Name: atomic force microscopy cantilever-based nanoindentation: mechanical property measurements at the nanoscale in air and fluid
Uploaded: 2022-12-02T13:10:00
Description: Watch this Scientific Journal Video about Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid at JoVE.com

所有AFM实验均在博伊西州立大学表面科学实验室（SSL）进行。SEM表征在博伊西州立材料表征中心（BSCMC）进行。本出版物中报道的有关生物燃料原料的研究得到了美国能源部、能源效率和可再生能源办公室、生物能源技术办公室作为原料转换接口联盟 （FCIC） 的一部分以及美国能源部爱达荷州运营办公室合同 DE-AC07-051ID14517 的部分支持。细胞力学研究得到了美国国立卫生研究院（美国）AG059923、AR075803和P20GM109095的资助，以及美国国家科学基金会（美国）的1929188和2025505资助。模型脂质双层系统工作得到了美国国立卫生研究院（美国）R01 EY030067资助的支持。作者感谢Elton Graugnard博士制作了 图11所示的合成图像。

注意：由于商用AFM种类繁多，并且基于悬臂的纳米压痕存在多种样品类型和应用，因此以下方案有意设计为相对通用的性质，侧重于所有悬臂式纳米压痕实验所需的共享步骤，无论仪器或制造商如何。正因为如此，作者假设读者至少对操作用于执行悬臂式纳米压痕的特定仪器具有基本的熟悉程度。然而，除了下面概述的一般协议外，还包括特定于AFM和此处使用的软件的详细分步标准操作程序（SOP?...

样品制备 对于空气中的纳米压痕，常见的制备方法包括冷冻切片（例如，组织样品）、研磨和/或抛光，然后进行超薄切片（例如，树脂嵌入的生物样品）、离子研磨或聚焦离子束制备（例如，不适合抛光的半导体、多孔或混合硬度样品）、机械或电化学抛光（例如，金属合金）或薄膜沉积（例如，原子层或化学气相沉积， 分子束外延）。目标是创建具有最小表面粗糙度的样品（?...

