All AFM experiments were performed in the Boise State University Surface Science Laboratory (SSL). SEM characterization was performed in the Boise State Center for Materials Characterization (BSCMC). Research reported in this publication regarding biofuel feedstocks was supported in part by the US Department of Energy, Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office as part of the Feedstock Conversion Interface Consortium (FCIC), and under DOE Idaho Operations Office Contract DE-AC07-051ID14517. Cell mechanics studies were supported by the National Institutes of Health (USA) under grants AG059923, AR075803, and P20GM109095, and by National Science Foundation (USA) grants 1929188 and 2025505. The model lipid bilayer systems work was supported by the National Institutes of Health (USA) under grant R01 EY030067. The authors thank Dr. Elton Graugnard for producing the composite image shown in Figure 11.

The authors have no conflicts of interest to disclose.

Sample preparation 
For nanoindentation in air, common preparation methods include cryosectioning (e.g., tissue samples), grinding and/or polishing followed by ultramicrotoming (e.g., resin-embedded biological samples), ion milling or focused ion beam preparation (e.g., semiconductor, porous, or mixed hardness samples not amenable to polishing), mechanical or electrochemical polishing (e.g., metal alloys), or thin film deposition (e.g., atomic layer or chemical vapor deposition, molecular beam epita...

Understanding the mechanical properties of materials is one of the most fundamental and essential tasks in engineering. For the analysis of bulk material properties, there are numerous methods available to characterize the mechanical properties of material systems, including tensile tests1, compression tests2, and three- or four-point bending (flexural) tests3. While these microscale tests can provide invaluable information regarding bulk material properties, they are generally conducted to failure, and are hence destructive. Additionally, they lack the spatial resolution necessary to accurately investigate the micro- and nanoscale properties of many material systems that are of interest today, such as thin films, biological materials, and nanocomposites. To begin addressing some of the problems with large-scale mechanical testing, mainly its destructive nature, microhardness tests were adopted from mineralogy. Hardness is a measure of the resistance of a material to plastic deformation under specific conditions. In general, microhardness tests use a stiff probe, usually made from hardened steel or diamond, to indent into a material. The resulting indentation depth and/or area can then be used to determine the hardness. Several methods have been developed, including Vickers4, Knoop5, and Brinell6 hardness; each provides a measure of microscale material hardness, but under different conditions and definitions, and as such only produces data that can be compared to tests performed under the same conditions.
Instrumented nanoindentation was developed to improve upon the relative values obtained via the various microhardness testing methods, improve the spatial resolution possible for the analysis of mechanical properties, and enable the analysis of thin films. Importantly, by utilizing the method first developed by Oliver and Pharr7, the elastic or Young&#39;s modulus, E, of a sample material can be determined via instrumented nanoindentation. Furthermore, by employing a Berkovich three-sided pyramidal nanoindenter probe (whose ideal tip area function matches that of the Vickers four-sided pyramidal probe)8, direct comparison between nanoscale and more traditional microscale hardness measurements can be made. With the growth in popularity of the AFM, AFM cantilever-based nanoindentation began receiving attention as well, particularly for measuring the mechanical properties of softer materials. As a result, as depicted schematically in Figure 1, the two most commonly employed techniques today to interrogate and quantify nanoscale mechanical properties are instrumented nanoindentation (Figure 1A) and AFM cantilever-based nanoindentation (Figure 1B)9, the latter of which is the focus of this work.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64497/64497fig01.jpg" /> 
Figure 1: Comparison of instrumented and AFM cantilever-based nanoindentation systems. Schematic diagrams depicting typical systems for conducting (A) instrumented nanoindentation and (B) AFM cantilever-based nanoindentation. This figure was modified from Qian et al.51. Abbreviation: AFM = atomic force microscopy. <a href="https://www.jove.com/files/ftp_upload/64497/64497fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Both instrumented and AFM cantilever-based nanoindentation employ a stiff probe to deform a sample surface of interest and monitor the resultant force and displacement as a function of time. Typically, either the desired load (i.e., force) or (Z-piezo) displacement profile is specified by the user via the software interface and directly controlled by the instrument, while the other parameter is measured. The mechanical property most often obtained from nanoindentation experiments is the elastic modulus (E), also referred to as the Young&#39;s modulus, which has units of pressure. The elastic modulus of a material is a fundamental property relating to the bond stiffness and is defined as the ratio of tensile or compressive stress (&#963;, the applied force per unit area) to axial strain (&#949;, the proportional deformation along the indentation axis) during elastic (i.e., reversible or temporary) deformation prior to the onset of plastic deformation (equation [1]):
<img alt="Equation 1" src="/files/ftp_upload/64497/64497eq01.jpg" style="margin: auto;" />&#160; &#160; (1)
It should be noted that, because many materials (especially biological tissues) are in fact viscoelastic, in reality, the (dynamic or complex) modulus consists of both elastic (storage, in phase) and viscous (loss, out of phase) components. In actual practice, what is measured in a nanoindentation experiment is the reduced modulus, E*, which is related to the true sample modulus of interest, E, as shown in equation (2):
