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 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

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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2.7K Views.  Boise State University. 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.

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

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

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. Thus, understanding of spatial variations in composition as well as electrical properties of OPV materials is of paramount importance for moving PV technology forward.1,2 In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution. Currently, materials properties measurements performed using commercially available AFM-based techniques (PeakForce, conductive AFM) generally provide only qualitative information. The values for resistance as well as Young's modulus measured using our method on the prototypical ITO/PEDOT:PSS/P3HT:PC61BM system correspond well with literature data. The P3HT:PC61BM blend separates onto PC61BM-rich and P3HT-rich domains. Mechanical properties of PC61BM-rich and P3HT-rich domains are different, which allows for domain attribution on the surface of the film. Importantly, combining mechanical and electrical data allows for correlation of the domain structure on the surface of the film with electrical properties variation measured through the thickness of the film.

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution.

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

Fabrication of Uniform Nanoscale Cavities via Silicon Direct Wafer Bonding

Measurements of the heat capacity and superfluid fraction of confined 4He have been performed near the lambda transition using lithographically patterned and bonded silicon wafers. Unlike confinements in porous materials often used for these types of experiments3, bonded wafers provide predesigned uniform spaces for confinement. The geometry of each cell is well known, which removes a large source of ambiguity in the interpretation of data.
Exceptionally flat, 5 cm diameter, 375 &#181;m thick Si wafers with about 1 &#181;m variation over the entire wafer can be obtained commercially (from Semiconductor Processing Company, for example). Thermal oxide is grown on the wafers to define the confinement dimension in the z-direction. A pattern is then etched in the oxide using lithographic techniques so as to create a desired enclosure upon bonding. A hole is drilled in one of the wafers (the top) to allow for the introduction of the liquid to be measured. The wafers are cleaned2 in RCA solutions and then put in a microclean chamber where they are rinsed with deionized water4. The wafers are bonded at RT and then annealed at ~1,100 &#176;C. This forms a strong and permanent bond. This process can be used to make uniform enclosures for measuring thermal and hydrodynamic properties of confined liquids from the nanometer to the micrometer scale.

A method for permanently bonding two silicon wafers so as to realize a uniform enclosure is described. This includes wafer preparation, cleaning, RT bonding, and annealing processes. The resulting bonded wafers (cells) have uniformity of enclosure ~1%1,2. The resulting geometry allows for measurements of confined liquids and gasses.

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

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 ...

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

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low friction at the interface. Nevertheless, these systems can be sensitive to wear due to interdigitation of the opposing brushes. In a recent publication, we have shown via molecular dynamics simulations and atomic force microscopy experiments, that using an immiscible polymer brush system terminating the substrate and the slider surfaces, respectively, can eliminate such interdigitation. As a consequence, wear in the contacts is reduced. Moreover, the friction force is two orders of magnitude lower compared to traditional miscible polymer brush systems. This newly proposed system therefore holds great potential for application in industry. Here, the methodology to construct an immiscible polymer brush system of two different brushes each solvated by their own preferred solvent is presented. The procedure how to graft poly(N-isopropylacrylamide) (PNIPAM) from a flat surface and poly(methyl methacrylate) (PMMA) from an atomic force microscopy (AFM) colloidal probe is described. PNIPAM is solvated in water and PMMA in acetophenone. Via friction force AFM measurements, it is shown that the friction for this system is indeed reduced by two orders of magnitude compared to the miscible system of PMMA on PMMA solvated in acetophenone.

The methodology to perform friction force microscopy experiments for contacting brushes is presented: Two polymer brushes that are grafted from (a) substrates and (b) colloidal probes are slid to show that, by using two contacting immiscible brush systems, friction in sliding contacts is reduced compared to miscible brush systems.

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

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 ...

Measurement of Quantum Interference in a Silicon Ring Resonator Photon Source

Silicon photonic chips have the potential to realize complex integrated quantum information processing circuits, including photon sources, qubit manipulation, and integrated single-photon detectors. Here, we present the key aspects of preparing and testing a silicon photonic quantum chip with an integrated photon source and two-photon interferometer. The most important aspect of an integrated quantum circuit is minimizing loss so that all of the generated photons are detected with the highest possible fidelity. Here, we describe how to perform low-loss edge coupling by using an ultra-high numerical aperture fiber to closely match the mode of the silicon waveguides. By using an optimized fusion splicing recipe, the UHNA fiber is seamlessly interfaced with a standard single-mode fiber. This low-loss coupling allows the measurement of high-fidelity photon production in an integrated silicon ring resonator and the subsequent two-photon interference of the produced photons in a closely integrated Mach-Zehnder interferometer. This paper describes the essential procedures for the preparation and characterization of high-performance and scalable silicon quantum photonic circuits.

Silicon photonic chips have the potential to realize complex integrated quantum systems. Presented here is a method for preparing and testing a silicon photonic chip for quantum measurements.