了解材料的机械性能是工程中最基本和最重要的任务之一。对于散装材料性能的分析，有许多方法可用于表征材料系统的机械性能，包括拉伸试验1，压缩试验2和三点或四点弯曲（弯曲）试验3。虽然这些微观测试可以提供有关散装材料特性的宝贵信息，但它们通常是在失败的情况下进行的，因此具有破坏性。此外，它们缺乏准确研究当今感兴趣的许多材料系统的微米和纳米级特性所需的空间分辨率，例如薄膜，生物材料和纳米复合材料。为了开始解决大规模机械测试的一些问题，主要是其破坏性，从矿物学中采用了显微硬度测试。硬度是衡量材料在特定条件下对塑性变形的抵抗力的指标。通常，显微硬度测试使用通常由硬化钢或金刚石制成的硬探头压入材料中。然后可以使用所得压痕深度和/或面积来确定硬度。已经开发了几种方法，包括维氏硬度4、努氏硬度 5 和布氏硬度 6;每个都提供了微观材料硬度的测量，但在不同的条件和定义下，因此仅产生可以与在相同条件下进行的测试进行比较的数据。
开发了仪器化纳米压痕，以改进通过各种显微硬度测试方法获得的相对值，提高用于分析机械性能的空间分辨率，并能够分析薄膜。重要的是，通过利用Oliver和Pharr7首先开发的方法，可以通过仪器化纳米压痕确定样品材料的弹性或杨氏模量E。此外，通过使用Berkovich三面锥体纳米压痕探头（其理想的尖端面积函数与维氏四面锥体探头相匹配）8，可以直接比较纳米级和更传统的微米级硬度测量。随着AFM的普及，基于AFM悬臂的纳米压痕也开始受到关注，特别是在测量较软材料的机械性能方面。因此，如图1所示，目前用于询问和量化纳米级机械性能的两种最常用的技术是仪器化纳米压痕（图1A）和基于AFM悬臂的纳米压痕（图1B）9，后者是本工作的重点。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64497/64497fig01.jpg"> 图 1：仪器化和基于 AFM 悬臂的纳米压痕系统的比较。 示意图描述了用于进行（A）仪器化纳米压痕和（B）基于AFM悬臂的纳米压痕的典型系统。这个数字是从钱等人51修改而来的。缩写：AFM = 原子力显微镜。 <a href="https://www.jove.com/files/ftp_upload/64497/64497fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
仪器化和基于AFM悬臂的纳米压痕都使用刚性探针使感兴趣的样品表面变形，并监测合力和位移作为时间的函数。通常，所需的负载（即力）或（Z压电）位移曲线由用户 通过 软件界面指定并由仪器直接控制，而另一个参数则被测量。最常从纳米压痕实验中获得的机械性能是弹性模量（E），也称为杨氏模量，它具有压力单位。材料的弹性模量是与粘结刚度有关的基本属性，定义为在塑性变形开始之前，拉伸或压应力（σ，每单位面积施加的力）与轴向应变（ε，沿压痕轴的比例变形）之比（即可逆或暂时）变形（方程 [1]）：
<img alt="Equation 1" src="/files/ftp_upload/64497/64497eq01.jpg" style="margin: auto;">（1）
应该注意的是，由于许多材料（特别是生物组织）实际上是粘弹性的，因此实际上，（动态或复杂）模量由弹性（存储，同相）和粘性（损失，异相）成分组成。在实际操作中，在纳米压痕实验中测量的是还原模量 E*，它与目标的真实样品模量 E有关，如公式（2）所示：
<img alt="Equation 2" src="/files/ftp_upload/64497/64497eq02.jpg" style="margin: auto;">（二）
其中 E 尖端和 ν 尖端分别是纳米压痕尖端的弹性模量和泊松比，ν 是样品的估计泊松比。泊松比是横向应变与轴向应变的负比，因此表示样品在承受轴向应变时（例如，在纳米压痕载荷期间）的横向伸长程度，如公式（3）所示：
<img alt="Equation 3" src="/files/ftp_upload/64497/64497eq03.jpg" style="margin: auto;">（三）
从降低模量转换为实际模量是必要的，因为a）压头尖端施加的一些轴向应变可以转换为横向应变（即，样品可能通过垂直于加载方向的膨胀或收缩而变形），以及b）压头不是无限硬的，因此压痕样品的行为导致尖端的一些（小）变形量。请注意，在E尖端>>E的情况下（即压头比样品硬得多，这在使用金刚石探头时通常是正确的），减少的样品模量和实际样品模量之间的关系大大简化为E ≈ E*（1 - v2）。 虽然仪器化纳米压痕在准确的力表征和动态范围方面具有优势，但基于AFM悬臂的纳米压痕速度更快，提供更高数量级的力和位移灵敏度，可实现更高分辨率的成像和改进的压痕定位，并且可以同时探测纳米级的磁性和电学特性9.特别是，基于AFM悬臂的纳米压痕在纳米级软材料（例如，聚合物，凝胶，脂质双层和细胞或其他生物材料），极薄（亚μm）薄膜（基底效应可能取决于压痕深度）10，11和悬浮二维（2D）材料12，13，14（例如石墨烯）的纳米级机械性能方面具有优越性15，16，云母17，六方氮化硼（h-BN）18或过渡金属硫族化物（TMDC;例如MoS2）19。这是由于其精细的力（sub-nN）和位移（sub-nm）灵敏度，这对于准确确定初始接触点并保持在弹性变形区域内非常重要。
在基于AFM悬臂的纳米压痕中，AFM探针向样品表面的位移由校准的压电元件驱动（图1B），柔性悬臂最终由于与样品表面接触时经历的电阻力而弯曲。悬臂的这种弯曲或偏转通常通过将激光从悬臂背面反射到光电探测器（位置敏感探测器 [PSD]）中来监测。再加上悬臂刚度（以nN / nm为单位）和偏转灵敏度（以nm/V为单位）的知识，可以将测量的悬臂挠度（以V为单位）转换为施加在样品上的力（以nN为单位）。接触后，Z-压电运动和悬臂偏转之间的差异会产生样品压痕深度。结合尖端面积功能的知识，可以计算尖端-样品接触面积。然后可以使用适当的接触力学模型（请参阅讨论的 数据分析 部分）拟合所得力-距离或力-位移（F-D）曲线的接触部分的斜率，以确定样品的纳米力学性能。虽然基于AFM悬臂的纳米压痕与上述仪器化纳米压痕相比具有一些明显的优势，但它也带来了一些实际实施挑战，例如校准，尖端磨损和数据分析，本文将对此进行讨论。基于AFM悬臂的纳米压痕的另一个潜在缺点是假设线性弹性，因为接触半径和压痕深度需要远小于压头半径，这在使用纳米级AFM探头和/或表现出显着表面粗糙度的样品时可能难以实现。
传统上，纳米压痕仅限于单个位置或小网格压痕实验，其中选择所需的位置（即感兴趣区域[ROI]）并且单个受控压痕，单个位置中的多个压痕由一些等待时间隔开，和/或以Hz量级的速率执行缩进的粗网格。然而，AFM的最新进展允许通过使用基于高速力曲线的成像模式（根据系统制造商的不同商品名称）同时获取机械性能和形貌，其中力曲线在负载控制下以kHz速率进行，最大尖端样品力用作成像设定点。还开发了傻瓜式方法，允许获取AFM形貌图像，然后在图像内的兴趣点进行选择性纳米压痕，从而对纳米压痕位置进行纳米级空间控制。虽然不是这项工作的主要重点，但代表性结果中提供了基于力曲线的成像和基于傻瓜悬臂的纳米压痕的特定 选定应用示例 ，并且可以与下面概述的协议结合使用（如果在采用的特定AFM平台上可用）。具体来说，这项工作概述了在任何有能力的AFM系统上实际实施基于AFM悬臂的纳米压痕的通用协议，并提供了该技术的四个用例示例（两个在空气中，两个在流体中），包括代表性结果和对细微差别的深入讨论，挑战和成功使用该技术的重要考虑因素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Atomic force microscope</td>
			<td>Bruker</td>
			<td>Dimension Icon</td>
			<td>Uses Nanoscope control software, including PeakForce Quantitative Nanomechanical Mapping (PF-QNM), FastForce Volume (FFV), and Point-and-Shoot Ramping experimental workspaces</td>
		</tr>
		<tr>
			<td>AtomicJ</td>
