Name: Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy (Video) | JoVE Journal
Uploaded: 2013-06-27T14:00:00.000+00:00
Duration: 8 min 41 s
Description: Learn to measure the elastic modulus of living cells using atomic force microscopy and force spectroscopy. This protocol enables researchers to create 2D stiffness maps and study cell mechanics in biophysics.

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5 minutes

The authors thank Dr. Paul Janmey at University of Pennsylvania for providing cell lines used in this paper. QW also acknowledge J.F. Byfield and Evan Anderson for their insightful discussions on AFM techniques.

1. Calibrate the Spring Constant of Cantilever
<ol>
	<li>Load the cantilever into the AFM according to the manufacturer's instructions. It is necessary to clean the cantilever holder with ethanol before any experiments. This will help limit bacterial contamination to culture during the AFM measurements.</li>
	<li>Calibrate InvOLS (Inverse Optical Lever Sensitivity). This parameter describes the amount of photodiode response (Volts) per nanometer of cantilever deflection.</li>
	<li>Load a clean glass slide onto the sample stage, then install the AFM head and adjust the laser beam and alignment according to the manufacturer's instructions. Engage the AFM tip on the glass slide.</li>
	<li>With the piezo withdrawn, realign the mirror to a photodiode reading of -2 V. Perform a force spectroscopy measurement with a trigger point (maximum photodiode response) of +2 V.</li>
	<li>With the piezo withdrawn, realign the mirror to a photodiode reading of -2 V. Perform a force spectroscopy measurement with a trigger point (maximum photodiode response) of +2 V. Note: The voltage values here are specific to the Asylum AFM. This value should be set according to the specifications provided by manufacturer. 
	After data acquisition is complete, zoom in to the firm-contact region of the force curve. Perform a linear fit to this region to find the slope, which will be in V/nm. The reciprocal of this value describes the optical sensitivity of the cantilever-photodiode ensemble.</li>
	<li>Reset the mirror alignment to a free deflection of 0 V.</li>
	<li>Calibrate the cantilever spring constant. A thermal-tune method is used to determine the spring constant of the cantilever 28.</li>
	<li>After calibrating the InvOLS, raise the scanner away from the sample stage such that there are no interactions between the tip and sample.</li>
	<li>Begin capturing thermal data. During this process, the thermal vibration of the cantilever beam is recorded. The AFM software analyzes a power spectrum of such a thermal vibration and plots it in a data window.</li>
	<li>After a few seconds of data acquisition, perform a fit to the data segment centered at the lowest-frequency (fundamental resonance) peak to determine the spring constant.</li>
</ol>
2. Loading the Sample
<ol>
	<li>Install the Dish Heater accessory on the AFM stage, if not already equipped.</li>
	<li>Set the temperature to 37 °C, and wait for 20 min for the system to reach a stable thermal equilibrium.</li>
	<li>Place the culture dish on the AFM stage and secure it using the clamp provided with the dish heater. It is important to minimize the time between removal of the dish from the incubator and placing it on the stage, to avoid trauma to the cells. For measurements longer than 30 min, CO2 independent medium should be used to replace the normal culture medium.</li>
	<li>Apply a small drop of 37 °C culture medium to the tip of the AFM cantilever, and lower the AFM head until the tip is just submerged in liquid.</li>
	<li>Using the top-view CCD camera, realign the laser beam on the cantilever (the alignment in liquid will be different than in air, because of the change in refractive index of the medium).</li>
	<li>Engage the AFM tip on a clean area of the culture dish.</li>
	<li>Perform calibration of the InvOLS as described above, for the cantilever sensitivity in the liquid environment.</li>
</ol>
Note: a) If cells are cultured on hydrogels, the calibration of InvOLS should be performed in advance against the bottom surface of a culture dish filled with cell culture media. When switching to cell samples, special attention has to be paid to not change the laser beam alignment with the cantilever. b) InvOLS has to be recalibrated whenever there is a change in the laser alignment. c) It is also recommended to take InvOLS as the average of value from several calibration curves, since each calibration generates a different InvOLS. The variation in InvOLS is, however, small comparing to the mean value. For example, calibrating an Bruker DNP-10 cantilever with spring constant 0.06 N/m in liquid by 100 times produce a mean InvOLS value of 66.3 nm/V, with standard deviation of only 0.5 nm/V.
3. Collecting Force Curves of Cell Indentation
<ol>
