This work was supported by the Max Planck Society, the German Cancer Research Center, the University of T&#252;bingen, and the Bundesministerium f&#252;r Bildung und Forschung (grant no. 0312040).
We thank Eduard Hermann for helping us automate the analysis of the force curve data and Dr. Jakob Suckale for careful reading of this manuscript.

1. Preparation of Supported Lipid Bilayers (SLB)11,12,21
<ol>
	<li>Preparation of Lipid Mixture and Multilamellar Vesicle Suspensions
	<ol>
		<li>Prepare the following buffers beforehand.
		<ol>
			<li>Prepare PBS buffer at concentrations of 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, and 137 mM NaCl, pH 7.2.</li>
			<li>Prepare SLB (supported lipid bilayer) buffer at concentrations of 150 mM NaCl, 10 mM HEPES, pH 7.4.</li>
			<li>Prepare a solution of 1 M CaCl2<...

<ol>
	<li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscope.">Atomic Force Microscope.</a> Phys. Rev. Lett. 56, 930-933 (1986).</li><li>Hansma, P. K., Elings, V. B., Marti, O., Bracker, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+Tunneling+Microscopy+and+Atomic+Force+Microscopy:+Application+to+Biology+and+Technology.">Scanning Tunneling Microscopy and Atomic Force Microscopy: Application to Biology and Technology.</a> Science. 242, 209-216 (1988).</li><li>Gaczynska, M., Osmulski, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM+of+biological+complexes:+What+can+we+learn.">AFM of biological complexes: What can we learn.</a> Curr, Opin. Colloid In. 13, 351-367 (2008).</li><li>Goksu, E. I., Vanegas, J. M., Blanchette, C. D., Lin, W. -. C., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM+for+structure+and+dynamics+of+biomembranes.">AFM for structure and dynamics of biomembranes.</a> BBA-Biomembranes. 1788, 254-266 (2009).</li><li>Muller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM:+A+Nanotool+in+Membrane+Biology.">AFM: A Nanotool in Membrane Biology.</a> Biochemistry-US. 47, 7986-7998 (2008).</li><li>Redondo-Morata, L., Giannotti, M. I., Sanz, F., Bar&#243;, A. M., Reifenberger, R. G., Sanz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atomic Force Microscopy in Liquid: Biological Applications. , (2012).</li><li>Castellana, E. T., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+supported+lipid+bilayers:+From+biophysical+studies+to+sensor+design.">Solid supported lipid bilayers: From biophysical studies to sensor design.</a> Surf. Sci. Rep. 61, 429-444 (2006).</li><li>Frederix, P. L. T. M., Bosshart, P. D., Engel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+of+Biological+Membranes.">Atomic Force Microscopy of Biological Membranes.</a> Biophys. J. 96, 329-338 (2009).</li><li>Mennicke, U., Salditt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Solid-Supported+Lipid+Bilayers+by+Spin-Coating.">Preparation of Solid-Supported Lipid Bilayers by Spin-Coating.</a> Langmuir. 18, 8172-8177 (2002).</li><li>Raviakine, I., Brisson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Supported+Phospholipid+Bilayers+from+Unilamellar+Vesicles+Investigated+by+Atomic+Force+Microscopy.">Formation of Supported Phospholipid Bilayers from Unilamellar Vesicles Investigated by Atomic Force Microscopy.</a> Langmuir. 16, 1806-1815 (2000).</li><li>Chiantia, S., Ries, J., Kahya, N., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+AFM+and+Two-Focus+SFCS+Study+of+Raft-Exhibiting+Model+Membranes.+.">Combined AFM and Two-Focus SFCS Study of Raft-Exhibiting Model Membranes. .</a> ChemPhysChem. 7, 2409-2418 (2006).</li><li>Unsay, J., Cosentino, K., Subburaj, Y., Garcia-Saez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+effects+on+membrane+structure+and+dynamics.">Cardiolipin effects on membrane structure and dynamics.</a> Langmuir. 29, 15878-15887 (2013).</li><li>Dom&#232;nech, &. #. 2. 1. 0. ;., Sanz, F., Montero, M. T., Hern&#225;ndez-Borrell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+and+structural+study+of+the+main+phospholipid+components+comprising+the+mitochondrial+inner+membrane.">Thermodynamic and structural study of the main phospholipid components comprising the mitochondrial inner membrane.