我们感谢他们正在进行的合作A. Tager和B。乳木果，并提供用于演示目的的肺组织。这项工作是由国家卫生补助HL - 092961研究院的支持。这项工作是在纳米系统（CNS），国家纳米技术基础设施网络的成员，这是由国家科学基金会的支持下国家科学基金会奖ECS - 0335765中心部分。中枢神经系统是在哈佛大学艺术和科学学院的一部分。

1。肺组织地带准备<ol><li>肺组织是高度兼容，难以切成条状原子力显微镜表征。瞬时稳定切割肺结构，膨胀小鼠离体肺，气管，50毫升/公斤体重2％的低凝点琼脂糖（用PBS配制）加热到37℃铁气管和冷却在4浴的PBS膨胀肺° 60分钟的彗星。琼脂糖凝胶和变硬空域轻轻稳定在这个区间5的肺结构。琼脂糖浓度较高，即3-4％，可用于进一步加强组织切割稳定。 </li><li>成5 × 5毫米的长度和宽度和厚度在400微米条用剃刀或解剖刀刀片剪切琼脂糖稳定的小鼠肺组织，然后洗一个PBS水浴预热至37条℃用100毫升5分钟以去除残留的琼脂糖台式加热搅拌盘上的玻璃烧杯。为了排除大的航空公司和船舶，切条从遥远从主茎支气管胸膜地区。如果航空公司和大血管进行成像，切条近端主茎支气管肺组织。 </li></ol> 2。的原子力显微镜Microindentation和荧光成像<ol><li>为了隔离感兴趣的领域的AFM microindentation，组织可以进行可视化相衬显微镜或免疫染色，荧光显微镜观察。免疫，组织带块与5％，从2小时或在室温下没有固定通透源所需的二次抗体（PBS）的血清。 </li><li>与相应的抗体孵育组织地带（如兔抗Ⅰ型胶原的可视化间质胶原（1:250稀释），兔抗层粘连蛋白（1:250稀释）的可视化基底膜）在12孔板以及中在1小时轻轻摇动摇杆，洗5分钟，用PBS样品的3倍，其次是适当的二级抗体结合荧光标记（如的Alexa Fluor 546山羊抗兔二抗（1:250稀释））1在室温小时。 </li><li>广泛组织带用PBS和存储在PBS清洗4 ° C </li><li>紧接原子力显微镜表征前，将组织地带到15毫米的聚- L -赖氨酸包盖玻片盖玻片解除低于浮动组织，确保组织地带盖玻片表面均匀传播，如有必要，三明治的地带第二清洁，无涂层的盖玻片和应用温和的压力，以协助组织附着的聚- L -赖氨酸包被的盖玻片。 </li><li>每一轮microindentation实验前制造的指令立即校准AFM系统。确定两个关键参数:1）悬臂弹簧常数使用热空气中的6的波动方法;，2）悬臂偏转灵敏度，这是用于规模的光电二极管的输出信号，以实际悬臂偏转距离参数，δD 。获得一个干净的载玻片在PBS一个标准的力 - 位移曲线，然后计算出的力 - 位移曲线（图1B）的斜率校准偏转灵敏度。力 - 位移曲线测量，延长对针尖和样品表面在一个位置（不包括在XY方向光栅）缩回，悬臂的偏转，δD尖位移，ΔZ功能监测， 。 </li></ol>我们建议使用一个直径为5微米的硼硅球形提示（Novascan）氮化硅三角形悬臂。使用AFM探针弹簧常数为0.06牛顿/米，我们有机械的特点与软质材料剪切模量跨越100至50,000霸。 <ol start="6"><li>样品盖玻片用纸巾擦拭底部表面，固定到一个真空润滑脂的标准载玻片，安装在载玻片上的原子力显微镜样品台上，和封面用500μlPBS（室温）的组织。 </li><li>设置显微镜目镜观看对齐针尖在视野的中心，通过调整显微镜样品台上。选择组织的利益方面，通过移动的原子力显微镜样品台上，然后切换到CCD摄像头，查看记录相衬图像和/或组织的荧光图像，根据需要。 </li><li>移动针尖慢慢向下，直到它与样品接触的是执行标准的AFM参与操作。 </li><li>精确的原子力显微镜表征microindentation软样品需要小压痕深度，以避免大的地方株无效赫兹的弹性模量计算模型。要避免大的菌株，进行触发模式设置悬臂最大挠度（触发点）到500 nm压痕。这偏转限制将抑制最大压痕力小于30 NN（F =悬臂弹簧常数*位移，或F MAX = 0.06 NN / NM * 500纳米= 30 nN个）。 </li></ol>应选择压痕速度足够慢的探索弹性而软的样品 7粘弹性能。一个2-20微米每秒的速度范围内是适用于肺组织。 <ol start="10"><li>对于感兴趣的领域从单一测量，原子力显微镜的探针移动感兴趣的位置，并执行一个单一的缩进收集一个标准的力 - 位移曲线（XY扫描探头，只有在Z轴方向移动没有）。 </li><li>自动映射一个感兴趣的区域，切换到部队的地图模式，选定的区域内，选择扫描尺寸和采样点。在部队的地图模式，整个样品表面之间的缩进运动针尖栅格，并在每个点的范围内收集个人力 - 位移曲线，一个定义的采样网格。更多的采样点，将提供更高的空间分辨率，但延长的映射所需的时间。