	<li>Select a cell for indentation. With the aid of the optical microscope, move the stage to position the cantilever above the cell so that the tip is located in the peri-nuclei region. Precise adjustment of the cantilever position may be accomplished by applying offsets to the X and Y scanners. 
	Note: The AFM cantilever has to be withdrawn from the sample surface while moving the sample stage to select a target cell. This protects the cantilever from knocking into the sample, since the sample surface may not be flat.</li>
	<li>Switch to Force Spectroscopy mode. Set the Indentation rate to within the range of 1-10 μm/sec, low enough to avoid hydrodynamic effects.</li>
	<li>Set the deflection trigger point, which limits the maximum indenting force to avoid damage to cells. Select the Relative triggering option, which will correct for any drift in the deflection signal. A maximum force of 2 nN is a good starting point for most samples. This value, however, should be adjusted according to the sample stiffness. For soft samples a lower value should be used to avoid excessive indentation to the sample. For stiff samples a high value should be used to generate measurable indentation.</li>
	<li>Set the force distance large enough to ensure that the tip will be fully detached from the cell between force measurements. Usually, the force distance is set at 5 μm.</li>
	<li>Command the AFM to take a single force curve.</li>
	<li>Collect at least three force curves at different locations in the peri-nuclei region of each cell. Although it is beneficial to take multiple curves on each cell for reliable statistical data, taking too many force curves can lead to changes in cell stiffness due to stress from the AFM probe.</li>
	<li>When data collection is complete, withdraw the tip, and repeat steps 3.1-3.6 for as many cells as needed for good statistical data on cell stiffness under a certain sample condition. Usually, 30 cells are measured for each condition.</li>
</ol>
To characterize the distribution of stiffness within a single cell, force-map mode is applied. In the Force-map mode, set a scan size to include the region of interest; set an appropriate resolution; set the indentation parameters as those selected for single force curves; the AFM will then raster across the defined sample area and take single force curves at each pixel in the sample region.
4. Data Analysis
The recorded force curves are analyzed using a custom MATLAB procedure to calculate the cell stiffness. The following is a brief description of MATLAB procedure:
<ol>
	<li>The MATLAB program identifies the contact point coordinates z0 and d0 (see Figure 1) using an algorithm adopted from a published method by Lin et al. 29:</li>
	<li>For each data point in the force curve, perform a linear fit of the data to the left of the point of interest, and a Hertz model fit to the right (using the selected point as the initial point of contact), up to the set maximum indentation (200-300 nm recommended).</li>
	<li>For each point, calculate the relative RMS error of both fits and sum these values.</li>
	<li>The point which attains the minimum total fitting error is selected as the initial point of contact. 
	Note: Computation time can be reduced by implementing a golden-section search rather than linearly scanning the entire force curve.</li>
	<li>Sample deformation δ and indenting force F are calculated as: 
	<img alt="Static equilibrium equations, δ and F, detailing conditions for z in equilibrium analysis." fo:content-width="3.073in" fo:src="/files/ftp_upload/50497/50497eq1highres.jpg" src="/files/ftp_upload/50497/50497eq1.jpg" data-altupdatedat="1787630408000" data-alt="Equation 1"></li>
	<li>A least squares fitting is applied to fit the F vs. δ data in the post-contact region, z ≥ z0 , to the Hertz model to extract the Young's modulus, E of the cell: 
	<img alt="Contact mechanics formula, indentation force equations, diagram explaining spherical and conical tips." fo:content-width="2.773in" fo:src="/files/ftp_upload/50497/50497eq2highres.jpg" src="/files/ftp_upload/50497/50497eq2.jpg" data-altupdatedat="1787630408000" data-alt="Equation 2"> 
	, where v is the Poisson's ratio. 
	Note: When δ is more than 10% of the sample thickness (cell height), the measured cell stiffness is affected by the substrate stiffness. The thickness of peri-nuclear region is usually on the order of a few micrometers. Therefore, only the first 200-300 nm of F-δ curve is fit to the Hertz model.</li>
</ol>

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<li>Lim, S. M., Kreipe, B. A., Trzeciakowski, J., Dangott, L., Trache, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix+effect+on+RhoA+signaling+modulation+in+vascular+smooth+muscle+cells%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells.</a> Exp. Cell Res. 316, 2833-2848 (2010).</li>
</ol>

No conflicts of interest declared.