<img alt="Equation 2" src="/files/ftp_upload/64497/64497eq02.jpg" style="margin: auto;" />&#160; &#160; (2)
Where Etip and &#957;tip are the elastic modulus and Poisson&#39;s ratio, respectively, of the nanoindenter tip, and &#957; is the estimated Poisson&#39;s ratio of the sample. The Poisson&#39;s ratio is the negative ratio of the transverse to axial strain, and hence indicates the degree of transverse elongation of a sample upon being subjected to axial strain (e.g., during nanoindentation loading), as shown in equation (3):
<img alt="Equation 3" src="/files/ftp_upload/64497/64497eq03.jpg" style="margin: auto;" />&#160; &#160; (3)
The conversion from reduced to actual modulus is necessary because a) some of the axial strain imparted by the indenter tip may be converted to transverse strain (i.e., the sample may deform via expansion or contraction perpendicular to the direction of loading), and b) the indenter tip is not infinitely hard, and thus the act of indenting the sample results in some (small) amount of deformation of the tip. Note that in the case where Etip &#62;&#62; E (i.e., the indenter tip is much harder than the sample, which is often true when using a diamond probe),&#160;the relationship between the reduced and actual sample modulus simplifies greatly to E &#8776; E*(1 -&#160;v2). While instrumented nanoindentation is superior in terms of accurate force characterization and dynamic range, AFM cantilever-based nanoindentation is faster, provides orders of magnitude greater force and displacement sensitivity, enables higher resolution imaging and improved indentation locating, and can simultaneously probe nanoscale magnetic and electrical properties9. In particular, AFM cantilever-based nanoindentation is superior for the quantification of mechanical properties at the nanoscale of soft materials (e.g., polymers, gels, lipid bilayers, and cells or other biological materials), extremely thin (sub-&#181;m) films (where substrate effects can come into play depending upon indentation depth)10,11, and suspended two-dimensional (2D) materials12,13,14 such as graphene15,16, mica17, hexagonal boron nitride (h-BN)18, or transition metal dichalcogenides (TMDCs; e.g., MoS2)19. This is due to its exquisite force (sub-nN) and displacement (sub-nm) sensitivity, which is important for accurately determining the initial point of contact and remaining within the elastic deformation region.
In AFM cantilever-based nanoindentation, displacement of an AFM probe toward the sample surface is actuated by a calibrated piezoelectric element (Figure 1B), with the flexible cantilever eventually bending due to the resistive force experienced upon contact with the sample surface. This bending or deflection of the cantilever is typically monitored by reflecting a laser off the back of the cantilever and into a photodetector (position sensitive detector [PSD]). Coupled with the knowledge of the cantilever stiffness (in nN/nm) and deflection sensitivity (in nm/V), it is possible to convert this measured cantilever deflection (in V) into the force (in nN) applied to the sample. Following contact, the difference between the Z-piezo movement and the cantilever deflection yields the sample indentation depth. Combined with the knowledge of the tip area function, this enables calculation of the tip-sample contact area. The slope of the in-contact portions of the resulting force-distance or force-displacement (F-D) curves can then be fit using an appropriate contact mechanics model (see the Data Analysis section of the discussion) to determine the nanomechanical properties of the sample. While AFM cantilever-based nanoindentation possesses some distinct advantages over instrumented nanoindentation as described above, it also presents several practical implementation challenges, such as calibration, tip wear, and data analysis, which will be discussed here. Another potential downside of AFM cantilever-based nanoindentation is the assumption of linear elasticity, as the contact radius and indentation depths need to be much smaller than the indenter radius, which can be difficult to achieve when working with nanoscale AFM probes and/or samples exhibiting significant surface roughness.
Traditionally, nanoindentation has been limited to individual locations or small grid indentation experiments, wherein a desired location (i.e., region of interest [ROI]) is selected and a single controlled indent, multiple indents in a single location separated by some waiting time, and/or a coarse grid of indents are performed at a rate on the order of Hz. However, recent advances in AFM allow for the simultaneous acquisition of mechanical properties and topography through the utilization of high-speed force curve-based imaging modes (referred to by various tradenames depending on the system manufacturer), wherein force curves are conducted at a kHz rate under load control, with the maximum tip-sample force utilized as the imaging setpoint. Point-and-shoot methods have also been developed, allowing for the acquisition of an AFM topography image followed by subsequent selective nanoindentation at points of interest within the image, affording nanoscale spatial control over nanoindentation location. While not the primary focus of this work, specific selected application examples of both force curve-based imaging and point-and-shoot cantilever-based nanoindentation are presented in the representative results, and can be used in conjunction with the protocol outlined below if available on the particular AFM platform employed. Specifically, this work outlines a generalized protocol for the practical implementation of AFM cantilever-based nanoindentation on any capable AFM system and provides four use case examples (two in air, two in fluid) of the technique, including representative results and an in-depth discussion of the nuances, challenges, and important considerations to successfully employ the technique.

