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With the piezo withdrawn, realign the mirror to a photodiode reading of -2 V. Perform a force spectroscopy measurement with a trigger point (maximum photodiode response) of +2 V.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With the piezo withdrawn, realign the mirror to a photodiode reading of -2 V. Perform a force spectroscopy measurement with a trigger point (maximum photodiode response) of +2 V. Note: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 &deg;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 &deg;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 &mu;m/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 &mu;m.</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 &delta; and indenting force F are calculated as: 
	<img alt="Equation 1" fo:content-width="3.073in" fo:src="/files/ftp_upload/50497/50497eq1highres.jpg" src="/files/ftp_upload/50497/50497eq1.jpg" /></li>
	<li>A least squares fitting is applied to fit the F vs. &delta; data in the post-contact region, z &ge; z0 , to the Hertz model to extract the Young's modulus, E of the cell: 
	<img alt="Equation 2" fo:content-width="2.773in" fo:src="/files/ftp_upload/50497/50497eq2highres.jpg" src="/files/ftp_upload/50497/50497eq2.jpg" /> 
	, where v is the Poisson's ratio. 
	Note: When &delta; 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-&delta; curve is fit to the Hertz model.</li>
</ol>

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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 &mu;m 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="Figure 1" fo:content-width="4.75in" fo:src="/files/ftp_upload/50497/50497fig1highres.jpg"/> 
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 &delta; 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>). 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

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...

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Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

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, ...

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Research

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Bioengineering

39.9K Views.  Worcester Polytechnic Institute. This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Video: Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

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

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.

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 ...

Engineering

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

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

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.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Biology

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

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 ...

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

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

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

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.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

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

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live 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.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

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

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

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.

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

Immunology and Infection

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

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Chapters in this video

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

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

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

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

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

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

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

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

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants