The authors thank the DFG (Hu 997/2-2) for financial support. FS acknowledges the Hanns-Seidel-Stiftung (HSS). SKr was supported by the Elitenetzwerk Bayern in the framework of the doctorate program Material Science of Complex Interfaces. SKi thanks the SFB 863 for financial support.

注:参见图2的处理流程，其包括制备，数据采集和数据分析的步骤的概述。 1.试剂设置注:所有化学品必须小心处理，因而白大褂，手套和保护眼睛应该被使用。所有操作都必须在实验室罩中进行。尤其特殊的手套时，应佩戴情况下，氯仿使用。 <ol><li>使用具有低水含量的化学物质，如无水氯仿迅速和存储干燥，但不超过一个星期。同时存储的化学品在-20℃和在氮气或氩气，因为APTES（（3-氨基丙基）triethoxysilan）（ 见表1）和PEG（聚乙二醇）具有吸湿性，PEG是受氧化在空气中。 </li><li>避免了库存到大气中的氧气和水分的频繁接触，制备小等分，理想手套箱系统用氮气情调内重。 </li></ol> 2.设备安装注:为避免交叉污染，使用新鲜的，干净的船只每一步。 <ol><li>清洁玻璃器皿和在30分钟在超声波浴中，在60℃的洗涤剂溶液镊子。 </li><li>冲洗，并从步骤2.1两次用超纯水充分声处理设备。 </li><li>热设备从RCA溶液步骤2.1（超纯水，过氧化氢和氨（5:1:1）），以75℃的烘箱中45分钟，随后用超纯水冲洗。 </li><li>最后，干燥的玻璃器皿和镊子在干燥氮气流下或者在烘箱（100℃，3小时）。 </li></ol> 3.提示功能化注意:使用镊子，容器等由不锈钢，聚四氟乙烯，玻璃或它是在有机溶液中，如果适用的化学稳定的任何其它材料制成。除非另有规定，做好各项步骤为RT.所需的温育溶液的量取决于悬臂的芯片的数量。确保该悬臂浸渍在各时间的各解决方案。 注:使用遮光罩，以避免吸入有机蒸气。 <ol><li>形成于悬臂表面（&#39;激活&#39;）（约0.5小时）OH-组: <ol><li>用镊子把新鲜的悬臂式芯片（材质:单，弹簧常数:10-100 PN /纳米）在一个干净的玻片上，并把它们在等离子室（100 W）。 </li><li>抽空腔室（〜0.1毫巴）。 </li><li>洪水室氧气，并再次撤离。 </li><li>激活等离子体处理（功率:20％，持续时间:15分钟，处理压力:0.25毫巴）。 </li></ol></li><li>氨基硅烷化悬臂（约1小时）: <ol><li>制备2.5毫升APTES溶液（ 见表1）中的玻璃培养皿-在等离子体处理执行此理想。 </li><li>立即出现等离子处理后Y，浸每个悬臂1秒丙酮，并将它们随即在APTES解决方案。 </li><li>孵育15分钟，在RT。 </li><li>在10毫升丙酮，一次在10毫升氯仿中仔细冲洗悬臂芯片的两倍。 </li><li>任选最终步骤:从在干净的载玻片上一步中放置悬臂芯片和烘烤它们30分钟，在70℃。注意，也有表面活化， 例如，UV处理12的替代策略。 </li></ol></li><li> PEG化（约2小时）: 注:执行过程中的氨基硅烷化步骤3.3.1-3.3.4。 NHS和马来酰亚胺基团受到水解在含水环境和PEG本身受氧化在空气中。因此定时（特别是在步骤之间）是一个关键的参数。请参阅13获取更多信息。 <ol><li>制备的氯仿溶液（ 见表1）。 </li><li>为避免结露温暖PEG粉末达RT打开等分，体重适量之前。 </li><li>对于聚合物或脂质与氨基的偶联，分别解决的NHS-PEG-NHS（6 kDa的）和甲基-PEG-NHS（5 kDa的），在氯仿溶液通过涡旋直到它们被完全解决。请参阅表1的浓度。 <ol><li>另外。用于与硫醇基团的聚合物的偶联分别解决MAL-NHS-PEG和甲基PEG-NHS的氯仿溶液中。 </li></ol></li><li>以调节NHS-或马来酰亚胺和甲基封端的PEG分子（:500通常为1）之间有一定的数量比需要混合的解决方案。需要注意的是理想的比例要被迭代的一系列制备-实验周期来确定。 </li><li>孵育悬臂芯片在氯仿饱和气氛中的PEG溶液1小时，以防止蒸发出氯仿。 </li></ol></li><li>探针分子共轭（&gt; 1小时）: 注意:在下面的3.4.1-3.4.3，三个例子为不同的探针分子的AFM针尖的共价偶联了描述。对于每一个分子的协议必须被调整。进一步注意到，与实施例1和3的NHS-化学中使用，而实施例2中的巯基官能化的聚合物PI- 克 -PS经由马来酰亚胺化学偶联于PEG。有关详情请参阅第14。 <ol><li>对聚（氨基酸）聚-D-酪氨酸（40-100 kDa）的<ol><li>溶解在1M的NaOH将聚酪氨酸缀合探针分子。调至浓度为1毫克/毫升。 </li><li>使用旋转脱盐立即柱（7 kDa的MWCO）的功能化之前交换的NaOH溶液硼酸钠缓冲液（pH 8.1） </li><li>用5毫升的氯仿中，然后用5毫升乙醇和最后用5毫升硼酸盐缓冲液的第一清洗悬臂。 </li><li>孵育悬臂芯片1小时的聚酪氨酸硼酸盐缓冲溶液，然后用清水冲洗硼酸盐缓冲剂和超纯水。储存在超纯水中，直到测量。 </li></ol></li><li>对于线性骨干（聚异戊二烯，119 kDa的），具有嫁接侧链（聚苯乙烯，88 kDa的）14聚合物<ol><li>溶解PI- 克 -PS无水氯仿。调节偶联溶液至4毫克/毫升的浓度。 </li><li>冲洗用5毫升氯仿悬臂孵育至少1小时，在缀合溶液。 </li><li>在5毫升氯仿中再次冲洗并在氯仿存储在氯仿 - 饱和的环境（防止氯仿蒸发），直到测量。 </li></ol></li><li>对于脂质教皇<ol><li>在干燥的氯仿溶解教皇。浓度调整至20mM。 </li><li>冲洗用5ml乙醇和5ml超纯水的悬臂1小时，在脂质溶液的温育前的悬臂。 </li><li>温育后，冲洗悬臂用5毫升氯仿，加入5ml ethano的L和5毫升的温，超纯水（按顺序），以除去未结合的探针分子，并摆脱三氯甲烷残留物。立即使用功能化的提示。可替代地，直到需要将它们存储在氯仿中。 </li></ol></li></ol></li></ol><img alt="图1" src="/files/ftp_upload/52456/52456fig1.jpg" /> 图1（A）的示意图表示使用NHS化学的例子的尖端官能化方法。采用通过氨基连接探针分子给小费（B）化学键。 <a href="https://www.jove.com/files/ftp_upload/52456/52456fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.表面处理<ol><li>自组装单层（SAM）的 注意:作为表面的第一个例子，一个自组装单层被选择。请参考文献15 </s达&gt;详细信息。 <ol><li>干净的载玻片用洗涤剂溶液，然后两次在超纯水中在超声浴中对每个30分钟。然后，作为附加的清洗步骤，将它们放置在RCA溶液（参见步骤2.3），在75℃下进行15分钟。 </li><li>外套与10纳米的铬镍，并在真空涂布机100纳米金的幻灯片。然后前直接进行下一步再存放在冰箱和清洁，在RCA溶液。 </li><li>孵育前一步骤的幻灯片在12小时为2mM 1-十二/乙醇溶液。该硫醇基团结合到金表面和疏水单层自组装。用氮气流冲洗，用乙醇和超纯水中，然后干燥的幻灯片。确认制得的表面的静态接触角测量的疏水性在用CCD照相机10一个测角器。 </li></ol></li><li>氢终止金刚石 注意:作为该第二个例子的表面上，一氢终止金刚石被选择。如先前16所述进行表面处理。 </li><li>支持的脂质双层 注意:在最后的例子中，表面支持的脂质双层（SLB）用作表面。为了获得这样的表面上，大的单层囊泡（的LUV）选自磷脂P​​OPC的溶液（0.1毫克/毫升）通过挤出法形成。然后将50μl的LUV溶液被放在一个新鲜剥离云母片（A = 1平方厘米），并孵育30分钟。最后该溶液漂洗用20ml超纯水。对于SLBs的编制的详细说明，请参阅17,18。 </li></ol> 5.数据采集注:对于实验中，使用AFM，它提供的液体测量能力。数据采集​​和数据分析方法都适用，无论使用AFM模式。此外，在一些实验中，有利的是要控制。温度的可能性液晶单元内即通过将激光束偏转方法19检测悬臂偏转。弹簧常数分别与热噪声的方法20。 <ol><li>将官能化的悬臂成的AFM悬臂支架，适用于测量流体。 </li><li>放表面（制备见上文）被采样到AFM和用液体覆盖。两者，悬臂和表面现在应浸渍在与悬臂尖端指向表面的液体。注意，在许多情况下，一滴液体就足够了。在流体中的任何情况下，蒸发应该避免， 例如，通过使用封闭的流体单元。 </li><li>如果适用的话，调整控制器上所需的温度。 </li><li>让系统平衡至少一个半小时。 </li><li>为了排除任何表面阻尼效应记录与所述悬臂的悬臂的热噪声频谱远离表面。需要注意的是，通常10以上的光谱必须累积，以获得足够的信噪比。 </li><li>接近表面。需要注意的是，为了节省末梢几何结构和小费官能的方法处理，必须仔细地进行。这可以通过以下方式实现，例如，在接近断续接触模式。 </li><li>确定纳米/伏测量逆光杠杆灵敏度（InvOLS）。通过推动悬臂刀片针对硬表面做到这一点。 <ol><li>通过测量压电行驶距离与在光电二极管电压的变化的斜率确定InvOLS。拟合线的力 - 距离曲线，其中所述悬臂刀片是在与表面接触的部分。在涉及柔软的表面像脂质双层实验，开展对未涵盖与脂质双层（&#39;缺陷点&#39;）的区域这一步。 </li></ol></li><li>通过安装一个谐振子的热噪声谱确定弹簧常数（PN /纳米）。使用自动化的软件功能弹簧常数测定;否则，查阅文献20。 </li><li>开始实验。 <ol><li>记录无数力 - 距离曲线在每个实验条件。通常情况下，用于该实验的参数以下值:5千赫，1微米/秒的尖端速度的采样率，回缩距离为1微米。 注:有时，根据所研究的实验的动力学，可能有必要等待一定量的时间，在与表面接触的前端（&#39;停留时间&#39;）。 注:本实验的具体参数可从以上给出的数值基本上偏离。 </li><li>在长期的测量，通过重新定位所述光电二极管零上的光电二极管上的激光位置不时。 </li><li>可选:每个力曲线后，重新定位AFM尖到另一个位置坐标。 </li><li>在实验结束时，确定灵敏度和弹簧CONSTA再次新台币检查系统的一致性和稳定性。 注:前和实验后的弹簧常数和灵敏度比较也是一种手段，以确保该系统的性能和尖端的特性保持不变在实验的过程中。如果弹簧常数之差不是太大（&lt;= 15％），它们可以被平均化，由于弹簧常数确定的固有的不确定性也是15％左右。对于较大的差别，最好是先找到并与进一步的实验，然后再继续消除病因的改变表观弹簧常数。 </li></ol></li></ol> 6.数据准备注:在其通常进行独立实验的具体类型的转换单位牛顿纳米以及以校正数据中描述了本节的一般数据准备步骤。实验的具体数据分析是BRiefly下面进一步描述，在各自的有代表性的例子一节。 <ol><li>通常情况下，进行所有数据准备和分析由家用写算法， 例如自动步骤，基于所述软件IGOR Pro 6的。 </li><li>原始偏转信号（DEFL）[V]的转换成力通过与InvOLS [纳米/ V]和弹簧常数[NN / nm]的相乘。 </li><li>抵消在这样一种方式，在从表面（空载悬臂）在足够的距离的悬臂上的力变为0 NN的力。 </li><li>计算实际尖端位置z *，这需要在悬臂的弯曲计（测量时相对于表面也称为分离）: <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/52456/52456eq1.jpg" /></li><li>偏移Z *在于Z * = 0对应于表面的z轴位置的方式。 注意:在这里，F是作 ​​用在前端，因此弯曲的力S中的悬臂（在步骤6.2确定的。）中，z是测量的z传感器位置（由仪器直接给出）和k表示弹簧常数（在步骤5.8确定）。 </li></ol><img alt="图2" src="/files/ftp_upload/52456/52456fig2.jpg" /> 展示样品制备，数据采集和数据分析图2.工艺流程图。 <a href="https://www.jove.com/files/ftp_upload/52456/52456fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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S., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nanomechanical+Properties+of+Lipid+Membranes+are+Significantly+Influenced+by+the+Presence+of+Ethanol.">The Nanomechanical Properties of Lipid Membranes are Significantly Influenced by the Presence of Ethanol.</a> Biophysical Journal. 104 (5), 1049-1055 (2013).</li><li>Putman, C. A. J., De Grooth, B. G., Hulst, N. F., Greve, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+analysis+of+the+optical+beam+deflection+technique+for+use+in+atomic+force+microscopy.">A detailed analysis of the optical beam deflection technique for use in atomic force microscopy.</a> Journal of Applied Physics. 72 (1), 6-12 (1992).</li><li>Hutter, J. L., Bechhoefer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+atomic-force+microscope+tips.">Calibration of atomic-force microscope tips.</a> Review of Scientific Instruments. 64 (7), 1868 (1993).</li><li>Krysiak, S., Liese, S., Netz, R. R., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+desorption+kinetics+from+single+molecule+force+spectroscopy+studies.">Peptide desorption kinetics from single molecule force spectroscopy studies.</a> Journal of the American Chemical Society. 136 (2), 688-697 (2014).