我们发现它实际使用了一个16 x 16个样品的网格地图缩进速度在每秒20微米，可以在大约10分钟完成一个80 × 80微米的区域。 </li></ol> 3。 AFM数据提取<ol><li>要计算的杨氏模量，适应力-位移曲线赫兹的球形压痕模型，利用最小二乘拟合非线性弯曲 8（图1，C ）: <img src="/files/ftp_upload/2911/2911eq1.jpg" alt="公式1" /> 其中F = k 彗星 *Δ 剐 ，的力量来弯曲的悬臂，K C是的悬臂弹簧常数，ř是领域的尖端半径，δ=Δž - Δð是缩进和υ是样品的泊松比率（υ= 0.4肺组织9）。 </li><li>为了评估适合的质量，计算出的SSR值，或数据和拟合值之间的差异的平方的总和，在非线性曲线拟合。消除"坏"的曲线，与大SSR值丢弃的数据不可靠或联合国解释测量。 </li><li>如果需要，弹性模量（E）转换为剪切模量（G），使用关系:E = 2 ×（1 +υ）× G.要可视化的空间刚度力的地图模式中收集的图案，等高线图图模量数据（例如，在16 × 16的样本电网覆盖一个80 × 80微米的面积）。 </li><li>要处理大量的力曲线数据，可能会被写入一个自定义的算法自动适应力 - 位移曲线，提取模量，和/或使用相同的程序和参数，在3.2和3.3（如MATLAB）中所述的情节elastographs。 </li></ol><img src="/files/ftp_upload/2911/2911fig1.jpg" alt="图1" /> 图1（a）使用球形探头（Dimitriadis大肠杆菌等2002生物物理学J 8许可转载）一个刚性基板上的软样品的原子力显微镜microindentation的原理图。 （b）代表力 - 位移曲线，收集在PBS洁净的载玻片，以确定悬臂偏转灵敏度。 （c）代表力 - 位移曲线从软（蓝色）和僵硬的（红色），显示相应的参数（一）样品收集。 4。代表性的成果: 图2A显示了肺实质带附着在涂层聚- L -赖氨酸的盖玻片在PBS代替上述地区的利益直接与原子力显微镜的探针和准备microindentation映射。图2b显示，在适当的剪裁和染色的组织，肺实质的肺泡微架构是保存完好，没有固定或通透的组成部分基底膜层粘连蛋白的免疫荧光染色观察。图2 CD显示我在收获以前，用PBS（图2C）处理的小鼠，或与博莱霉素（图2D）处理，诱导间质纤维化 10新鲜不固定的肺胶原蛋白的免疫荧光染色。 图3a显示样品的力 - 位移，从原子力显​​微镜microindentation获得的地块。随着同样适用于力量，针尖会产生一个大的缩进于软的区域（蓝线），在一个相对的"扁平化"的一个小的压痕和地区的围追堵截"陡峭"的力 - 位移曲线与力 - 位移曲线（红线）。为了获得清洁力曲线，每个缩进后针尖需要完全缩回从样品的表面和接触之前，未来的缩进。这种接触的自由状态，对应于图3A，提示没有悬臂偏转转换曲线的平坦区域。图3B显示了一个典型的虚假曲线不平坦的地区，发生笔尖的时候，不能完全缩回从样品表面（如软样品ttaches到TIP）使其无法确定接触点。如果提示是完全被困在软样品，曲线看起来可能如图3C所示，没有明确的偏转，但噪音小。 图4显示了剪切模量提取的数据空间作为刚度地图（elastographs），剪切模量的色阶显示力 - 位移曲线。 Elastographs表现出显着的差异，在正常的范围和组织剪切模量的分布（图4A）和纤维化（图4B），特别是在肺纤维化样品肺实质和剪切模量较大的空间变化，。 <img src="/files/ftp_upload/2911/2911fig2.jpg" alt="图2" /> 图2:（一）相衬显微鼠标肺实质条显示了原子力显微镜的探针。比例尺，50微米。 （二）在正常小鼠肺组织层粘连蛋白的免疫染色显示正常肺组织的肺泡微架构。白框表示典型的由80 -80μm的弹性映射区域。比例尺:20微米。 （C和D）我在生理盐水（C）和博莱霉素（四）治疗小鼠肺实质的胶原蛋白免疫染色。比例尺，100微米。 <img src="/files/ftp_upload/2911/2911fig3.jpg" alt="图3" /> 图3（一）代表解释力-位移曲线，分别收集液（蓝色）和博莱霉素治疗（红色）小鼠肺实质。黑线是从球形赫兹模型的最佳契合，并计算其相应的杨氏模量的标记。 （B，C）代表联合国解释力 - 位移曲线。 <img src="/files/ftp_upload/2911/2911fig4.jpg" alt="图4" /> 图4。生理盐水（A）和博莱霉素治疗（二）小鼠肺组织收集的代表elastographs 。彩条显示在千帕的剪切模量。轴标签显示的空间尺度在微米。比例尺:20微米