<table><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&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/64497/64497eq05.jpg&quot; style=&quot;margin: auto;&quot;/&gt;, &amp;ldquo;AtomicJ: An open source software for analysis of force curves&amp;rdquo; &lt;em&gt;Rev. Sci. Instrum.&lt;/em&gt; &lt;strong&gt;85&lt;/strong&gt;: 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 (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>S271 (&gt;99% purity NaCl), P9541 (&gt;99% purity KCl), BP332(&gt;99% purity Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;), BP362 (&gt;99% purity KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</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 Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, and 1.8 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 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 (&lt;em&gt;E&lt;/em&gt; = 1140 GPa) tip AFM probe (nominal &lt;em&gt;R&lt;/em&gt; = 40 nm) with a stainless steel cantilever (nominal &lt;em&gt;k&lt;/em&gt; = 225 N/m, &lt;em&gt;f&lt;sub&gt;0&lt;/sub&gt;&lt;/em&gt; = 50 kHz). Spring constant is measured at the factory (&lt;em&gt;k&lt;/em&gt; = 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&amp;deg;</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. &quot;Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis&quot; &lt;em&gt;Int J Mol Sci&lt;/em&gt; &lt;strong&gt;22&lt;/strong&gt;: 6580 (2021) doi:10.3390/ijms22126580 and Peister, A. et al. &quot;Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential&quot; &lt;em&gt;Blood &lt;/em&gt;&lt;strong&gt;103&lt;/strong&gt;: 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 (&lt;em&gt;E&lt;/em&gt; = 18 GPa) and PS (&lt;em&gt;E&lt;/em&gt; = 2.7 GPa). Also contains 2x PDMS (Tack 0, &lt;em&gt;E&lt;/em&gt; = 2.5 MPa; Tack 4, &lt;em&gt;E&lt;/em&gt; = 3.5 MPa), PS-LDPE (&lt;em&gt;E&lt;/em&gt; = 2.0/0.2 GPa), fused silica (&lt;em&gt;E&lt;/em&gt; = 72.9 GPa), sapphire (&lt;em&gt;E&lt;/em&gt; - 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).&amp;nbsp;</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 (&lt;em&gt;E&lt;/em&gt; = 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; &lt;em&gt;k&lt;/em&gt; = 42 N/m (nominal), &lt;em&gt;f&lt;sub&gt;0&lt;/sub&gt;&lt;/em&gt; = 350 kHz. Nominal &lt;em&gt;R&lt;/em&gt; = 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&amp;nbsp;</td><td>Rotated tip etched silicon probes with reflective aluminum backside coating; &lt;em&gt;k&lt;/em&gt; = 200 N/m (nominal), &lt;em&gt;f&lt;sub&gt;0&lt;/sub&gt; &lt;/em&gt;= 525 kHz. Nominal &lt;em&gt;R&lt;/em&gt; = 8 nm for RTESPA-525, &lt;em&gt;R&lt;/em&gt; = 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 (&lt;em&gt;R&lt;/em&gt; = 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, &lt;em&gt;k&lt;/em&gt; = 0.07 N/m; B, &lt;em&gt;k&lt;/em&gt; = 0.02 N/m; C, &lt;em&gt;k&lt;/em&gt; = 0.01 N/m; D, &lt;em&gt;k&lt;/em&gt; = 0.03 N/m; E, &lt;em&gt;k&lt;/em&gt; = 0.1 N/m; F, &lt;em&gt;k&lt;/em&gt; = 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, &lt;em&gt;k&lt;/em&gt; = 0.35 N/m; B, &lt;em&gt;k&lt;/em&gt; = 0.12 N/m; C, &lt;em&gt;k&lt;/em&gt; = 0.24 N/m; D, &lt;em&gt;k&lt;/em&gt; = 0.06 N/m. Nominal radii of curvature, &lt;em&gt;R&lt;/em&gt; = 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, &lt;em&gt;f&lt;sub&gt;0&lt;/sub&gt; &lt;/em&gt;= 70 kHz; spring constant, &lt;em&gt;k&lt;/em&gt; = 0.4 N/m; radius of curvature, &lt;em&gt;R&lt;/em&gt; = 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 N&lt;sub&gt;2&lt;/sub&gt;), 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 &amp;ge;18.2 M&amp;Omega;*cm ultrapure water with 1-5 ppb TOC (total organic content), per inline UV monitoring. Includes 0.2 &amp;micro;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&quot; x 30&quot; vibration isolation table with optional air damping (disabled). Used with Bruker MultiMode AFM. Herzan TS-140 &quot;Table Stable&quot; 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