</li><li>Li, I. T. S., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+free+energy+governs+single+polystyrene+chain+collapse+in+water+and+aqueous+solutions.">Interfacial free energy governs single polystyrene chain collapse in water and aqueous solutions.</a> Journal of the American Chemical Society. 132 (18), 6530-6540 (2010).</li><li>Dudko, O. K., Hummer, G., Szabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory,+analysis,+and+interpretation+of+single-molecule+force+spectroscopy+experiments.">Theory, analysis, and interpretation of single-molecule force spectroscopy experiments.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15755-15760 (2008).</li><li>Stetter, F. W. S., Cwiklik, L., Jungwirth, P., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lipid+Extraction:+The+Anchoring+Strength+of+Cholesterol+in+Liquid-Ordered+and+Liquid-Disordered+Phases.">Single Lipid Extraction: The Anchoring Strength of Cholesterol in Liquid-Ordered and Liquid-Disordered Phases.</a> Biophysical Journal. 107 (5), 1167-1175 (2014).</li><li>Horinek, D., 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=Peptide+adsorption+on+a+hydrophobic+surface+results+from+an+interplay+of+solvation,+surface,+and+intrapeptide+forces.">Peptide adsorption on a hydrophobic surface results from an interplay of solvation, surface, and intrapeptide forces.</a> Proc Natl Acad Sci U S A. 105 (8), 2842-2847 (2008).</li><li>Ebner, 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=Functionalization+of+probe+tips+and+supports+for+single-molecule+recognition+force+microscopy.">Functionalization of probe tips and supports for single-molecule recognition force microscopy.</a> Topics in current chemistry. 285 (April), 29-76 (2008).</li><li>Geisler, M., Balzer, B. N., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Adhesion+at+the+Solid-Liquid+Interface+Probed+by+a+Single-Molecule+Force+Sensor.">Polymer Adhesion at the Solid-Liquid Interface Probed by a Single-Molecule Force Sensor.</a> Small. 5 (24), 2864-2869 (2009).</li><li>Morfill, J., 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=Affinity-matured+recombinant+antibody+fragments+analyzed+by+single-molecule+force+spectroscopy.">Affinity-matured recombinant antibody fragments analyzed by single-molecule force spectroscopy.</a> Biophysical journal. 93 (10), 3583-3590 (2007).</li></ol>