			<td>American Institute of Physics</td>
			<td>https://doi.org/10.1063/1.4881683</td>
			<td>Flexible, powerful, free open source Java-based force curve analysis software package. Supports numerous contact mechanic models, such as Hertz, Sneddon DMT, JKR, Maugis, and cone or pyramid (including blunt and truncated). Also includes a variety of initial contact point estimation methods to choose from. Supports batch processing of data and subsequent statistical analysis (e.g., averages, standard deviations, histograms, goodness of fit, etc.). Literature citation is: P. Hermanowicz, M. Sarna, K. Burda, and H. Gabry<img alt="Equation 1" src="/files/ftp_upload/64497/64497eq05.jpg" style="margin: auto;" />, &#8220;AtomicJ: An open source software for analysis of force curves&#8221; Rev. Sci. Instrum. 85: 063703 (2014), https://doi.org/10.1063/1.4881683</td>
		</tr>
		<tr>
			<td>Buffer solution (PBS)</td>
			<td>Fisher Chemical (NaCl), Sigma Aldrich (KCl), Fisher BioReagents (Na2HPO4 and KH2PO4)</td>
			<td>S271 (&#62;99% purity NaCl), P9541 (&#62;99% purity KCl), BP332(&#62;99% purity Na2HPO4), BP362 (&#62;99% purity KH2PO4)</td>
			<td>Phosphate buffered saline (PBS) was prepared in the laboratory as an aqueous solution consisting of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 dissolved in ultrapure water. Reagents were measured out using an analytical balance, and glassware was cleaned with soap and water followed by autoclaving immediately prior to use.</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond tip AFM probe</td>
			<td>Bruker</td>
			<td>PDNISP</td>
			<td>Pre-mounted factory-calibrated cube corner diamond (E = 1140 GPa) tip AFM probe (nominal R = 40 nm) with a stainless steel cantilever (nominal k = 225 N/m, f0 = 50 kHz). Spring constant is measured at the factory (k = 256 N/m for the probe, Serial #13435414, used here) and calibration data (including AFM images of indents showing probe geometry) is provided with the probe.</td>
		</tr>
		<tr>
			<td>Diamond ultramicrotome blade</td>
			<td>Diatome</td>
			<td>Ultra 35&#176;</td>
			<td>2.1 mm width. Also used a standard glass blade for intial rough cut of sample surface before transitioning to diamond blade for final surface preparation</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Gorilla Glue</td>
			<td>26853-31-6</td>
			<td>Epoxy resin and hardner were mixed in a 1:1 ratio, a small drop was placed on a stainless steel sample puck (Ted Pella), and V1 grade muscovite mica (Ted Pella) was attached to create an atomically flat surface for preparation of phospholipid membranes.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LR white resin, medium grade (catalyzed)</td>
			<td>Electron Microscopy Sciences</td>
			<td>14381</td>
			<td>500 mL bottle, Lot #150629</td>
		</tr>
		<tr>
			<td>Mesenchymal stem cells (MSCs)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>MSCs for nanomechanical studies were primary cells harvested from 8-10 week old male C57BL/6 mice as described in Goelzer, M. et al. &#34;Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis&#34; Int J Mol Sci 22: 6580 (2021) doi:10.3390/ijms22126580 and Peister, A. et al. &#34;Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential&#34; Blood 103: 1662-1668 (2004), doi:10.1182/blood-2003-09-3070.</td>
		</tr>
		<tr>
			<td>Modulus standards</td>
			<td>Bruker</td>
			<td>PFQNM-SMPKIT-12M</td>
			<td>Used HOPG (E = 18 GPa) and PS (E = 2.7 GPa). Also contains 2x PDMS (Tack 0, E = 2.5 MPa; Tack 4, E = 3.5 MPa), PS-LDPE (E = 2.0/0.2 GPa), fused silica (E = 72.9 GPa), sapphire (E - 345 GPa), and tip characterization (titanium roughness) sample. All samples come pre-mounted on a 12 mm diameter steel disc (sample puck).</td>
