Name: atomic force microscopy imaging and force spectroscopy of supported lipid bilayers
Uploaded: 2015-07-22T14:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers at JoVE.com

这项工作是由马普学会，图宾根大学的德国癌症研究中心，以及德国联邦联邦教育与研究部（批准号:0312040）的支持。 我们感谢爱德华·赫尔曼帮助我们自动力曲线数据和雅各布Suckale博士仔细阅读本手稿的分析。

1.准备支持的脂质双层（SLB）11,12,21 <ol><li>脂质混合物和多层囊泡悬浮液的制备<ol><li>事先准备以下的缓冲区。 <ol><li>制备PBS缓冲液以2.7毫米氯化钾，浓度为1.5mM KH 2 PO 4，8毫的Na 2 HPO 4，和137 mM氯化钠，pH值7.2。 </li><li>制备SLB（支持的脂质双层）缓冲液，在150 mM氯化钠，的10mM HEPES，pH 7.4中的浓度。 </li><li>制备的1M的CaCl 2的溶液中。 </li></ol></li><li>溶解在氯仿以下脂质在期望的浓度:例如，二油酰基磷脂酰胆碱（DOPC），在25毫克/毫升，鞘磷脂（SM）在25毫克/毫升和胆固醇（CHOL）以10毫克/毫升。 </li><li>取18.38微升DOPC的，17.10微升的SM和11.30微升澈的，使用1mg总脂质中一个2的溶液:2:1 DOPC:缅澈的摩尔比。改变卷基础上相应的conce1.1.2制备的脂质ntrations。但是，保持的摩尔比为2:2:1 DOPC:缅澈，如改变的摩尔比将改变膜25的相位特性。 </li><li>通过涡旋彻底混合溶液。添加荧光染料脂质在0.05％（摩尔）如果希望通过荧光显微镜可视化的双层。 注意:长链dialkylcarbocyanines家庭，像那样，的DiI和DIO跨越大范围的波长的，并且可以理想地用于标记脂质膜。替代地，荧光标记的脂质，如若丹明 - 磷脂或BODIPY胆固醇都可以使用。在任何情况下，染料的浓度应根据显微镜的设置被优化，同时考虑到高浓度结合到脂质的荧光团可以改变脂质的行为19。 </li><li>擦干采用N 2气的氯仿（或任何可用的惰性气体，如Ar和He）。 </li><li>此外牛逼干他的脂质混合物在真空下至少1小时。 注意:如果存储该脂质混合物，净化空气用惰性气体（N 2，Ar或He），并在-20℃存储。分装多余的脂肪，在氯仿成小棕瓶。干燥它们以类似的方式为所述脂质混合物，置换空气用惰性气体（N 2，Ar或He），并储存在-20℃。 </li><li>溶解在PBS缓冲液中的干燥脂质的10毫克/毫升的浓度。 </li><li>涡大力为1分钟的混合物约20秒，以得到浑浊悬浮液，含有多层囊泡。确保没有脂质膜粘到闪烁小瓶的侧面。 </li><li>发布这一悬挂成10等份微升（1毫克脂质，使10等分）。 </li><li>储存在-20°C，直到进一步使用。 </li></ol></li><li>单层囊泡的制备 注意:各种尺寸单层囊泡的可使用超声处理法或挤压法来制备。超声使用sound能量搅拌该混合物中，并分离出多层悬浮的层。这是通过清理朦胧暂停指示。挤压迫使多层囊泡穿过膜过滤器具有确定孔径。 <ol><li>超声法<ol><li>取10微升的多层脂质悬浮液，加入150微升SLB缓冲区。 </li><li>转移混合物成小（2ml）中加帽的玻璃小瓶中并密封用Parafilm。 </li><li>超声处理在室温下将混合物在水浴超声10分钟，或直至它变得清晰，得到小单层囊泡（SUV的，低于50纳米的直径）。 </li></ol></li><li>挤压法<ol><li>取10微升的多层脂质悬浮液，加入150微升SLB缓冲区。 </li><li>转移悬浮液送入低温管中。 </li><li>通过浸没在低温管在液氮几秒冻结的脂质悬浮液。 </li><li>解冻通过浸没在低温管在水浴中在室温下或37℃下进行数分钟的脂质悬浮液。重复冻融步骤的至少10倍。 </li><li>为了挤出脂质悬浮液，使用聚碳酸酯膜具有200nm的孔的尺寸（其他孔径可用等，100纳米或400纳米）和市售挤出装置26。 注意:目前，有两个挤出机的设备可商购的:一个手动挤出机可用于小体积（200μl到几毫升）。在这种情况下，将样品通过膜通过手动推悬浮来回两个注射器之间挤出。根据制造商，脂质等份将需要被组合以达到设定的最高最小体积。对于更大的体积（最多50毫升）更复杂的设备用于在其中悬浮液是通过聚碳酸酯膜通过使用压缩气体被迫。设置和挤出条件可以根据国防部改变埃尔。在使用前从制造商参考说明书。这个协议是指手动挤出机为小体积。 </li><li>通过在挤出机中通过水平衡的挤出机中的水。浸泡在水中的膜。 </li><li>放置在膜的两个活塞之间，组装设备和平衡缓冲液中，通过使缓冲器通过挤出机，从一个针筒到另一。这个步骤允许额外地测试系统的泄漏。如果泄漏，拆卸和重新组装一遍改变膜。 </li><li>取使用挤出机中的一个注射器（"脏侧"）的脂质悬浮液。 </li><li>附加一个包含在一腔室中的脂质悬浮液并在相对腔室中的空的注射器的注射器。 </li><li>推动注射器的一端。穿过膜并进入对置注射器的解决方案。这是第1次传球。 </li><li>通过膜传递，共计31次这样的该溶液最终的对置注射器（&#39;干净侧"）。 注意:检查膜挤出后。如果它被打破，则挤出过程不成功并且需要重复。 </li><li>空注射器插入小管。大单层囊泡（的LUV）是稳定的1-2天，如果保存在4℃。 </li></ol></li></ol></li><li>云母和盖玻片的制备<ol><li>洗涤先在水中，然后盖玻片乙醇。空气干燥。 </li><li>取云母光盘和使用胶带剥离外层。 </li><li>附加云母盘上盖玻片。透明的光学胶粘剂是最优这样一方面可以检查双层用荧光显微镜。 </li><li>附加使用光学胶水/润滑脂塑料圆筒（2.5厘米外径，2厘米内径和0.5厘米高）。确保一切是密封的，而且没有泄漏。希望当重新使用气缸，因为它们可容易地从第超脱使用油脂Ë盖玻片和洗涤。该塑料缸的尺寸取决于AFM悬臂支架的尺寸，应作相应的调整。 </li></ol></li><li>单层囊泡的固体沉积支持<ol><li>吸管直接整个脂质体溶液涂布的云母。添加更多的SLB缓冲器达到300微升的体积。 </li><li>加入0.9微升的1M氯化钙溶液，以达到3毫米的Ca 2+的最终浓度。 </li><li>在37℃进行2分钟，然后在65℃下至少10分钟。总是覆盖缓冲区的双层。与空气和气泡接触会破坏它。如果必要的话在孵育，添加更多的缓冲液，尤其是当孵育温度较高。 注意:孵化温度根据所需的脂质混合物和相变化。至少10分钟温育时间是必要的，以形成支承双层，但它可能是更长的时间，这取决于温度和组成。 </li><li>洗用热（65℃）该双层SLB缓冲器15-20次，以去除钙和未融合的囊泡。在洗涤步骤格外小心，不断缓冲的双层轻轻吸管的清洗液，以免破坏双层。 </li><li>凉爽支承双层至室温AFM测量之前。这是必要的，以形成膜的不同的阶段，以及在仪器减少热漂移。 </li></ol></li></ol> 2.原子力显微镜测量11,12 <ol><li>成像 注:在接触模式或轻敲模式进行原子力显微镜成像。在溶液中的图像支持双层;不要让解决方案完全蒸发，因为这会破坏双层。作为一个将与缓冲区的工作，请遵守确保任何AFM部分由液体溢出保护的安全防范措施。 <ol><li>选择一个软悬臂（低于0.2牛顿/米弹簧常数决定），并安装它到t他AFM。悬臂刚度的选择是对力谱更关键（这将在后面讨论）。 </li><li>安装在原子力显微镜阶段的样品，并确保所有隔振模块被接通。等待至少15分钟以达到平衡，并降低了系统的热漂移。根据AFM的设置，调整和成像规格可能不同。咨询制造商的手册。 </li><li>接近与针尖接触，直到样品。 </li><li>由于脂质双层通常是高度4纳米，使用Z系列的仪器，将给予最高的Z分辨率（例如1.5微米）。 </li><li>选择一个地区的形象。为了获得脂质双层的概述，一个50微米×50微米或25微米×25微米的区域将是一个很好的起点，以形象。如果更小的特征出现，在较小的领域之一即可图像（10微米×10微米，或2微米×2微米）。成像区域的选择还取决于工作ř安格压电扫描器。请参考仪器手册此。 </li><li>作为双层是很脆弱的，在成像过程中调整针尖的设定点，以保持在最低限度的力和正确热漂移，同时保持与样品接触。在接触模式下，最小化的设定点。在轻敲模式，最大限度的设定点。 </li><li>处理图像拟合每个扫描线的多项式函数练级。对于失去与使用原子力显微镜制造公司的专有软件膜接触扫描线进行清除。 </li></ol></li><li>力谱<ol><li>校准根据制造商的协议以下的说明悬臂。校准允许光电二极管到力（ 图1B）的电信号的转换。 <ol><li>校准尖的灵敏度来测量偏转z-压电运动（长度单位），而不是电信号伏特。 <b转/&gt;注:AFM检测的变化在使用激光悬臂的偏转的悬臂的背部反射。激光到达光电二极管，可以在空间上探测激光点的位置。在它弯曲时与样品接触悬臂的偏转的改变是由该光电二极管为"垂直偏转"检测的，而这是在电压的单元。因为悬臂是由z-压电移动时，垂直偏转涉及在z-压电运动。垂直偏转和z运动之间的比率被称为灵敏度和转换电压的距离。 </li><li>计算使用的热噪声频率的方法，通常在AFM软件中包含的悬臂弹簧常数。在该方法中，标绘出由噪音振动的振幅相对于振荡频率，产生一个功率谱密度图。该曲线将显示谐振频率附近具有峰值。该频率上的幅度我S中最大的是共振频率。适应周围的峰的洛伦兹函数由软件自动计算弹簧常数。对于热噪声方法的理论一些有用的资源列在参考文献27-30。 </li></ol></li><li>成像双层的面积后，在双层选择一个5微米×5微米区域来执行力谱。 </li><li>设置了原子力显微镜，以使它能力曲线在一个16×16网格区域。这将导致在每个区域256的力曲线。两个相邻点之间的间距应足够，以避免膜面积被探测由一点会与下一个点相一致。这取决于尖端半径。两点之间的100nm的距离将是理想的。除以5微米×5微米区域分为16×16格。取决于相的大小，可以选择较小的或较大的区域，然后相应地调整网格大小。 </li><li>设置z-压电位移400纳米。这就决定了z-压电的运动的最大范围。它应当足够大，以确保该悬臂完全缩回远离样品在测量之间。 </li><li>设置方法速度800海里/秒的速度缩回到200nm /秒。使用400之间的接近速度到6000纳米/秒取决于双层成分和脂质阶段进行研究6,16。 </li><li>设置所有参数后，收购力曲线，按AFM软件的"运行"按钮。 </li><li>保存力曲线为力与高度曲线（z-压电运动的度量）。这并没有考虑到在悬臂的弯曲在与样品接触时。在数据处理步骤，在AFM软件允许校正悬臂弯曲得到力与针尖样品分离曲线（ 图1C），这将给予适当的值双层厚度。修正值在CALIBR通常掺入通货膨胀，这是保存与每个力曲线。通过在给定的点减去压电运动垂直偏转计算针尖 - 样品的分离。 注意:在该方法的力曲线的峰值（该力值下降，即使悬臂运动仍然在方法循环）是膜的突破力，而该峰的位置和点的位置之间的差其中力开始上升再次提供了膜的厚度。 </li><li>获得至少500力曲线每个条件以获得良好的统计数据。积使用像母或MatLab的方案的值的直方图，以及适合直方图高斯分布，以获得分布的峰值和宽度。 </li></ol></li></ol>

