The authors would like to thank Elena Lomakina, Richard Bauserman, Margaret Youngman, Shay Vaknin, Jessica Snyder, Chris Striemer, Nakul Nataraj, Hung Li Chung, Tejas Khire, and Eric Lam for their assistance with this project. This project was funded by NIH #PO1 HL 018208.

1. Methods
1.1 Cell Flow Chamber
A flow chamber, shown in Figure 1, was constructed so that cells could be grown under a shear of 1.0 Pa (10 dyn/cm2) and then transferred directly to an Asylum MFP3D AFM (Santa Barbara, CA).
<ol>
	<li>The flow chamber was prepared for the experiment by first cleaning the glass slides in piranha solution (3:1 H2SO4:H2O2) for 15 min and then washing them with distilled water. They were then baked to dry and coated with aminopropyl triethoxy silane (APTES) in a vacuum deposition chamber.</li>
	<li>A silicone gasket was cut using a Silhouette SD cutting tool. This allowed us to finely control the flow chamber dimensions for control of the flow rate and shear stress during cell growth. Typically, a channel was cut 6.4 mm wide by 19 mm long from a sheet of 0.4 mm silicone. The flow rate necessary to generate a shear stress of 1.0 Pa (10 dyn/cm2) is calculated assuming laminar flow in a rectangular channel with the equation:</li>
</ol>
<img alt="Equation 1" fo:content-width="0.737in" fo:src="/files/ftp_upload/50163/50163eq1highres.jpg" src="/files/ftp_upload/50163/50163eq1.jpg" /> 
where Q is the flow rate, &tau; is the shear stress, &mu; is the viscosity of the medium, assumed here to be 1.0 mPa (0.01 dyn*sec/cm2), h is the height and w is the width of the flow chamber.
<ol start="3">
	<li>The top piece of the flow chamber was aligned with the gasket in the cell culture dish and secured with a magnetic ring. The assembly was filled with isopropyl alcohol (IPA) for sterilization.</li>
	<li>The full flow system was assembled. The flow ports in the cell culture dish were connected to three-way valves. The valves were connected to open 30 ml syringes. IPA was flushed through the system, which was then washed with 30 ml of McCoy's medium with 4% Fetal Calf Serum (FCS). The system was then filled with 20 ml of the Vec Technologies cell growth medium. Covers were placed on the tops of the syringes. The catch reservoir syringe cap had a needle in place to move medium back to the feed reservoir. Sterile 0.2 &mu;m filters were attached to the air inlets in the covers to prevent contamination of the system. The flow chamber was then ready for cell seeding.</li>
</ol>
1.2 Cell Culture
<ol>
	<li>Human umbilical vein endothelial cells (HUVEC's) and growth medium were purchased from Vec Technologies (Rensselaer, NY) and grown to confluence in a T25 Flask.</li>
	<li>The growth medium was removed from the flask and the monolayer of cells released with 2 ml of 2.5% trypsin. Once the cells were in solution, the trypsinization was quenched with the addition of 10 ml of cell culture medium to the flask.</li>
	<li>The cell suspension was centrifuged for 5 min and the supernatant was removed. The cells were resuspended in 1 ml of cell culture medium (containing serum) for injection into the flow chamber.</li>
	<li>The cell suspension (0.5 ml, ~50,000 cells) was loaded into a syringe and injected into the flow chamber through a three-way valve.</li>
	<li>The cells were allowed to settle and adhere to the glass substrate for 2 hr before flow was started. The cells were grown under flow in an incubator at 37 &deg;C for 1 to 5 days until confluent.</li>
</ol>
1.3 Cantilever Preparation and Cell Indentation
<ol>
	<li>Tipless AFM cantilevers (NanoWorld, Switzerland) were cleaned in nitric acid for 5 min and functionalized with aminopropyl triethoxy silane (APTES) in a vapor deposition chamber.</li>
	<li>A solution of 5 mg/ml by weight NHS-sulfo-LC-biotin in Hank's Buffered Salt Solution (HBSS) was prepared. The cantilevers were submerged in the solution for 15 min to conjugate the silane with N-Hydroxysuccinimide (NHS) chemistry.</li>
	<li>A solution of biotin free medium was made by incubating 20 ml of Vec Technologies cell culture medium (including serum) with 200 &mu;l of streptavadin beads for 12 hr. The beads were removed from the medium with a magnet and the medium was filtered through a 0.22 &mu;m sterile filter.</li>
	<li>The flow chamber was removed from the cell culture dish and the cells were washed in 37 &deg;C biotin-free medium.</li>
	<li>A stock solution of 1 &mu;l of 2.4 &mu;m beads coated with streptavidin in 1 ml of biotin free medium was prepared, and 100 &mu;l of the stock was added to the cell culture dish.</li>
	<li>Streptavidin beads were picked up with the cantilever by landing the tip on the glass surface next to a bead, retracting, positioning the apex of the cantilever over the bead, and then pressing the cantilever down onto the bead and resting for several seconds.</li>
	<li>The sensitivity of the cantilever was measured by indenting onto a region of bare glass and using the slope of the curve to set the tip deflection as a function of voltage.</li>
	<li>The spring constant of the cantilever was then calculated from a thermal calibration in the MFP3D software.</li>
	<li>The calibrated cantilever was then used to indent the samples, as shown in Figure 2. These 2.4 &mu;m beads offer a larger contact area with the cell surface so that the mechanical properties of the soft glycocalyx layer can be detected. The cantilever was positioned over a cell near the cell nucleus and a soft approach of the tip onto the cell was used to set the cantilever height approximately 3 &mu;m above the cell surface. The software was set for 20 repeated indentations at a rate of 1 &mu;m/sec to a maximum force of 7 nN. Approximately 6 sec elapsed between successive contacts. It is possible to use different rates of indentation to test for time-dependent properties of the glycocalyx, although in these initial experiments, only a single indentation rate (1 &mu;m/sec) was used.</li>
</ol>
2. Indentation Theory
Indentation into an elastic half-space with a sphere of radius R can be described using Hertz theory where the force of indentation, F, is given by the equation:
<img alt="Equation 2" fo:content-width="1.157in" fo:src="/files/ftp_upload/50163/50163eq2highres.jpg" src="/files/ftp_upload/50163/50163eq2.jpg" /> 
Where &delta; is the indentation depth and E* is the reduced modulus of the material under test (Figure 3). In the case of an infinitely stiff indenter impinging a uniform elastic half-space, E* is given by the equation:
<img alt="Equation 3" fo:content-width="0.757in" fo:src="/files/ftp_upload/50163/50163eq3highres.jpg" src="/files/ftp_upload/50163/50163eq3.jpg" /> 
where E is the elastic modulus and &nu; is the Poisson ratio of the material. Recent work with polymer films has inspired the development of a two layer model for determining the modulus and thickness of thin films 1. We are applying this model to cell biology by treating the glycocalyx as a uniform thin soft film on the surface of the cell body. Using this model, the reduced modulus of the system becomes:
<img alt="Equation 4" fo:content-width="2.85in" fo:src="/files/ftp_upload/50163/50163eq4highres.jpg" src="/files/ftp_upload/50163/50163eq4.jpg" /> 
Where EGC is the modulus of the glycocalyx, Ecell is the modulus of the cell body, P, Q and n are constants which have been empirically determined from the polymer fits, and z is given by the equation:
<img alt="Equation 5" fo:content-width="1.257in" fo:src="/files/ftp_upload/50163/50163eq5highres.jpg" src="/files/ftp_upload/50163/50163eq5.jpg" /> 
Where t is the thickness of the glycocalyx layer. A schematic of these parameters is shown in Figure 3. The model has been shown to be an accurate way of determining the modulus and thickness of a thin film on stiffer substrate 1. This equation can be used to fit the curves obtained from indentation into cells to determine the modulus and thickness of the endothelial glycocalyx, as shown in Figure 4.