The AFM indentation method has advantages to characterize mechanical properties of living cells. Albeit less sensitive than the magnetic twisting cytometry and optical tweezers, which can measure forces on the piconewton level32, the AFM can detect resistance force from samples ranging from tens of pico-Newton to hundreds of nano-Newton, comparable to range of force that can be applied to cells using a micropipette19. This range of force fits the needs to create measurable deformations in all types ...

Mechanical properties, especially stiffness, of individual cells and their surrounding extracellular matrices (ECM) are critical for many biological processes including cell growth, motility, division, differentiation, and tissue homeostasis.1 It has been demonstrated that cell mechanical stiffness is mainly determined by the cytoskeleton, especially the networks of actin and intermediate filaments and other proteins associated with them.2 Results from mechanical tests on in vitro networks of actin and intermediate filaments suggest that the cell mechanics is largely dependent on the cytoskeletal structure and the pre-stress in the cytoskeleton.3-5 Stiffness of live cells is then regarded as an index to evaluate the cytoskeletal structure6, myosin activity7 and many other cellular processes. More importantly, changes in cell mechanical properties are also often found to be closely associated with various disease conditions such as tumor formation and metastasis.8-10 Monitoring the mechanical stiffness of living cells can therefore provide a novel way to monitor cell physiology; to detect and diagnose diseases8 ; and to evaluate the effectiveness of drug treatments.11,12
Multiple methods including particle-tracking microrheology,13-16 magnetic twisting cytometry,17 micropipette aspiration18,19 and microindentation20-22 have been developed to measure the elasticity of cells. Particle tracking microrheology traces the thermal vibrations of either submicron fluorescent particles injected into cells or fiducial markers inside the cell cytoskeleton.23 Elastic and viscous properties of cells are calculated from the measured particle displacements using the fluctuation-dissipation theorem.14,23 This method allows simultaneous measurements of local mechanical properties with high spatial resolution at different places in a cell. However, injecting fluorescent particles into cells may lead to changes in cellular function, cytoskeleton structure, and hence the cell mechanics. The micropipette aspiration method applies negative pressure in a micropipette of diameter ranging from 1 to 5 μm to suck a small piece of cell membrane into the pipette. Cell stiffness is calculated from the applied negative pressure and cell membrane deformation.18 This method, however, cannot detect the heterogeneous distribution of stiffness across the cell. Magnetic twisting cytometry (MTC) applies magnetic field to generate torque on super paramagnetic beads attached to the cell membrane.17 Cell stiffness is derived in this method from the relationship between the applied torque and the twisting deformation of the cell membrane. It is difficult to control the location of magnetic beads in the MTC method, and it is also challenging to characterize the twisting deformation with high resolution. Microindentation applies an indenter with well-defined geometry to punch into the cell. The indenting force and the resulting indentation in cells often follow the prediction of the Hertz model. Young's moduli of cells can be calculated from the force-indentation curves by fitting them to the Hertz model. This method has been widely applied to test the mechanical properties of tissue and cells despite of its limitations such as uncertainty in contact point determination, applicability of the Hertz model, and the potential to physically damage the cells. Among the many devices for microindentaion20, the Atomic Force Microscope (AFM) is commercially available and has been widely applied to characterize mechanical properties of living cells and tissues21,24-27.
This paper demonstrates the procedure of using an Asylum MFP3D-Bio AFM to characterize cell mechanics. AFM not only provides high-resolution topography of cells but also has been widely applied to characterize the mechanical properties of tissue cells. The principle of AFM indentation is illustrated in Figure 1. The AFM cantilever approaches the cell from a few micrometers above; makes contact with the cell; indents the cell so that the cantilever deflection reaches a preselected set point; and pulls away from the cell. During this process the cantilever deflection is recorded as a function of its location as shown in Figure 1. Before making contact with the cell, the cantilever moves in the medium without any apparent deflection. When indenting on the cell, the cantilever bends and the deflection signal increases. The cantilevers are modeled as elastic beams so that their deflection is proportional to the force applied to the cell. By setting the maximum cantilever deflection, the maximum magnitude of force applied to the sample is limited to avoid damage to cells. The portion of the force curve from point b to point c in Figure 1, where the tip indents into the cell, is fit to the hertz model to extract the cell stiffness.
<img src="/files/ftp_upload/50497/50497fig1.jpg" alt="Cantilever deflection process in atomic force microscopy; diagram with deflection vs. position graph." fo:content-width="4.75in" fo:src="/files/ftp_upload/50497/50497fig1highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Illustration of AFM microindentation and interpretation of the force curve. The top panel shows the motion of AFM cantilever driven by the piezo scanner. The vertical location of cantilever z and the cantilever deflection signal d is recorded during the process. The cantilever starts from point a, a few micrometers above the cell. While approaching the cell, the sample indentation δ remains zero until it reaches point b, where the tip comes into contact with the cell. The coordinates of point b in the plot are critical values for data analysis, denoted by (z0, d0&gt;). From b to c, the cantilever indents into the cell until the cantilever deflection reaches a set point, which is set to be the ratio between the targeted maximum indenting force and the cantilever spring constant. Once the deflection signal reaches the preset maximum value, the cantilever is then withdrawn from the cell to point d, where it often be pulled downwards due to tip-sample adhesion, detaches from the cell and returns to its initial location at e. The right panel illustrates the relationship between the indentation and the recorded z and d signal. In on the lower left panel is a plot of a representative force curve, the maximum indentation of a cantilever, of which the spring constant is measured to be 0.07N/m, is set to be 17 nm so that the maximum indenting force applied to sample is 1.2 nN. The key locations during the indentation are marked.

