实验。在mesos凯尔，我们执行使用的是基于钟摆仪器化压压痕冲击实验。在宏观尺度，我们进行平行板流变量化该频率依赖剪切弹性模量。我们还讨论了与每个方法相关的挑战和限制。这些技术共同使脑组织，可用于更好地理解脑的结构和工程师仿生材料进行了深入的机械特性。

图4示出代表压痕和力与时间的反应（ 图4B，C）用于蠕变柔和力松弛实验中，给定分别施加的力或压痕深度（ 图4A中的D）。利用这些数据和系统的几何形状，蠕变柔量J C（t）和强制松弛模量ģR（T）可被计算为大脑（ 图4C中的F）的不同的区域。虽然以前的研究表明大脑23的不同区域的弹性模量之间的差，粘弹性特性以这种方式对小鼠脑组织切片不显示给定的组织切片中的区域间的变化进行测定。 冲击压痕在空间和时间上concentra高速率测量组织的机械性质特德加载。这些实验的结果提供了关于组织响应于外伤性损伤或与手术相关故意的变形如何消耗能量的信息。冲击压痕探针的阻尼振荡运动（ 图2B）提供的信息来计算最大穿入深× 最大 （ 图5A），能量耗散率k（ 图5B）和所述组织的能量耗散率Q（ 图5C）。穿透深度测量抗变形能力，有力地与组织的弹性模量密切相关:更严厉的组织表现出对于给定的冲击速度和冲击能量更小的穿透深度。耗能能力是其中第一冲击循环期间组织耗散冲击能量的程度的一个无单位的量度。耗散品质因数的措施有多少次我从振荡发生前MPACT被显著阻尼 - 这直接涉及能量耗散率，虽然这是不以时间为单位来表示。这三种冲击响应参数可以在不同的冲击速度，这提供研究的组织的速率依赖特性的装置进行定量。 图6示出宏观G&#39;和G"为频率范围从0.1弧度/秒至50弧度/秒。储能模量几乎要比在低频损耗模量大一个数量级，然而，存储和损耗模量之间的比值随频率的增加而增加。这表明弹性性质支配脑组织的行为，因为储能模量描述弹性性能和损耗模量描述该材料的粘滞度损失。在一个足够高的装载次数，所述存储和损耗模量都等同，指示在该点该材料开始流动（ 即 ，粘性性质支配样品的行为）。如本文所示测量脑组织的情况下，仪器仪表的物理限制不允许我们在更高的频率测量的材料特性。 <img alt="图1" src="/files/ftp_upload/54201/54201fig1.jpg" /> 图1. 启用AFM-蠕变柔的插图，并迫使松弛实验。（A）中启用的AFM-缩进使用柔性悬臂具有纳米的球状珠附着到自由端微尺度半径进行。 （B）在缩进，悬臂偏转使用激光反射离开悬臂的并到达光电二极管结束时测量。 （C）力松弛实验被缩进悬臂恒定的应用进行了深入的，而力衰减与资源PECT时间被测量。 （D）的蠕变柔措施以恒定作用力悬臂的变化压痕深度。 （C）和（D）已经被分为五个区域（绿色文本）:（1）原子力显微镜探针与样品表面的方法，（2）与样品接触，并上升到设定值压痕/力，（3）维护设定压痕/力，（4）减速并（5）从样品表面的原子力显微镜探针的回缩。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54201/54201fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54201/54201fig2.jpg" /> 图2. 插图影响压痕实验。（A）冲击压痕示意图，说明在充分水化条件进行实验的能力。 （B）Repres<html> entative探针位移分布作为来自小鼠脑切片和相应的速度分布收集时间的函数。关键测量位移，用来量化耗能指示计算速度参数， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54201/54201fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54201/54201fig3.jpg" /> 图3.的平行板流变仪的实验。（A）的平行板流变仪实验和与施加的振荡剪切应变定义的示意图。 （B）的代表性的外加应变和所得应力作为时间的函数。剪切储能模量G&#39;和剪切损耗模量G"经由应变振幅计算54201 / 54201eq12.jpg"/&gt;，扭矩幅度T&#39;0，相位滞后<img alt="式（1）" src="/files/ftp_upload/54201/54201eq13.jpg" /> ，探针和样品半径R，和样品高度h。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54201/54201fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54201/54201fig4.jpg" /> 图4.从蠕变柔代表性数据，并迫使松弛实验。（A，B）从图1中的原始数据，感兴趣区域为蠕变柔如那里施加力时定义保持不变（A）中 ，而压痕深度测定（B）中 。 （A）中的插图显示了一个不成功的实验，其中AFM压电无法数据保持所施加的力，并在（B）中的插图显示了对应的压痕反应，这是定性类似于在（B）中示出的成功实验的数据。 （c）与从所施加的力的数据，测量的压痕，以及探针的几何形状的知识，蠕变柔量J C（t）被计算出来。 （D，E）在力松弛，压痕深度保持恒定（D），而力与时间的测定（E）。 （F）的利用这些数据，力松弛模量G R（T）可以被计算出来。蠕变柔并迫使松弛实验可对大脑的解剖不同的区域，如胼胝体（红色）和皮质（蓝色）进行。在（C，F）数据是从N = 5只小鼠测量的平均值。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54201/54201fig4large.jpg" target="_blank">请CLIC</a>ķ此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/54201/54201fig5.jpg" /> 图5.影响压痕实验的代表性数据。最大穿透深x 最大值 ，耗能能力K，和耗散品质因子小鼠脑组织Q的从不同的冲击速度获得的原始位移范围计算。数据表示为平均值±标准偏差（n =每18点重复测量）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54201/54201fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54201/54201fig6.jpg" /> 图6.流变实验的代表性数据。存储G&#39;和损失G&#39;&#39;猪大脑冠状切片的模量。量的tanδ计算为丧失对储能模量的比值。数据表示为平均值±标准偏差（n =每4点重复测量）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54201/54201fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry at JoVE.com