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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. Chem. 278, 28109-28115 (2003).</li><li>Akbarzadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome:+classification,+preparation+and+applications.">Liposome: classification, preparation and applications.</a> Nanoscale Research Letters. 8, 102 (2013).</li><li>Butt, H. -. J., Jaschke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+thermal+noise+in+atomic+force+microscopy.">Calculation of thermal noise in atomic force microscopy.</a> Nanotechnology. 6, 1-7 (1995).</li><li>Chon, J. W. M., Mulvaney, P., Sader, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+validation+of+theoretical+models+for+the+frequency+response+of+atomic+force+microscope+cantilever+beams+immersed+in+fluids.">Experimental validation of theoretical models for the frequency response of atomic force microscope cantilever beams immersed in fluids.</a> Journal of Applied Physics. 87, 3973 (2000).</li><li>Sader, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+response+of+cantilever+beams+immersed+in+viscous+fluids+with+applications+to+the+atomic+force+microscope.">Frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope.</a> Journal of Applied Physics. 84, 64 (1998).</li><li>Sader, J. E., Pacifico, J., Green, C. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+Pore+Formation+Induced+by+Membrane+Active+Peptides.">Energetics of Pore Formation Induced by Membrane Active Peptides.</a> Biochemistry-US. 43, 3590-3599 (2004).</li><li>Henriksen, J. R., Ipsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+membrane+elasticity+by+micro-pipette+aspiration.">Measurement of membrane elasticity by micro-pipette aspiration.</a> The European physical journal. E, Soft matter. 14, 149-167 (2004).</li><li>Nichols-Smith, S., Teh, S. -. Y., Kuhl, T. L. <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+mechanical+properties+of+model+mitochondrial+membranes.">Thermodynamic and mechanical properties of model mitochondrial membranes.</a> BBA-Biomembranes. 1663, 82-88 (2004).</li><li>Tian, A., Johnson, C., Wang, W., Baumgart, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Line+Tension+at+Fluid+Membrane+Domain+Boundaries+Measured+by+Micropipette+Aspiration.">Line Tension at Fluid Membrane Domain Boundaries Measured by Micropipette Aspiration.</a> Phys. Rev. Lett. 98, (2007).</li><li>Rigaud, 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=Membrane+proteins:+functional+and+structural+studies+using+reconstituted+proteoliposomes+and+2-D+crystals.">Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals.</a> Brazilian Journal of Medical and Biological Research. 35, 753-766 (2002).</li></ol>