		</tr>
		<tr>
			<td>Muscovite mica</td>
			<td>Ted Pella</td>
			<td>50-12</td>
			<td>12 mm diameter, V1 grade muscovite mica</td>
		</tr>
		<tr>
			<td>Nanscope Analysis</td>
			<td>Bruker</td>
			<td>Version 2.0</td>
			<td>Free AFM image processing and analysis software package, but designed for, and proprietary/limited to Bruker AFMs; similar functionality is available from free, platform-independent AFM image processing and analysis software packages such as Gwyddion, WSxM, and others. Has built-in capabilities for force curve analysis, but AtomicJ is more flexible/full featured (e.g., more built-in contact mechanics models to choose from, statistical analysis of force curve fitting results, etc.) for force curve analysis and handles batch processing of force curves.</td>
		</tr>
		<tr>
			<td>Phospholipids: POPC, Cholesterol (ovine)</td>
			<td>Avanti Polar Lipids</td>
			<td>POPC: CAS # 26853-31-6, Cholesterol: CAS # 57-88-5</td>
			<td>POPC lipid dissolved in chloroform (25 mg/mL) was obtained from vendor and used without further purification. Cholesterol powder from the same vendor was dissolved in chloroform (20 mg/mL).&#160;</td>
		</tr>
		<tr>
			<td>Probe holder (fluid, lipid bilayers)</td>
			<td>Bruker</td>
			<td>MTFML-V2</td>
			<td>Specific to the particular AFM used; MTFML-V2 is a glass probe holder for scanning in fluid on a MultiMode AFM.</td>
		</tr>
		<tr>
			<td>Probe holder (fluid, MSCs)</td>
			<td>Bruker</td>
			<td>FastScan Bio Z-scanner</td>
			<td>Used with Dimension FastScan head (XY flexure scanners). Serial number MXYPOM5-1B154.</td>
		</tr>
		<tr>
			<td>Probe holder (standard, ambient)</td>
			<td>Bruker</td>
			<td>DAFMCH</td>
			<td>Specific to the particular AFM used; DAFMCH is the standard contact and tapping mode probe holder for the Dimension Icon AFM, suitable for nanoindentation (PF-QNM, FFV, and point-and-shoot ramping)</td>
		</tr>
		<tr>
			<td>Sample Puck</td>
			<td>Ted Pella</td>
			<td>16218</td>
			<td>Product number is for 15 mm diameter stainless steel sample puck. Also available in 6 mm, 10 mm, 12 mm, and 20 mm diameters at https://www.tedpella.com/AFM_html/AFM.aspx#anchor842459</td>
		</tr>
		<tr>
			<td>Sapphire substrate</td>
			<td>Bruker</td>
			<td>PFQNM-SMPKIT-12M</td>
			<td>Extremely hard surface (E = 345 GPa) for measuring deflection sensitivity of probes (want all of the deflection to come from the probe, not the substrate). Part of the PF-QNM/modulus standards kit.</td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Hitachi</td>
			<td>S-3400N-II</td>
			<td>Located at Boise State. Used to perform co-localized SEM/EDS on all samples except additively manufactured (AM) Ti-6Al-4V.</td>
		</tr>
		<tr>
			<td>Silicon AFM probes (standard)</td>
			<td>NuNano</td>
			<td>Scout 350</td>
			<td>Standard tapping mode silicon probe with reflective aluminum backside coating; k = 42 N/m (nominal), f0 = 350 kHz. Nominal R = 5 nm. Also available uncoated or with reflective gold backside coating. Probes with similar specifications are available from other manufacturers (e.g., Bruker TESPA-V2).</td>
		</tr>
		<tr>
			<td>Silicon AFM probes (stiff)</td>
			<td>Bruker</td>
			<td>RTESPA-525, RTESPA-525-30&#160;</td>
			<td>Rotated tip etched silicon probes with reflective aluminum backside coating; k = 200 N/m (nominal), f0 = 525 kHz. Nominal R = 8 nm for RTESPA-525, R = 30 nm for RTESPA-525-30. Spring constant of each RTESPA-525-30 is measured individually at the factory via laser Doppler vibrometry and supplied with the probe.</td>