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没有利益冲突的声明。

肺组织使用原子力显微镜microindentation的机械特性，提供了前所未有的空间分辨率（图4），提供一个独特的视角对微观组织的刚度变化。由于其效用的一个例子，以前的宏观尺度的测量表明在正常和纤维化的肺组织条近似倒电容与纤维化11,12增加2 3倍。相比之下，原子力显微镜microindentation显示，一些地区参展〜30倍，在正常肺组织10中观察到的中位数以上的剪切模量的增加，该组织变硬是高度本地化的。作为基体刚度是目前已知看房影响细胞功能，这些地方的测量提供了宝贵的参数，以提高细胞肺癌细胞培养研究biofidelity。 几个实际问题的产生与使用细条状的肺组织。条表面完全平坦，为组织配置文件如下的基本肺泡架构。 AFM系统自动调整在压痕探针在Z方向的位置时，样品表面的高度变化超过15微米的小，以帮助克服这一挑战。测量是在室温，37℃，所以不能在这种温度变化所造成的机械性能偏差评估，尽管他们会预计将轻微。底层的肺泡壁结构上观察到的机械性能的影响是难以确定，与目前的光学显微镜设置。举例来说，将是可取的，以确定是否肺泡壁时，表现出的各向异性和不同的力学性能符合或横墙的平面的方向缩进。然而，当前的标本厚成像系统没有3D功能，因此它是不可能在每个接触点，以确定当地的肺泡壁方向。最后，细胞成分的测量机械性能的影响仍有待完全阐明。在这里详细的方法，没有作出努力，特别是消除组织的细胞成分。然而，细胞表面压痕可用目前是不大可能是可行的，自组织收获和切割组织进入到肺泡的必要性所经过的时间。具体的实验，去除细胞，或与活细胞重新填充矩阵，评估组织刚度产生的变化会出现必要的。 由于这些测量需要新鲜不固定的组织，从组织收获已过测量时间应尽量减少和样品应保存在4 ° C，以避免在机械性能的变化。每当组织条在洗涤或染色，以便产生最小的失真或损坏容器之间转移支付，应特别注意。原子力显微镜在液体中的应用，一个关键步骤是削减尽可能扁平的组织和支持盖玻片固定的样品。如果可用，作为vibratome或组织切片机自动切片机，可以用来切割高度厚度均匀的片。重要的是立即组织带附加AFM测量前，尽量减少AFM测量经过的时间，作为样品将最终脱离的盖玻片。一个有用的观察，出现较大条连接更加稳定盖玻片，并留在原地，持续时间较长的PBS比较小条。 原子力显微镜的microindentation可以表征样本，涵盖了广泛的范围从100帕到50千帕（剪切模量）一起使用时，一个直径5微米的球形提示一个标准的0.06牛顿/米的悬臂。这个范围可以扩大使用与不同的弹簧常数探针;原子力显微镜探针与球面玻璃从0.6至12微米范围从0.01至0.58牛顿/米的直径和弹簧常数不等市售（例如Novascan），常用3的提示。 5微米的球形提示，提示和组织之间的理论的接触面积约5-9微米为400-700纳米压痕（ 图 1A ）2。更大或更小的技巧可以被用来提供更大或更小尺度的空间分辨率。锥体提示也被用于在原子力显微镜microindentation 13-16，提供更小的接触面积，从而增加映射的空间分辨率，虽然数据拟合这个尖几何形状更为复杂。 这种方法应注意的几个限制。肺历来被机械的非侵入性的特点，例如使用压力-容积分析 17或整个肺部穿孔缩进 19,20 。这里描述的改变肺结构的重要途径之一通过空气液体INT的损失，如侵入性的方法erface，通常在充满空气的肺和预应力的损失，保持呼吸道肌肉放松后肺局部通胀存在。这些限制是在肺组织带18的所有测量的共同。然而，值得注意的是，中位数刚度测量在正常肺组织的实质（剪切模量〜0.5kPa）不大幅不同于基于 19,20休息卷完好肺部穿孔缩进的估计。虽然肺组织表现出越来越变形的非线性劲，它是不可能的，在严格的方式进行测试，这个属性是否仍然存在，这里的方法，以微观尺度。赫兹模型假设样本的同质性。但是，大多数生物材料，包括肺实质，越来越多地在减少空间尺度上的异构。样本的异质性，可能会导致文物如根据压痕深度，即取决于层或元件，是变形的杨氏模量的变化。