The authors declare that they have no competing financial interests.

在过去的几十年中，单分子实验提供了前所未有的见解的分子机制，竟然是在生命科学及以后的宝贵途径。以实现从SMFS实验，理想地1，并在同一分子用在实验的全过程良好的和有意义的统计数据。与此相反，以与分子的合奏实验，SMFS实验能够检测罕见事件和隐藏的分子状态。的单分子实验的另一个优点是，它们可以通过分子动力学模拟24,25手段进行建模。 上述悬臂?...

在过去的30年中，原子力显微镜（AFM）已被证明是一个有价值的成像技术来研究生物学1,2和3的合成材料和表面，因为它提供了分子的空间分辨率在三个维度，可以在不同的溶剂进行操作环境。此外，AFM单分子力谱（SMFS）使测量力范围从PN为μN政权，并给予前所未有的洞察到例如蛋白质折叠4,5，高分子物理6 - 8，和单分子表面相互作用9 -学习背后的单个分子，而不是分子的集合体12 .The理由是为了避免影响平均，往往掩盖了罕见的事件或隐藏的分子状态。此外，众多的分子参数，如轮廓长度，库恩长度，粘合自由能等可以获得。此详述在下面的实施例。在一个典型的AFM-SMFS实验，探针分子通过接头分子偶联到一个非常尖锐的尖端。尖端本身位于一可弯曲的悬臂的端部。如果针尖被带入与该表面接触的探针分子将与该表面相互作用。通过在尖端，力，因此自由能缩回观察悬臂的偏转，以从表面分离的分子可以被确定。以获得有意义的统计数据，大量的所谓的力 - 距离曲线已被收购。此外，能有真正的单分子实验（ 即 ，使用一个和在整个实验期间相同的探针分子）探针分子应当共价偶联到所述针尖。这里，一个实验协议悬臂官能经由共价键的单个分子呈现。单分子既可以通过氨基或硫基耦L组的针尖。缀合方法可以在广泛的各种溶剂（有机相和水）占所用的聚合物的溶剂化性质来进行。 在第一部分中，一个通用的方案来共价通过接头分子与AFM针尖描述附加一个单一的分子（"探针分子"）。为此目的，有机NHS-或马来酰亚胺化学过程中使用13。随着协议3例如分子，数据采集和数据分析的方法描述和提供了用于进一步阅读参考。的例子的分子是:（线性）聚合物酪氨酸，接枝聚合物PI- 克 -PS和脂质POPE。这包括协议的微小变化，例如共价连接半胱氨酸。另外，一个段是专用不同的表面的制备诸如金刚石表面，CH 3 -self组装单层和脂质双层。这些接口都省恩是很好的参考和实例。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III alkaline liquid concentrate (detergent solution)</td>
			<td>Hellma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RCA (ultrapure water, hydrogen peroxide (35 %), ammonia (32%); 5:1:1(v/v/v))</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vectabond reagent / APTES (3-Aminopropyl)triethoxysilane</td>
			<td>Vectorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dry acetone (&#60; 50 ppm H2O)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dry chloroform (&#62; 99.9 %)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Biochrom, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Di-sodium tetraborate (&#62; 99.5 %)</td>
			<td>Biochrom, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Biochrom, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monofunctional &#945;-methoxy-&#969;-NHS PEG, 5kDa, &#8220;methyl-PEG-NHS&#8221;</td>
			<td>Rapp, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heterobifunctional &#945;,&#969;-bis-NHS PEG, 6 kDa, &#8220;NHS-PEG-NHS&#8221;</td>
			<td>Rapp, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heterobifunctional &#945;-maleimidohexanoic- &#969;-NHS PEG, 5 kDa, &#8220;Mal-PEG-NHS&#8221;</td>
			<td>Rapp, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Probe molecule (polymer, lipid, etc.)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sufficient amount of glass crystallising dishes with spout (10 ml), glass petri dishes (500 &#181;l) and glass lids</td>
			<td>VWR International GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory oven model UF30</td>
			<td>Memmert, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controlled sonicator</td>
			<td>VWR International GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma system &#34;Femto&#34;, 100 W</td>
			<td>Diener, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>One separate glass syringe for each organic solvent</td>
			<td>VWR International GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>VWR International GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (0.5 ml or 1.5 ml)</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes: 10-100 &#181;l, 50-200 &#181;l and 100-1000 &#181;l</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AFM with temperature controlled fluid cell (e.g. MFP-3D with BioHeater)</td>
			<td>Asylulm Research, Santa Barbara</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soft SiN cantilevers cantilever, typically made from silicon nitride (SiN) (spring constant less than 100 pN/nm, e.g. MLCT)</td>
			<td>Bruker AXS, Santa Barbara</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating single molecule adhesion by atomic force spectroscopy