</a> BBA-Biomembranes. 1758, 213-221 (2006).</li><li>Dom&#232;nech, &. #. 2. 1. 0. ;., Morros, A., Caba&#241;as, M. E., Teresa Montero, M., Hern&#225;ndez-Borrell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+planar+bilayers+from+hexagonal+phases.">Supported planar bilayers from hexagonal phases.</a> BBA-Biomembranes. 1768, 100-106 (2007).</li><li>Garcia-Saez, A. J., Chiantia, S., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+line+tension+on+the+lateral+organization+of+lipid+membranes.">Effect of line tension on the lateral organization of lipid membranes.</a> J Biol Chem. 282, 33537-33544 (2007).</li><li>Alessandrini, A., Seeger, H. M., Caramaschi, T., Facci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Force+Spectroscopy+on+Supported+Lipid+Bilayers:+Effect+of+Temperature+and+Sample+Preparation.">Dynamic Force Spectroscopy on Supported Lipid Bilayers: Effect of Temperature and Sample Preparation.</a> Biophys. J. 103, 38-47 (2012).</li><li>Butt, H. -. J., Franz, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rupture+of+molecular+thin+films+observed+in+atomic+force+microscopy+I.+Theory.">Rupture of molecular thin films observed in atomic force microscopy I. Theory.</a> Physical Review E. 66, 031601 (2002).</li><li>Garcia-Manyes, S., Oncins, G., Sanz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Temperature+on+the+Nanomechanics+of+Lipid+Bilayers+Studied+by+Force+Spectroscopy.">Effect of Temperature on the Nanomechanics of Lipid Bilayers Studied by Force Spectroscopy.</a> Biophys. J. 89, 4261-4274 (2005).</li><li>Chiantia, S., Kahya, N., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raft+domain+reorganization+driven+by+short-+and+long-chain+ceramide:+a+combined+AFM+and+FCS+study.">Raft domain reorganization driven by short- and long-chain ceramide: a combined AFM and FCS study.</a> Langmuir. 23, 7659-7665 (2007).</li><li>Canale, C., Jacono, M., Diaspro, A., Dante, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+spectroscopy+as+a+tool+to+investigate+the+properties+of+supported+lipid+membranes.">Force spectroscopy as a tool to investigate the properties of supported lipid membranes.</a> Microsc. Res. Techniq. 73, 965-972 (2010).</li><li>Garc&#237;a-S&#225;ez, A. J., Chiantia, S., Salgado, J., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore+Formation+by+a+Bax-Derived+Peptide:+Effect+on+the+Line+Tension+of+the+Membrane+Probed+by+AFM.">Pore Formation by a Bax-Derived Peptide: Effect on the Line Tension of the Membrane Probed by AFM.</a> Biophys. J. 93, 103-112 (2007).</li><li>Moreno Flores, S., Toca-Herrera, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+future+of+scanning+probe+microscopy:+Combining+atomic+force+microscopy+with+other+surface-sensitive+techniques,+optical+microscopy+and+fluorescence+techniques.">The new future of scanning probe microscopy: Combining atomic force microscopy with other surface-sensitive techniques, optical microscopy and fluorescence techniques.</a> Nanoscale. 1, 40-49 (2009).</li><li>Simons, K., Vaz, W. L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+Systems,+Lipid+Rafts,+and+Cell+Membranes1.">Model Systems, Lipid Rafts, and Cell Membranes1.</a> Annu. Rev. Bioph. Biom. 33, 269-295 (2004).</li><li>Pike, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rafts+defined:+a+report+on+the+Keystone+symposium+on+lipid+rafts+and+cell+function.">Rafts defined: a report on the Keystone symposium on lipid rafts and cell function.</a> The Journal of Lipid Research. 47, 1597-1598 (2006).</li><li>Kahya, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Lipid+Mobility+of+Raft-exhibiting+Model+Membranes+by+Fluorescence+Correlation+Spectroscopy.">Probing Lipid Mobility of Raft-exhibiting Model Membranes by Fluorescence Correlation Spectroscopy.</a> J. Biol. 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The authors declare that they have no competing financial interests.