作者宣称，他们有没有竞争的财务权益。

的SLBs DOPC组成:缅澈（2:2:1）中诱导氯化钙囊泡吸附和破裂后形成在云母。这种脂质成分分离为L- D和LØ阶段。的1。·相位富含鞘磷脂和胆固醇，小于流体/更粘（ 图1A）比l D同时相11。 1。·了由L D相分离的表现高于周围（ 图1B，C）升高为圆形结构。该平台是由l D同时相包围1。·阶段。膜缺陷（或在膜...

原子力显微镜（AFM）由扫描用悬臂具有非常尖锐的端部1产生在样品的一个区域的表面的图像。悬臂的运动探测样品的表面拓扑结构。 AFM已经广泛应用于生物分子-包括蛋白质，DNA，和膜，由于它的多功能性在分析中液体2-5在空气中或接近天然状态固定的样品。 除了 ​​其在纳米范围内的高分辨率成像能力，原子力显微镜悬臂充当弹簧探测的相互作用力（粘附力和斥力）和样品5,6的机械性能。这就是所谓的力谱。在这种模式下，探针先靠近样品和施加在其上的力，然后缩回，直到它失去接触与样品（ 图1A）。所产生的曲线显示力作为距离悬臂的两个应用程序的函数蟑螂和回缩。几个属性，包括弹性模量测量材料的硬度和粘合力可以得到。 支持的脂质双层的生物模型膜躺在了坚实的支撑顶部-通常是云母，硼硅玻璃，熔融石英，或氧化硅7。他们使用的是一样的囊泡沉积的各种技术制备，朗缪尔-布洛杰特法和旋涂8,9。原子力显微镜成像已被用来按照下列支持双层10的形成，并探测由不同的组合物11-15的膜形成不同的结构。 执行力谱上支持双层结果的方法曲线的峰值。这表明峰值刺穿双层所需的力，被称为突破力。该双层厚度，也可以使用力曲线6测量。双层典型的突破力1-50之间范围NN 6。这些特性取决于脂质填料（液体或凝胶相）和结构（酰基链长和不饱和度），并通过膜活性剂16改变。破裂背后的理论已经解释17等实验参数，如悬臂柔软度，刀尖半径和进近速度也影响了突破力15,16,18。力光谱已被用来分析不同的脂质相11,19，组成依赖变化12,20，以及其它生物分子的效果，例如，肽的性质，在膜21的稳定性。 支持双层的平面取向是有利的原子力显微镜和其他方法，例如表面等离子共振22和荧光显微镜11,19组合以更好地表征结构和膜的特性。 这种详细的原始视频COL旨在利用囊泡沉积制备支持的脂质双层和他们AFM和力谱分析。虽然可以使用各种尺寸的囊泡以制备双层，该协议的重点是小的和大单层囊泡。支持双层相分离成液体下令（L O）和液体紊乱（L D）阶段即进行了表征11,15。 2:1的比例将膜在2由二油酰 - 磷脂酰胆碱（DOPC），鞘磷脂（SM）和胆固醇（CHOL）的。该组合物车型脂筏，这是建议的行为作为平台蛋白质运输和分拣，细胞信号和其他细胞的过程23,24重要。