		</tr>
		<tr>
			<td>Silicon carbide grit paper (abrasive discs)</td>
			<td>Allied</td>
			<td>50-10005</td>
			<td>120 grit</td>
		</tr>
		<tr>
			<td>Silicon nitride AFM probes (soft, large radius hemispherical tip)</td>
			<td>Bruker</td>
			<td>MLCT-SPH-5UM, MLCT-SPH-5UM-DC</td>
			<td>Also MLCT-SPH-1UM-DC. New product line of factory-calibrated (probe radius and spring constants of all cantilevers) large radius (R = 1 or 5 mm) hemispherical tip (at the end of a 23 mm long cylindrical shaft) probes. DC = drift compensation coating. 6 cantilevers/probe (A-F). Nominal spring constants: A, k = 0.07 N/m; B, k = 0.02 N/m; C, k = 0.01 N/m; D, k = 0.03 N/m; E, k = 0.1 N/m; F, k = 0.6 N/m.</td>
		</tr>
		<tr>
			<td>Silicon nitride AFM probes (soft, medium sharp tip)</td>
			<td>Bruker</td>
			<td>DNP</td>
			<td>4 cantilevers/probe (A-d). Nominal spring constants: A, k = 0.35 N/m; B, k = 0.12 N/m; C, k = 0.24 N/m; D, k = 0.06 N/m. Nominal radii of curvature, R = 10 nm.</td>
		</tr>
		<tr>
			<td>Silicon nitride AFM probes (soft, sharp tip)</td>
			<td>Bruker</td>
			<td>ScanAsyst-Air</td>
			<td>Nominal values: resonance frequency, f0 = 70 kHz; spring constant, k = 0.4 N/m; radius of curvature, R = 2 nm. Designed for force curve based AFM imaging.</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Henkel</td>
			<td>Loctite 495</td>
			<td>Cyanoacrylate based instant adhesive. Lots of roughly equivalent products are readily available.</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td>NE1000US</td>
			<td>One channel syringe pump system with infusion and withdrawal capacity</td>
		</tr>
		<tr>
			<td>Tip characterization standard</td>
			<td>Bruker</td>
			<td>PFQNM-SMPKIT-12M</td>
			<td>Titanium (Ti) roughness standard. Part of the PF-QNM/modulus standards kit.</td>
		</tr>
		<tr>
			<td>Ultrahigh purity nitrogen (UHP N2), 99.999%</td>
			<td>Norco</td>
			<td>SPG TUHPNI - T</td>
			<td>T size compressed gas cylinder of ultrahigh purity (99.999%) nitrogen for drying samples</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>EM UC6</td>
			<td>Equipped with a glass blade (standard, for intial sample preparation) and a diamond blade (for final preparation)</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Thermo Fisher</td>
			<td>Barnstead Nanopure Model 7146</td>
			<td>Model has been discontinued, but equivalent products are available. Produces &#8805;18.2 M&#937;*cm ultrapure water with 1-5 ppb TOC (total organic content), per inline UV monitoring. Includes 0.2 &#181;m particulate filter, ion exchange columns, and UV oxidation chamber.</td>
		</tr>
		<tr>
			<td>Variable Speed Grinder</td>
			<td>Buehler</td>
			<td>EcoMet 3000</td>
			<td>Used with silicon carbide grit papers during hand polishing.</td>
		</tr>
		<tr>
			<td>Vibration isolation table (active)</td>
			<td>Herzan</td>
			<td>TS-140</td>
			<td>Used with Bruker MultiMode AFM. Sits on a TMC 65-531 vibration isolation table. Bruker Dimension Icon AFM utilizes strictly passive vibration isolation (comes from manufacturer with custom acoustic hood, air table, and granite slab).</td>
		</tr>
		<tr>
			<td>Vibration isolation table (passive)</td>
			<td>TMC</td>
			<td>65-531</td>
			<td>35&#34; x 30&#34; vibration isolation table with optional air damping (disabled). Used with Bruker MultiMode AFM. Herzan TS-140 &#34;Table Stable&#34; active vibration control table is located on top.</td>
		</tr>
	</tbody>
</table>