<table><tbody><tr><td>Atomic Force Microscope </td><td>Asylum Research</td><td>MFP3D-BIO</td><td></td></tr></tbody></table>

measuring the mechanical properties of living cells using atomic force microscopy

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.

Figure 2a shows three representative force curves taken from 3T3 fibroblasts cultured on plastic surface, polyacrylamide gel of Young's moduli 3,000 Pa and 17,000 Pa, respectively. After carefully identifying the contact points in the curves, the indenting force as function of cell deformation is plotted in Figure 2b. Under a force of magnitude smaller than 0.3 nN, a pyramid shape tip indents 3 micrometers into a cell cultured on a 3 kPa polyacrylamide gel. In contrast, a force more than...

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, ...

The overall goal of the following experiment is to measure the elastic modulus of living cells using an atomic force microscope. This is achieved by first performing a FM force spectroscopy measurements at various locations on the cell. As a second step, every forced distance curve is analyzed to determine the point where the A FM tip initially makes contact with the cell.Next, starting at the point of contact, the first 200 to 300 nanometers of indentation data are fitted to the hertz model. In order to extract the elastic modulus, the results yield a 2D map of the cell stiffness obtained by using the force mapping mode where each pixel represents a single forced distance curve Demonstrating the procedure will be Guen Thomas, a graduate student from my laboratory. To begin this procedure, clean the cantilever holder with 70%ethanol and allow it to dry.Then load a bru DNP 10 cantilever into the A FM according to the manufacturer's instructions. Next, calibrate the inverse optical lever sensitivity. This parameter describes the amount of photodiode response in volts per nanometer of cantilever deflection.To accomplish this first load a clean glass slide onto the sample stage. Install the a FM head and adjust the laser beam and alignment according to the manufacturer's instructions. Once aligned, engage the A FM tip on the glass slide with the P eight O withdrawn.Realign the mirror to a photo diode reading of negative two volts. Then perform a force spectroscopy measurement with a maximum photo diode response or trigger point of positive two volts. After data acquisition is complete, zoom into the firm contact region of the force curve and perform a linear fit to this region.To find the slope in volts per nanometer, then reset the mirror alignment to a free deflection of zero volts. Next, calibrate the cantilever spring constant using the thermal tune method described by levy and Malam. To begin, raise the scanner away from the sample stage so that there are no interactions between the tip and the sample.Then capture the thermal data by recording the thermal vibration of the cantilever beam. The A FM software analyzes a power spectrum of such a thermal vibration and plots it in a data window. Next, perform a fit to the data segment centered at the lowest frequency, also known as the fundamental resonance peak to determine the spring constant.In order to prepare the a FM for the culture plates, install a dish heater accessory on the a FM stage and set the temperature to 37 degrees Celsius. Wait 20 minutes for the system to reach a stable thermal equilibrium. Once preheated, place a culture dish on the a FM stage and secure it using the clamp provided with the dish heater for measurements longer than 30 minutes, carbon dioxide independent medium should be used to replace the normal culture medium.Next, apply a small drop of 37 degrees Celsius culture medium to the tip of the A FM cantilever and lower the a FM head until the tip is just submerged in liquid. Using a top view CCD camera realign the laser beam on the cantilever. The alignment in liquid will be different than an air because of the change in refractive index of the medium.Once aligned, engage the A FM tip on a cell-free area of the culture dish and perform calibration of the inverse optical lever sensitivity in the liquid environment. To begin mechanical property measurements of cells, position the cantilever tip above the perle eye region of a cell with the aid of an optical microscope. Precise adjustments of the cantilevers position are accomplished by applying offsets to the X and Y scanners.Then switch the a FM into four spectroscopy mode and set the indentation rate at five micrometers per second, which is low enough to avoid hydrodynamic effects. Next, set the deflection trigger