<table><tbody><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-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&amp;nbsp;&amp;deg;C. Visit &lt;a href=&quot;http://www.avantilipids.com/index.php?option=com_content&amp;amp;view=article&amp;amp;id=1679&amp;amp;Itemid=398&quot;&gt;here&lt;/a&gt; for more information on storage and handling.</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&amp;nbsp;&amp;deg;C. Visit &lt;a href=&quot;http://www.avantilipids.com/index.php?option=com_content&amp;amp;view=article&amp;amp;id=1679&amp;amp;Itemid=398&quot;&gt;here&lt;/a&gt; for more information on storage and handling.</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&amp;nbsp;&amp;deg;C. Visit &lt;a href=&quot;http://www.avantilipids.com/index.php?option=com_content&amp;amp;view=article&amp;amp;id=1679&amp;amp;Itemid=398&quot;&gt;here&lt;/a&gt; for more information on storage and handling.</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 (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;), &gt;99%</td><td>AppliChem GmbH</td><td>A1046</td><td></td></tr><tr><td>Potassium dihydrogenphosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;), 99%</td><td>Carl Roth GmbH + Co. KG</td><td>3904.1</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;), 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, 24&amp;nbsp;x&amp;nbsp;60&amp;nbsp;mm, 1&amp;nbsp;mm thickness</td><td>Duran Group</td><td>2355036</td><td></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.&amp;nbsp;</td></tr><tr><td colspan="4">[header]</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 ranging 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.&amp;nbsp; Always handle mica with gloves or tweezers.</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. (&lt;a href=&quot;http://www.bandelin.com/datenblaetter/dt/DT_31_H_1798d_DE_GB_FR_BANDELIN.pdf&quot;&gt;Data sheet&lt;/a&gt;)</td></tr><tr><td>Silicon Nitride AFM Cantilever&amp;nbsp;</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&amp;nbsp;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.

DOPC构成支持的脂质双层:缅澈（2:2:1）进行成像的原子力显微镜（ 图2的AC）。由于脂质成分，SM /澈丰富1。·和DOPC丰富l D同时相观察。从原子力显​​微镜成像高度轮廓可以提供在膜结构的重要信息。通过查看高度轮廓，该双层厚度可在缺陷在膜中（ 图2B），或的Lø/ L D-相之间的高度差存在待测量可以提供。此外，AFM允许代表这些阶段具体选择区?...

Watch this Scientific Journal Video about Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers at JoVE.com

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.

原子力显微镜成像和支持的脂质双层力谱

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

atomic-force-microscopy-imaging-force-spectroscopy-supported-lipid

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

14.6K 次观看.  Interfaculty Institute for Biochemistry. We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

视频: Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

科研

生物化学

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

调查单分子粘附的原子力谱

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

工程学

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

亚纳米级分辨率成像与调幅原子力显微镜在液体

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

生物工程

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Visualizing Solution Structure at Solid-Liquid Interfaces using Three-Dimensional Fast Force Mapping

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the solution conditions such as ion concentrations, pH, ligands, and trace additives, as well as the underlying crystallography and chemistry. In this context, three-dimensional fast force mapping (3D FFM) has emerged as a promising tool for investigating solution structure at interfaces. This capability is based on atomic force microscopy (AFM) and allows the direct visualization of interfacial regions in three spatial dimensions with sub-nanometer resolution. Here we provide a detailed description of the experimental protocol for acquiring 3D FFM data. The main considerations for optimizing the operating parameters depending on the sample and application are discussed. Moreover, the basic methods for data processing and analysis are discussed, including the transformation of the measured instrument observables into tip-sample force maps that can be linked to the local solution structure. Finally, we shed light on some of the outstanding questions related to 3D FFM data interpretation and how this technique can become a central tool in the repertoire of surface science.

Here, we present a protocol for using three-dimensional fast force mapping - an atomic force microscopy technique - for visualizing solution structure at solid-liquid interfaces with the subnanometer resolution by mapping the tip-sample interactions within the interfacial region.

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the ...

化学

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.

原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

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

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

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

凸出力显微镜: 一种量化细胞突起力的方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...