atomic force microscopy cantilever-based nanoindentation: mechanical property measurements at the nanoscale in air and fluid

原子力显微镜（AFM）从根本上测量纳米级AFM探针尖端与样品表面之间的相互作用。如果探针尖端施加的力及其与样品的接触面积可以量化，则可以确定被探测表面的纳米级机械性能（例如，弹性或杨氏模量）。这里提供了进行定量AFM悬臂式纳米压痕实验的详细程序，并提供了如何应用该技术来确定从kPa到GPa的各种样品类型的弹性模量的代表性示例。这些包括生理缓冲液中的活间充质干细胞（MSCs）和细胞核，树脂嵌入的脱水松横截面以及不同成分的Bakken页岩。
此外，基于AFM悬臂的纳米压痕用于探测磷脂双层的断裂强度（即突破力）。讨论了重要的实际考虑因素，例如方法选择和开发、探头选择和校准、感兴趣区域识别、样品异质性、特征尺寸和纵横比、尖端磨损、表面粗糙度以及数据分析和测量统计，以帮助正确实施该技术。最后，证明了AFM衍生的纳米力学图与电子显微镜技术的共定位，该技术提供了有关元素组成的附加信息。

力-位移曲线 图 7 显示了在树脂嵌入的枇杷松样品（图 7A）和间充质干细胞 （MSC） 细胞核上的液体（磷酸盐缓冲盐水 [PBS]）上进行的纳米压痕实验中获得的具有代表性的、近乎理想的 F-D 曲线（图 7B）。任何接触力学模型的使用都依赖于对初始针尖-样品接触点的准确可靠测定。因此，初始接触点之前相对平坦、低?...