point to two nano newtons. In order to avoid damaging the cells, adjust this value according to the sample stiffness.Additionally, select the relative triggering option, which will correct for any drift in the deflection signal. Then set the force distance at five micrometers to ensure that the tip will be fully detached from the cell between force measurements. Collect three force curves at different locations in the nuclei region of at least 30 cells for each condition.Although it is beneficial to take multiple curves on each cell. For reliable statistical data, taking too many can lead to changes in cell stiffness to distress from the A FM probe. Further characterize the distribution of mechanical properties throughout a single cell using force map mode.In force map mode, set the scan size to 30 micrometers the resolution to 32 by 32, and the indentation parameters are the same as those selected for single force curves. The A FM will then take single force curves at each pixel in the sample region and provide a stiffness map of the entire region. Next, analyze the recorded force curves using MATLAB to calculate the cell stiffness.Begin by adopting an algorithm first published by Lynette Al, which computes both a linear fit and hertz fit from each point using matlab. Calculate the relative RMS error of both fits and sum these values at each point. The point which attains the minimum total fitting error is selected as the initial point of contact locating this point is crucial for accurate measurement of the cells young's modulus.Next, calculate the sample deformation delta. Using the following equations with the deformation is equal to zero prior to cell contact where Z is less than Z zero and is equal to the difference between the cantilever deflection D and total distance Z past the contact point. Then calculate the indenting force F using the following equations where the force is equal to zero prior to cell contact where Z is less than Z zero and is equal to the spring constant K of the cantilever multiplied by the cantilever deflection past the point of contact.Elise square's fit is then applied to the sample deformation versus indenting force data in the post contact region of the hertz model in order to extract the Young's modulus of the cell based on the shape of tip used shown. Here are representative force curves taken from three T three fiberblast cells cultured on three different surfaces. As you can see here, different surfaces can greatly influence the stiffness of the cell cytoskeleton.The average deformation of cells cultured on a hard plastic surface are shown in red. Those grown on a stiff poly acrylamide gel are shown in purple, and those grown on an elastic poly acrylamide gel are shown in blue. After carefully identifying the contact points in the curves, the indenting forces as a function of cell deformation can be accurately shown.Additionally, their Young's modular, I can accurately be calculated from the first 300 nanometers of indentation after contact. Shown here is a fibroblast that has been transfected with green fluorescent protein tagged menton, a type of intermediate filament. The brighter the image, the more menton is in that region.This can be related to the stiffness of the cell cytoskeleton. Using the A FM force mapping technique presented here, it is possible to show a similar stiffness map of a fiberblast with a resolution of 32 by 32 pixels where each pixel represents 2.5 by 2.5 microns of cell surface area. After watching this video, it will have a good idea on how to use the atomic force microscopy to measure the elasticity of living tissue cells.

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Research

JoVE Journal

Bioengineering

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Here, we present a protocol for the simultaneous use of Förster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

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.

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

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

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

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

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.

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

Learn to measure the elastic modulus of living cells using atomic force microscopy and force spectroscopy. This protocol enables researchers to create 2D stiffness maps and study cell mechanics in biophysics.

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

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