SLBs composed of DOPC:SM:Chol (2:2:1) were formed on mica after vesicle adsorption and rupture induced by calcium chloride. This lipid composition separated into Ld and Lo phases. The Lo phase is enriched in sphingomyelin and cholesterol and is less fluid/more viscous (Figure 1A) than the Ld phase11. The separation of Lo from Ld phase manifests as circular structures elevated above the surrounding (Figure 1B, C)...

Atomic Force Microscopy (AFM) generates an image of a surface by scanning across an area of the sample using a cantilever with a very sharp tip1. The movement of the cantilever probes the surface topology of the sample. AFM has been widely applied to biological molecules &#8212; including proteins, DNA, and membranes, owing to its versatility in analyzing fixed samples in air or near-native state in liquid2-5.
Apart from its high-resolution imaging capability in the nanometer range, the AFM cantilever acts as a spring to probe interaction forces (adhesion and repulsion) and mechanical properties of the sample5,6. This is known as force spectroscopy. In this mode, the probe first approaches the sample and exerts a force on it, then is retracted until it loses contact with the sample (Figure 1A). The generated curves show force as a function of distance of the cantilever for both the approach and retraction. Several properties including the elastic modulus to measure the stiffness of a material, and adhesion forces can be derived.
Supported lipid bilayers are biological model membranes lying on top of a solid support &#8212; usually mica, borosilicate glass, fused silica, or oxidized silicon7. They are prepared using various techniques like vesicle deposition, Langmuir-Blodgett method and spin-coating8,9. AFM imaging has been used to follow the formation of these supported bilayers10, and probe different structures formed by membranes of different compositions11-15.
Performing force spectroscopy on supported bilayers results in a peak in the approach curve. This peak indicates the force needed to pierce the bilayer, and is called breakthrough force. The bilayer thickness can also be measured using the force curve6 . The typical breakthrough force of bilayers range between 1-50 nN6. These properties depend on lipid packing (liquid or gel phase) and structure (acyl chain length and degree of unsaturation) and altered by membrane-active agents16. The theory behind the rupture has been explained17 and other experimental parameters such as cantilever softness, tip radius and approach speed also affect the breakthrough force15,16,18. Force spectroscopy has been used to analyze properties of different lipid phases11,19, composition-dependent changes12,20, as well as effects of other biomolecules, like peptides, on the stability of the membrane21.
The flat orientation of supported bilayers is advantageous for combining AFM with other methods such as surface plasmon resonance22 and fluorescence microscopy11,19 to better characterize structure and properties of membranes.
This detailed video protocol is intended to prepare supported lipid bilayers using vesicle deposition and analyze them with AFM and force spectroscopy. While vesicles of various sizes may be used to prepare bilayers, this protocol focuses on small and large unilamellar vesicles. Supported bilayers that phase separate into liquid ordered (Lo) and liquid disordered (Ld) phases were characterized11,15. The membrane is composed of di-oleoyl-phosphatidylcholine (DOPC), sphingomyelin (SM), and cholesterol (Chol) at 2:2:1 ratio. This composition models the lipid rafts, which are proposed to behave as platforms important for protein trafficking and sorting, cell signaling and other cellular processes23,24.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850375P</td>
			<td>Comes as lyophilized powder in sealed vials. Dissolve all powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20&#176;C. For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&#38;view=article&#38;id=1679&#38;Itemid=398</td>