Watch this Scientific Journal Video about Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid at JoVE.com

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

量化原子力显微镜（AFM）探针尖端施加到样品表面的接触面积和力，可以确定纳米级机械性能。讨论了在软硬样品的空气或流体中实施基于AFM悬臂的纳米压痕的最佳实践，以测量弹性模量或其他纳米力学性能。

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 ...

原子力显微镜或基于AFM悬臂的纳米压痕可用于确定材料在空气和流体中从千帕斯卡到千兆帕斯卡的模量的纳米级机械性能。基于AFM悬臂的纳米压痕能够在各种材料和相关环境中以纳米级精度和分辨率进行共定位形貌成像和原位定量机械性能测量。基于AFM悬臂的纳米压痕可用于区分表现出不同机械性能的健康与疾病结构，组织或细胞。准确确定尖端样品接触面积和在悬臂式纳米压痕过程中施加的力需要仔细校准AFM探头，这对于定量纳米级机械性能测量具有挑战性，但至关重要。首先，根据介质、预期模量、样品形貌和相关特征尺寸选择合适的原子力显微镜或AFM探针，对目标样品进行纳米压痕。将探头装载到探头支架上，并将探头支架连接到AFM扫描头。在AFM软件中选择适当的纳米压痕模式，使用户能够控制各个斜坡。将激光对准探头悬臂的背面，与探头尖端的位置相对，并进入位置敏感检测器或PSD。通过最大化总电压，将激光束光斑居中悬臂背面。接下来，通过将垂直和水平偏转信号调整为尽可能接近零，将反射的激光束光斑居中在PSD上，从而提供最大的可检测偏转范围，以产生与悬臂偏转成比例的输出电压。校准探头 AFM 系统的偏转灵敏度或 DS。为此，请在 Sapphire 上设置并执行 DS 校准压痕，以实现与计划样品压痕大致相同的探头偏转，因为测量的位移是尖端偏转角的函数，并且对于大挠度变为非线性。重复斜坡五次。在产生的力位移或FD曲线中的初始接触点之后，确定以纳米/伏特为单位的DS或反向光杠杆灵敏度和每纳米伏特。使用值的平均值以获得最大精度。如果相对标准偏差超过1%重新测量DS，因为有时由于最初引入粘附力，前几条FD曲线是不理想的。如果探头悬臂的弹簧常数 K 未经过工厂校准，请校准弹簧常数。如果探头没有工厂校准的尖端半径测量，请测量有效尖端半径R.如果使用盲尖重建方法，请使用慢扫描速率和高反馈增益对尖端特征或粗糙度样品进行成像，以帮助优化对非常尖锐特征的跟踪。根据预期的尖端半径选择图像大小和像素密度。接下来，使用AFM图像分析软件对探针尖端进行建模，并在预期的样品压痕深度估计其末端半径和有效尖端直径。完成探头校准后，在软件中输入 DS、K 和 R 值。最后，输入样品泊松比的估计值，以将测量的降低模量转换为实际样本模量。如果采用基于尖端形状和压痕深度的圆锥形或圆锥形接触力学模型，则需要输入尖端半角。在AFM头下导航样品，并在所需的感兴趣区域接合。监测垂直偏转信号或执行一个小的初始斜坡，以验证吸头和样品是否接触。稍微向上调整AFM头部位置，然后再次斜坡。重复直到吸头和样品刚好不接触，几乎平坦的斜坡和悬臂的最小垂直偏转证明了这一点。一旦不存在明显的尖端样品相互作用，将AFM头降低相当于斜坡尺寸的约50%，以确保探头在手动移动AFM头时不会撞到样品中。再次斜坡，重复直到观察到良好的曲线。调整斜坡参数。根据样品和所需的压痕深度选择合适的斜坡尺寸。然后，选择适当的斜坡速率。对于大多数样本来说，一赫兹是一个很好的起点。设置每个斜坡的样本数，以达到所需的测量分辨率。设置 X 旋转以减少样品和尖端上的剪切力，方法是同时在 X 方向上稍微移动探头，同时在 Z 方向上缩进。对“X 旋转”（X Rotate） 使用的值等于探头支架相对于表面法线的偏移角度。接下来，选择是采用触发坡道还是未触发斜坡。如果选择了触发斜坡，请设置触发阈值以在样品中产生所需的压痕。在所选位置执行缩进。在软件中选择并加载要分析的数据。输入弹簧常数、DS 或反向光杆灵敏度和探头尖端半径的校准值，以及样品泊松比的估计值。选择适合针尖和样品的纳米压痕接触力学模型。运行拟合算法。检查FD曲线是否正确拟合。对应于平均 R 平方接近单位的低残差表明与所选模型的良好拟合。如果需要，抽查单个曲线，以目视检查曲线、模型拟合和计算的接触点。显示了在树脂嵌入的枇杷松样品和间充质干细胞核上的磷酸盐缓冲盐溶液的空气中获得的近乎理想的FD曲线。使用硅探头，在整个成像过程中，尖端相对于其初始原始状态经历了显着磨损。对于每个后续图像，尖端逐渐变得更加圆润。与金刚石探头相比，尖端半径在盲尖重建方法的极限内没有变化，突出了金刚石的极端耐磨性。此处显示了覆盖多个细胞的AFM形貌图像，具有伪3D描绘和Loblolly松样品的相应模量图。在获得AFM图像时生成的AFM衍生模量图显示，图像中心的矿物内含物明显比周围的有机基质更硬。显示了探针尖端半径和形状对Bakken页岩样品高纵横比特征外观的影响。悬臂式纳米压痕在间充质干细胞和分离的细胞核上表现出弹性模量无统计学差异。这里展示了AFM研究的含脂质双层胆固醇的形态和机械性能。在接触力学模型中进行适当的探头校准和选择合适的斜坡参数对于精确的纳米力学测量至关重要。除了实现弹性模块化测量外，基于AFM悬臂的纳米压痕还可用于探测生理相关条件下磷脂双层的断裂强度或突破力。基于AFM悬臂的纳米压痕能够研究结构敲除，药物治疗和低强度振动的影响，以模拟运动对间充质干细胞核的机械性能。