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.

Force-displacement curves 
Figure 7 shows representative, near-ideal F-D curves obtained from nanoindentation experiments performed in air on resin-embedded loblolly pine samples (Figure 7A) and in fluid (phosphate-buffered saline [PBS]) on mesenchymal stem cell (MSC) nuclei (Figure 7B). The use of any contact mechanics model relies on the accurate and reliable determination of the initial tip-sample contact po...

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

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.

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

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2.9K Views.  Boise State University. Atoomkrachtmicroscopie, of AFM cantilever-gebaseerde nano-indentatie, kan worden gebruikt om de mechanische eigenschappen op nanoschaal te bepalen van materialen variërend van modulus, van kilopascals tot gigapascals in zowel lucht als vloeistof. AFM cantilever-gebaseerde nano-indentatie maakt co-gelokaliseerde topografische beeldvorming en in situ kwantitatieve mechanische eigenschappen metingen mogelijk met nanoschaal precisie en resolutie op een breed scala aan materialen en relevante omg...

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

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Chemistry

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.

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 ...

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).

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

Bioengineering

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.

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

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.

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

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.

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

Measuring the Interaction Force Between a Droplet and a Super-hydrophobic Substrate by the Optical Lever Method

The goal of this paper is to investigate the interaction force between droplets and super-hydrophobic substrates in the air. A measurement system based on an optical lever method is designed. A millimetric cantilever is used as a force sensitive component in the measurement system. Firstly, the force sensitivity of the optical lever is calibrated using electrostatic force, which is the critical step in measuring interaction force. Secondly, three super-hydrophobic substrates with different grid fractions are prepared with nanoparticles and copper grids. Finally, the interaction forces between droplets and super-hydrophobic substrates with different grid fractions are measured by the system. This method can be used to measure the force on the scale of sub-micronewton with a resolution on the scale of nanonewton. The in-depth study of the contact process of droplets and super-hydrophobic structures can help to improve the production efficiency in coating, film and printing. The force measurement system designed in this paper can also be used in other fields of microforce measurement.

The protocol aims to investigate the interaction between droplets and super-hydrophobic substrates in the air. This includes calibrating the measurement system and measuring the interaction force at super-hydrophobic substrates with different grid fractions.

The goal of this paper is to investigate the interaction force between droplets and super-hydrophobic substrates in the air. A measurement system based on ...

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.

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

Developmental Biology

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.

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

Biology

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.

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

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

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