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

characterizing-multiscale-mechanical-properties-brain-tissue-using

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

12.2K 次观看.  Massachusetts Institute of Technology. We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

视频: Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

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, ...

科研

生物工程

Quantitative Hardness Measurement by Instrumented AFM-indentation

In this work, a combination of amplitude-modulated non-contact atomic force microscopy and atomic force spectroscopy is applied for instrumented hardness measurements on an Au(111) surface with atomistic resolution of single plasticity events. A careful experimental procedure is described that includes the force sensor selection, its calibration, the calibration of the cantilever deflection detection system, and the minimization of instrumental drift for accurate and reproducible force-distance measurements. Also, a method for the data analysis is presented that allows the extraction of force-penetration curves from recorded force-distance curves. A typical curve displays a clear elastic deformation regime up to the first plasticity event, or pop-in, with a length in the range of one to two Burger's vectors. Later plasticity events exhibit the same magnitude. The work of plasticity is further extracted from the measurements. Finally, the hardness is determined in combination with the indentation curve using non-contact atomic force microscopy images of the remaining indents.

This experimental protocol describes how atomic force microscopy can be used to measure hardness at the true nanometer scale and to detect single atomistic plasticity events.

通过仪表AFM压痕硬度定量测量

In this work, a combination of amplitude-modulated non-contact atomic force microscopy and atomic force spectroscopy is applied for instrumented hardness ...

工程学

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

表征多层鱼鳞（中<em&gt; Atractosteus锅铲</em&gt;）基于纳米压痕技术，X射线CT，红外光谱和SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

微型机械的肺组织利用原子力显微镜表征

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

生物学

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

近同步激光扫描在活细胞上的共和和原子力显微镜（康波卡尔）

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

软物质纳米压痕的实验和数据分析工作流程

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

基于原子力显微镜的人关节软骨外植体微压痕研究的实际问题

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young&#39;s modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young&#39;s modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young&#39;s moduli, and generation of Young&#39;s modulus maps. Finally, it shows how to determine and isolate Young&#39;s modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

This protocol visualizes how to prepare cryosections of murine lung tissue, perform atomic force microscopy force map experiments, and analyze the data to determine Young's modulus of the native murine pulmonary basement membrane.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

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