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Education

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Unit 1

Deflection of Beams

SCIENTISTS IN ACTION

Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM

并行定量的导电性和力学性能测试的有机光伏材料利用原子力显微镜

Organic photovoltaic  (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale  inhomogeneity of OPV materials affects performance ...

科研

工程学

Fabrication of Uniform Nanoscale Cavities via Silicon Direct Wafer Bonding

通过硅直接晶圆粘接制造均匀纳米级腔

Measurements of the heat capacity and superfluid fraction of confined 4He have been performed near the lambda transition using lithographically patterned ...

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

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 ...

Preparation and Friction Force Microscopy Measurements of Immiscible, Opposing Polymer Brushes

互溶的测量准备和摩擦力显微镜，反对聚合物刷

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low ...

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

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 ...

Measurement of Quantum Interference in a Silicon Ring Resonator Photon Source

量子干涉效应在硅谐振环光子源测量

Silicon photonic chips have the potential to realize complex integrated quantum information processing circuits, including photon sources, qubit ...

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

通过冷冻聚焦离子束铣削与扫描电子显微镜和光谱学耦合，对液固界面进行纳米级表征

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar ...

Optimizing Magnetic Force Microscopy Resolution and Sensitivity to Visualize Nanoscale Magnetic Domains

优化磁力显微镜分辨率和灵敏度，以可视化纳米级磁畴

Magnetic force microscopy (MFM) enables mapping local magnetic fields across a sample surface with nanoscale resolution. To perform MFM, an atomic force ...

Parallel Active Cantilever Arrays in AFMS to Enable High-Throughput Inspections

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

Author Spotlight: Introduction to Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. ...

Bio-Hybrid AFM Probe with Mosquito Labrum for Studying Biting Behaviors

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms

基于生物杂化蚊虫叮咬的原子力显微镜探针的研制

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The ...