<ol>
	<li>Clifford, C., Seah, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+measurement+of+young's+modulus+for+compliant+layers+on+stiffer+substrates+including+the+effect+of+poisson's+ratios.">Nanoindentation measurement of young's modulus for compliant layers on stiffer substrates including the effect of poisson's ratios.</a> Nanotechnology. , (2009).</li><li>Gouverneur, M., Spaan, J. A. E., Pannekoek, H., Fontijn, R. D., Vink, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shear+stress+stimulates+incorporation+of+hyaluronan+into+endothelial+cell+glycocalyx.">Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx.</a> Am. J. Physiol. Heart. Circ. Physiol. 290 (1), 458-452 (2006).</li><li>Hocde, S. A., Hyrien, O., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+distribution+relative+to+neutrophil+surface+topography+assessed+by+tirfm.">Cell adhesion molecule distribution relative to neutrophil surface topography assessed by tirfm.</a> Biophysical Journal. 97 (1), 379-387 (2009).</li><li>Lipowski, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+as+a+barrier+to+leukocyte+adhesion+and+its+mediation+by+extracellular+proteases.">The endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases.</a> Annals of biomedical engineering. 40 (4), 840-848 (2012).</li><li>Lu, L., Oswald, S. J., Ngu, H., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+actin+stress+fibers+in+living+cells.">Mechanical properties of actin stress fibers in living cells.</a> Biophysical Journal. 95 (12), 6060-6071 (2008).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+surface+layer.">The endothelial surface layer.</a> Pflugers Archiv. European Journal of Physiology. 440 (5), 653-666 (2000).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophysical Journal. 87 (6), 4237-4245 (2004).</li><li>vanden Berg, B. M., Vink, H., Spaan, J. A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+protects+against+myocardial+edema.">The endothelial glycocalyx protects against myocardial edema.</a> Circulation Research. 92 (6), 592-594 (2003).</li><li>Williams, T. E., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+impact+of+membrane+microtopology+on+effective+two-dimensional+affinity.">Quantifying the impact of membrane microtopology on effective two-dimensional affinity.</a> J. Biol. Chem. 276 (16), 13283-138 (2001).</li><li>Vink, H., Duling, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Luminal+Domains+for+Macromolecules,+Erythrocytes,+and+Leukocytes+Within+Mammalian+Capillaries.">Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries.</a> Circulation Research. 79, 581-589 (1996).</li></ol>

No conflicts of interest declared.