		</tr>
		<tr>
			<td>Sphingomyelin (Brain, Porcine)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>860062P</td>
			<td>Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20&#176;C.&#160; For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&#38;view=article&#38;id=1679&#38;Itemid=398</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>700000P</td>
			<td>Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20&#176;C.&#160; For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&#38;view=article&#38;id=1679&#38;Itemid=398</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl), 99.8%</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl), 88%</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogenphosphate (Na2HPO4), &#62;99%</td>
			<td>AppliChem GmbH</td>
			<td>A1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogenphosphate (KH2PO4), 99%</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2), molecular biology grade</td>
			<td>AppliChem GmbH</td>
			<td>A4689</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, molecular biology grade</td>
			<td>AppliChem GmbH</td>
			<td>A3724</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip, 24x60 mm, 1mm thickness</td>
			<td>Duran Group</td>
			<td>2355036</td>
			<td></td>
		</tr>
		<tr>
			<td>Mica blocks</td>
			<td>NSC Mica Exports Ltd.</td>
			<td></td>
			<td>These are mica pieces at least 1 sq. Inches in area and thickness randing from 0.006 inches to 0.016 inches. They are cut to a specific size by the company for shipping. Small mica discs can be punched from the mica blocks using the punch and die set.&#160; Always handle mica with gloves or tweezers.</td>
		</tr>
		<tr>
			<td>Punch and Die Set</td>
			<td>Precision Brand Products, Inc</td>
			<td>40105</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Adhesive</td>
			<td>Norland Products, Inc.</td>
			<td>NOA 88</td>
			<td>Liquid adhesive that hardens when cured under long wavelength UV light.&#160;</td>
		</tr>
		<tr>
			<td>Laboratory Equipment Grease</td>
			<td>Borer Chemie AG</td>
			<td>Glisseal N</td>
			<td></td>
		</tr>
		<tr>
			<td>Liposome Extruder</td>
			<td>Avestin</td>
			<td>LiposoFast-Basic</td>
			<td>As an alternative one can also look at offers from Northern Lipids, Inc.</td>
		</tr>
		<tr>
			<td>Adhesive Tape</td>
			<td>3M</td>
			<td>Scotch(R) Magic (TM) Tape 810 (1-inch)</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath Sonicator</td>
			<td>Bandelin Sonorex Digitec</td>
			<td>DT-31</td>
			<td>No heating, Frequency: 35 kHz, Ultrasonic Peak Output: 160 W, HF Power: 40 W. http://www.bandelin.com/datenblaetter/dt/DT_31_H_1798d_DE_GB_FR_BANDELIN.pdf</td>
		</tr>
		<tr>
			<td>Silicon Nitride AFM Cantilever&#160;</td>
			<td>Bruker AFM Probes</td>
			<td>DNP-10</td>
			<td>Each cantilever has four tips and their nominal tip radius is 20 nm (with possible maximum at 60 nm). Based on the specifications, we use tip D with resonance frequency of 18 kHz, and nominal spring constant of 0.06 N/m.</td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>JPK</td>
			<td>JPK Nanowizard II mounted on Zeiss Axiovert 200</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Supported lipid bilayers composed of DOPC:SM:Chol (2:2:1) were imaged in AFM (Figure 2 A-C). Because of the lipid composition, SM/Chol-rich Lo and DOPC-rich Ld phases were observed. The height profile from the AFM imaging can provide important information on the membrane structure. By looking at the height profile, the bilayer thickness can be measured in presence of defects in the membrane (Figure 2B), or the difference in height between the Lo/Ld</...

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Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

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 ...

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Research

JoVE Journal

Bioengineering

14.5K Views.  Interfaculty Institute for Biochemistry. We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.