在XY平面的异质性，可以通过仔细选择合适的球形针尖大小，取决于Dimitriadis EK等提出的生物材料的微观结构有限8这是更难以预测或不正确的赫兹模式错误，由于材料。在z方向的异质性。 Azeloglu等。最近提出了一种混合计算模型来表征异构基板的弹性性能与离散嵌入式夹杂21。他们的新技术提供了潜在的手段来克服赫兹分析的局限性的非均质材料计算列入属性。 赫兹模型还假定绝对的弹性行为，而生物材料通常显示随时间变化的粘弹性行为。一个完整的组织粘弹特性，可通过改变所使用的压痕速度。更重要的是，以前的宏观尺度的机械测试显示正常和纤维化的肺组织肺组织的力学性能弱的频率的依赖，并保存所有频率的正常和纤维化组织的力学性能之间的差异测试11。这些研究结果有力地表明，使用一个单一的压痕速度与原子力显微镜的机械性能的测量捕获组织的力学性能的变化，伴随着肝纤维化的一个重要方面。 0.4肺组织在这项工作中使用的泊松比是从宏观的测量9。不幸的是，在微观尺度的泊松比，并根据病情的任何变化都没有在文献中。 22作为替代品E，E /（1 -υ2）或（1 -υ2）/πE（表示弹性常数k）可以计算出原子力显微镜microindentation和报告时的泊松比是未知的。对于大多数生物材料的泊松比是在0.4至0.5的范围内，由于其含水量高。在0.3-0.5的范围内，只有从1.10-1.33因子1 /（1 -υ2）不同，这样合理的泊松比的变化对报告模量的影响有限。 ，我们为纤维组织相对正常组织报告的剪切模量的增加幅度的数倍，这意味着与泊松比的变化相关联的错误是轻微的相对力学性能的变化观察。 具体应用条件和后续力 - 位移曲线的不同人群的特点是受力 - 位移数据的分析，可以使用实际的算法和代码。如果感兴趣的是更复杂的分析，一个可咨询林等人的工作。23 。作者编译成一系列的协同战略的一个算法，克服了许多先前已阻碍了努力自动化赫兹压痕数据模型的拟合并发症。 其他几个领域的进一步发展，并利用此方法可用。其中一个是在可视化没有抗体标记的肺泡壁有兴趣的情况下，弹性蛋白和胶原蛋白可以从他们在绿色光谱autofluorescent信号的可视化。另一方面，更好的成像，使用或者稀释剂的组织切片，三维成像技术，或两者，可以加强相互联系的能力与组织架构的基本力学性能。虽然目前的方法使细胞外基质成分，如胶原蛋白和层粘连蛋白的染色和可视化，更多的努力可以针对染色细胞表面标志确定具体的细胞群，并在这些人群中附近机械的微环境特征。 Alt键ernatively，组织可收获小鼠表达谱系标记或细胞的特定蛋白质荧光标记的追求相同的目标。最后，该方法的详细，出现在这里非常适合描述其他解剖特点，如改造中高血压的船只，并改造在哮喘气道，肺，。根据其目前的发展和进一步提高的潜力状态，原子力显微镜microindentation似乎准备组织刚度的变化，陪在肺部疾病进展产生有价值的见解，无疑将是有价值的，在时空变化特征在刚度的各种其他软组织。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>针尖</td><td> Novascan </td><td> PT.GS </td><td> 5微米的高硼硅珠，0.06牛顿/米</td></tr><tr><td>聚- L -赖氨酸盖玻片</td><td> VWR </td><td> 354085 </td><td>屋宇署BioCoat 12毫米的圆形第一玻璃</td></tr><tr><td>琼脂糖凝胶温度低</td><td>西格玛</td><td> A0701 </td><td></td></tr><tr><td>兔抗- I型胶原（Rb的PAB）抗体</td><td> Abcam公司</td><td> ab34710 - 100 </td><td></td></tr><tr><td>的Alexa Fluor 546山羊抗兔抗体</td><td> Invitrogen公司</td><td> A11010 </td><td></td></tr><tr><td>兔抗-层粘连蛋白</td><td>西格玛</td><td> L9393 </td><td></td></tr></tbody></table>