We used values calculated from the two-layer model and Hertz theory to model the interaction of a leukocyte circulating in the blood with the endothelial wall. We have calculated that a microvillus on the leukocyte with a diameter of 50 nm under a 10 pN load would indent approximately 150 nm into the glycocalyx, only a fraction of the total thickness. This indicates that the glycocalyx, with properties as measured in this experiment, is a significant barrier to cell-cell interaction and can be a large steric hindrance wh...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>McCoy's Medium</td>
 <td>Gibco</td>
 <td>16600-082</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Calf Serum</td>
 <td>Hyclone</td>
 <td>SH30070</td>
 <td></td>
 </tr>
 <tr>
 <td>Endothelial Cell Growth Medium</td>
 <td>Vec Technologies</td>
 <td>MCDB-131</td>
 <td></td>
 </tr>
 <tr>
 <td>Pooled Human Umbilical Vein Endothelial Cells</td>
 <td>Vec Technologies</td>
 <td>PHUVEC/T-25</td>
 <td></td>
 </tr>
 <tr>
 <td>Sulfuric Acid</td>
 <td>JT Baker</td>
 <td>9681-02</td>
 <td></td>
 </tr>
 <tr>
 <td>Hydrogen Peroxide</td>
 <td>VWR</td>
 <td>BDH3742-1</td>
 <td></td>
 </tr>
 <tr>
 <td>(3-aminopropyl)triethoxysilane</td>
 <td>Aldrich</td>
 <td>440140-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td>Isopropyl Alcohol</td>
 <td>VWR</td>
 <td>BDH8999-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Cellgro</td>
 <td>25-054-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's Buffered Salt Solution</td>
 <td>Gibco</td>
 <td>14175-095</td>
 <td></td>
 </tr>
 <tr>
 <td>sulfo-NHS-LC-Biotin</td>
 <td>Thermo Scientific</td>
 <td>21335</td>
 <td></td>
 </tr>
 <tr>
 <td>Streptavadin beads</td>
 <td>Dynabeads</td>
 <td>112.06D</td>
 <td></td>
 </tr>
 <tr>
 <td>MFP-3D AFM</td>
 <td>Asylum Research</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tipless Cantilevers</td>
 <td>Nanoworld</td>
 <td>ARROW-TL1-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Silhouette SD</td>
 <td>Quickutz</td>
 <td>Silhouette-SD</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone Rubber</td>
 <td>Stockwell Elastomerics</td>
 <td>SE50-RS</td>
 <td></td>
 </tr>
 <tr>
 <td>30 ml Syringes</td>
 <td>Benton Dickinson</td>
 <td>309650</td>
 <td></td>
 </tr>
 <tr>
 <td>18 gauge needles</td>
 <td>Benton Dickinson</td>
 <td>305196</td>
 <td></td>
 </tr>
 <tr>
 <td>Extension Sets</td>
 <td>Hospira</td>
 <td>4429-48</td>
 <td></td>
 </tr>
 <tr>
 <td>4 way valves</td>
 <td>Teleflex</td>
 <td>W21372</td>
 <td></td>
 </tr>
 <tr>
 <td>Male/Female Port Caps</td>
 <td>Smith's Medical</td>
 <td>MX491B</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Watson-Marlow</td>
 <td>401U/D</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic Tubing</td>
 <td>Watson-Marlow</td>
 <td>903.0016.016</td>
 <td></td>
 </tr>
 <tr>
 <td>sterile filters</td>
 <td>Pall Life Science</td>
 <td>4652</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

In a typical experiment, 20 force-vs-distance curves were obtained from a given region of the cell, typically in the perinuclear region, near, but not on, the nucleus (within ~2 &mu;m). The curves were aligned to account for any sample drift over the duration of the measurement and then averaged to remove cantilever noise, as shown in Figure 4. The curves were analyzed and fit with the two layer model that was developed for determining the modulus and thickness of thin polymer films 1. From fi...

Watch this Scientific Journal Video about Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy at JoVE.com

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

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 ...

quantifying-mechanical-properties-endothelial-glycocalyx-with-atomic

Research

JoVE Journal

Bioengineering

13.1K 조회수.  University of Rochester. 내피 glycocalyx의 기계적 특성은 AFM의 cantilevers에 마이크론 크기의 영역을 사용하여 들여 쓰기에 의해 측정되었다. 내피 세포는 glycocalyx 식을 유도하기 위해 생리 흐름 조건에서 사용자 정의 챔버에 배양 하였다. 데이터는 glycocalyx 두께와 탄성 계수를 결정하기 위해 박막 모델을 사용 분석 하였다.

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

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

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

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

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...