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

摘要

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。

摘要

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。

引言

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Pharr

研究方案

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）（参见材料 表），重点是悬臂式样品在流体中的纳米压痕，作为 补充材料。

1. 样品制备和仪器设置

1. 在不改变感兴趣区域的机械性能的情况下，以最小化表面粗糙度（理想情况下为纳米级，比预期的压痕深度小~10倍）和污染的方式制备样品。
2. 根据介质（即空气或流体）、预期模量、样品形貌和相关特征尺寸选择合适的AFM探针，对目标样品进行纳米压痕（请参阅讨论中的 探针选择注意事项 ）。将探头装载到探头支架上（参见 材料表），并将探头支架连接到AFM扫描头。
3. 在AFM软件中选择合适的纳米压痕模式，使用户能够控制单个斜坡（即力-位移曲线）。
   注意：不同AFM制造商和单个仪器的具体模式会有所不同（有关更多详细信息和具体示例，请参阅 补充材料 中提供的SOP）。

结果

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?.......

讨论

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（?.......

材料

Name	Company	Catalog Number	Comments
Atomic force microscope	Bruker	Dimension Icon	Uses Nanoscope control software, including PeakForce Quantitative Nanomechanical Mapping (PF-QNM), FastForce Volume (FFV), and Point-and-Shoot Ramping experimental workspaces
AtomicJ	American Institute of Physics	https://doi.org/10.1063/1.4881683	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, “AtomicJ: An open source software for analysis of force curves” Rev. Sci. Instrum. 85: 063703 (2014), https://doi.org/10.1063/1.4881683
Buffer solution (PBS)	Fisher Chemical (NaCl), Sigma Aldrich (KCl), Fisher BioReagents (Na2HPO4 and KH2PO4)	S271 (>99% purity NaCl), P9541 (>99% purity KCl), BP332(>99% purity Na2HPO4), BP362 (>99% purity KH2PO4)	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.
Chloroform
Diamond tip AFM probe	Bruker	PDNISP	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.
Diamond ultramicrotome blade	Diatome	Ultra 35°	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
Epoxy	Gorilla Glue	26853-31-6	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.
Ethanol
LR white resin, medium grade (catalyzed)	Electron Microscopy Sciences	14381	500 mL bottle, Lot #150629
Mesenchymal stem cells (MSCs)	N/A	N/A	MSCs for nanomechanical studies were primary cells harvested from 8-10 week old male C57BL/6 mice as described in Goelzer, M. et al. "Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis" Int J Mol Sci 22: 6580 (2021) doi:10.3390/ijms22126580 and Peister, A. et al. "Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential" Blood 103: 1662-1668 (2004), doi:10.1182/blood-2003-09-3070.
Modulus standards	Bruker	PFQNM-SMPKIT-12M	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).
Muscovite mica	Ted Pella	50-12	12 mm diameter, V1 grade muscovite mica
Nanscope Analysis	Bruker	Version 2.0	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.
Phospholipids: POPC, Cholesterol (ovine)	Avanti Polar Lipids	POPC: CAS # 26853-31-6, Cholesterol: CAS # 57-88-5	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).
Probe holder (fluid, lipid bilayers)	Bruker	MTFML-V2	Specific to the particular AFM used; MTFML-V2 is a glass probe holder for scanning in fluid on a MultiMode AFM.
Probe holder (fluid, MSCs)	Bruker	FastScan Bio Z-scanner	Used with Dimension FastScan head (XY flexure scanners). Serial number MXYPOM5-1B154.
Probe holder (standard, ambient)	Bruker	DAFMCH	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)
Sample Puck	Ted Pella	16218	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
Sapphire substrate	Bruker	PFQNM-SMPKIT-12M	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.
Scanning electron microscope	Hitachi	S-3400N-II	Located at Boise State. Used to perform co-localized SEM/EDS on all samples except additively manufactured (AM) Ti-6Al-4V.
Silicon AFM probes (standard)	NuNano	Scout 350	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).
Silicon AFM probes (stiff)	Bruker	RTESPA-525, RTESPA-525-30	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.
Silicon carbide grit paper (abrasive discs)	Allied	50-10005	120 grit
Silicon nitride AFM probes (soft, large radius hemispherical tip)	Bruker	MLCT-SPH-5UM, MLCT-SPH-5UM-DC	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.
Silicon nitride AFM probes (soft, medium sharp tip)	Bruker	DNP	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.
Silicon nitride AFM probes (soft, sharp tip)	Bruker	ScanAsyst-Air	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.
Superglue	Henkel	Loctite 495	Cyanoacrylate based instant adhesive. Lots of roughly equivalent products are readily available.
Syringe pump	New Era Pump Systems	NE1000US	One channel syringe pump system with infusion and withdrawal capacity
Tip characterization standard	Bruker	PFQNM-SMPKIT-12M	Titanium (Ti) roughness standard. Part of the PF-QNM/modulus standards kit.
Ultrahigh purity nitrogen (UHP N2), 99.999%	Norco	SPG TUHPNI - T	T size compressed gas cylinder of ultrahigh purity (99.999%) nitrogen for drying samples
Ultramicrotome	Leica	EM UC6	Equipped with a glass blade (standard, for intial sample preparation) and a diamond blade (for final preparation)
Ultrapure water	Thermo Fisher	Barnstead Nanopure Model 7146	Model has been discontinued, but equivalent products are available. Produces ≥18.2 MΩ*cm ultrapure water with 1-5 ppb TOC (total organic content), per inline UV monitoring. Includes 0.2 µm particulate filter, ion exchange columns, and UV oxidation chamber.
Variable Speed Grinder	Buehler	EcoMet 3000	Used with silicon carbide grit papers during hand polishing.
Vibration isolation table (active)	Herzan	TS-140	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).
Vibration isolation table (passive)	TMC	65-531	35" x 30" vibration isolation table with optional air damping (disabled). Used with Bruker MultiMode AFM. Herzan TS-140 "Table Stable" active vibration control table is located on top.