micro-mechanical characterization of lung tissue using atomic force microscopy

矩阵刚度强烈地影响着1-3的贴壁细胞生长，分化和功能。在宏观尺度的组织和器官在人体内的刚度，跨越几个数量级4订单。更别提了解如何刚度空间内组织的变化，刚度变化的范围和空间尺度疾病过程中，在组织重构的结果。为了更好地了解如何在矩阵刚度的变化在健康和疾病的细胞生理学，在相关的空间尺度，居民细胞获得组织刚度​​的测量是必要的。这是肺，高度兼容和弹性纤维组织基质重塑是一个突出的特点，如哮喘，肺气肿，高血压和纤维化等疾病尤其如此。为了描述在空间尺度上，有关居民细胞肺实质本地的力学环境，我们已经开发出直接测量新鲜小鼠肺组织利用原子力显微镜（AFM）microindentation局部弹性性能的方法。有了适当的选择，AFM的压头，悬臂式，并压痕深度，这些方法允许相衬和感兴趣的区域的荧光成像并行局部组织的剪切模量的测量。组织带系统抽样提供了组织机械性能，剪切模量，揭示了当地的空间变化的地图。机械性能及相关的解剖和病理特征之间的相关性说明如何刚度与基质沉积在肝纤维化的变化。这些方法可以扩展到其它软组织疾病的进程，揭示局部组织的力学性能如何跨越空间和疾病的进展而异。

Watch this Scientific Journal Video about Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy at JoVE.com

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

细胞外基质的刚度强烈地影响着贴壁细胞的多种行为。矩阵刚度不同空间在整个组织，并在各种疾病的情况下发生的修改。在这里，我们开发方法来表征的空间变化，在正常和纤维化小鼠肺组织的刚度，使用原子力显微镜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 ...

micro-mechanical-characterization-lung-tissue-using-atomic-force

Nanyang Technological University

Research

JoVE Journal

Biology

22.8K 次观看.  Harvard School of Public Health. 细胞外基质的刚度强烈地影响着贴壁细胞的多种行为。矩阵刚度不同空间在整个组织，并在各种疾病的情况下发生的修改。在这里，我们开发方法来表征的空间变化，在正常和纤维化小鼠肺组织的刚度，使用原子力显微镜microindentation。

视频: Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

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

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 measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

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 ...

科研

工程学

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

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

亚纳米级分辨率成像与调幅原子力显微镜在液体

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

发育生物学

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 ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百个 C.白化病 细胞生物原子力显微镜测量自动化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

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 ...

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.

从原子力显微镜衍生的力图中提取天然小鼠肺基底膜的杨氏模量

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.
To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young&#39;s (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.
A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

We present a protocol to measure the elastic moduli of collagen-rich areas in normal and diseased liver using atomic force microscopy. The simultaneous use of polarization microscopy provides high spatial precision for localizing collagen-rich areas in the liver sections.

使用原子力显微镜结合偏振显微镜测量肝硬度

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

微型机械的肺组织利用原子力显微镜表征

摘要

探索更多视频

此视频中的章节

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

使用原子力显微镜测量活细胞的力学性能

亚纳米级分辨率成像与调幅原子力显微镜在液体

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

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

数百个 C.白化病细胞生物原子力显微镜测量自动化

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）

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

从原子力显微镜衍生的力图中提取天然小鼠肺基底膜的杨氏模量

使用原子力显微镜结合偏振显微镜测量肝硬度

微型机械的肺组织利用原子力显微镜表征

摘要

探索更多视频

此视频中的章节

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

使用原子力显微镜测量活细胞的力学性能

亚纳米级分辨率成像与调幅原子力显微镜在液体

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

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

数百个 C.白化病 细胞生物原子力显微镜测量自动化

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）

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

从原子力显微镜衍生的力图中提取天然小鼠肺基底膜的杨氏模量

使用原子力显微镜结合偏振显微镜测量肝硬度

数百个 C.白化病细胞生物原子力显微镜测量自动化