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

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes.
This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage devices.

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

Magnetic force microscopy (MFM) enables mapping local magnetic fields across a sample surface with nanoscale resolution. To perform MFM, an atomic force microscopy (AFM) probe whose tip has been magnetized vertically (i.e., perpendicular to the probe cantilever) is oscillated at a fixed height above the sample surface. The resultant shifts in the oscillation phase or frequency, which are proportional to the magnitude and sign of the vertical magnetic force gradient at each pixel location, are then tracked and mapped. Although the spatial resolution and sensitivity of the technique increases with decreasing lift height above the surface, this seemingly straightforward path to improved MFM images is complicated by considerations such as minimizing topographical artifacts due to shorter range van der Waals forces, increasing the oscillation amplitude to further improve sensitivity, and the presence of surface contaminants (in particular water due to humidity under ambient conditions). In addition, due to the orientation of the probe's magnetic dipole moment, MFM is intrinsically more sensitive to samples with an out-of-plane magnetization vector. Here, high-resolution topographical and magnetic phase images of single and bicomponent nanomagnet artificial spin-ice (ASI) arrays obtained in an inert (argon) atmosphere glovebox with &lt;0.1 ppm O2 and H2O are reported. Optimization of lift height and drive amplitude for high resolution and sensitivity while simultaneously avoiding the introduction of topographical artifacts is discussed, and detection of the stray magnetic fields emanating from either end of the nanoscale bar magnets (~250 nm long and &lt;100 nm wide) aligned in the plane of the ASI sample surface is shown. Likewise, using the example of a Ni-Mn-Ga magnetic shape memory alloy (MSMA), MFM is demonstrated in an inert atmosphere with magnetic phase sensitivity capable of resolving a series of adjacent magnetic domains each ~200 nm wide.

Magnetic force microscopy (MFM) employs a vertically magnetized atomic force microscopy probe to measure sample topography and local magnetic field strength with nanoscale resolution. Optimizing MFM spatial resolution and sensitivity requires balancing decreasing lift height against increasing drive (oscillation) amplitude, and benefits from operating in an inert atmosphere glovebox.

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

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. However, due to their limited imaging throughput, AFMs have not been widely adopted for large-scale inspection purposes. Researchers have developed high-speed AFM systems to record dynamic process videos in chemical and biological reactions at tens of frames per second, at the cost of a small imaging area of up to several square micrometers. In contrast, inspecting large-scale nanofabricated structures, such as semiconductor wafers, requires nanoscale spatial resolution imaging of a static sample over hundreds of square centimeters with high productivity. Conventional AFMs use a single passive cantilever probe with an optical beam deflection system, which can only collect one pixel at a time during AFM imaging, resulting in low imaging throughput. This work utilizes an array of active cantilevers with embedded piezoresistive sensors and thermomechanical actuators, which allows simultaneous multi-cantilever operation in parallel operation for increased imaging throughput. When combined with large-range nano-positioners and proper control algorithms, each cantilever can be individually controlled to capture multiple AFM images. With data-driven post-processing algorithms, the images can be stitched together, and defect detection can be performed by comparing them to the desired geometry. This paper introduces principles of the custom AFM using the active cantilever arrays, followed by a discussion on practical experiment considerations for inspection applications. Selected example images of silicon calibration grating, highly-oriented pyrolytic graphite, and extreme ultraviolet lithography masks are captured using an array of four active cantilevers (&#34;Quattro&#34;) with a 125 &#181;m tip separation distance. With more engineering integration, this high-throughput, large-scale imaging tool can provide 3D metrological data for extreme ultraviolet (EUV) masks, chemical mechanical planarization (CMP) inspection, failure analysis, displays, thin-film step measurements, roughness measurement dies, and laser-engraved dry gas seal grooves.

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

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

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

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The primary prevention strategy currently in use involves chemical repellents, which often prove ineffective as mosquitoes rapidly develop resistance. Consequently, the invention of new preventive methods is crucial. Such development hinges on a thorough understanding of mosquito biting behaviors, necessitating an experimental setup that accurately replicates actual biting scenarios with controllable testing parameters and quantitative measurements. To bridge this gap, a bio-hybrid atomic force microscopy (AFM) probe was engineered, featuring a biological stinger - specifically, a mosquito labrum - as its tip. This bio-hybrid probe, compatible with standard AFM systems, enables a near-authentic simulation of mosquito penetration behaviors. This method marks a step forward in the quantitative study of biting mechanisms, potentially leading to the creation of effective barriers against vector-borne diseases (VBDs) and opening new avenues in the fight against mosquito-transmitted illnesses.

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

Quantitative and controlled investigations into insect biting behaviors are crucial for devising effective strategies to combat vector-borne diseases. In this context, a method for fabricating a bio-hybrid atomic force microscopy (AFM) probe is introduced.

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