原子力谱是研究分子在表面和界面的​​理想工具。一个试验性协议，以夫妻种类繁多的单分子的共价键上的原子力显微镜针尖呈现。同时针尖被钝化，以防止尖端和基板，这是一个先决条件，研究附着在针尖单个分子之间的非特异性相互作用。分析，以确定的粘合力，粘合长度，并在固体表面上和生物界面这些分子不久提出并提供用于进一步阅读外部引用的自由能。实施例的分子是聚（氨基酸）聚酪氨酸，接枝聚合物PI- 克 -PS和磷脂POPE（1-棕榈酰-2-油酰- SN -glycero -3-磷酸）。这些分子是从不同的表面，如CH 3 -SAMs，氢封端的金刚石解吸并支持的各种溶剂的条件下的脂质双层。最后，该力分光单分子实验的优点进行了讨论，包括装置，以决定是否确实是一个单一分子已经研究了实验。

在下文中，其结果为上述实施例的分子，即该聚合物的聚（氨基酸）聚酪氨酸，接枝聚合物PI- 克 -PS和磷脂POPE，现介绍。首先对各实施例，实验的具体细节的数据采集和数据准备设置。然后，示例性结果实验，其中这些分子是从不同的表面上解吸（CH 3 -SAMs，氢封端的金刚石和脂质双层）被示出。的粘附力，粘合长度和自由能的测定进行了介绍。 实施例1:...

Watch this Scientific Journal Video about Investigating Single Molecule Adhesion by Atomic Force Spectroscopy at JoVE.com

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

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 ...

investigating-single-molecule-adhesion-by-atomic-force-spectroscopy

Research

JoVE Journal

Engineering

10.3K 次观看.  Technische Universität München. 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.

视频: Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

紧凑型量子点的单分子成像

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

科研

工程学

Scanning-probe Single-electron Capacitance Spectroscopy

The integration of low-temperature scanning-probe techniques and single-electron capacitance spectroscopy represents a powerful tool to study the electronic quantum structure of small systems - including individual atomic dopants in semiconductors. Here we present a capacitance-based method, known as Subsurface Charge Accumulation (SCA) imaging, which is capable of resolving single-electron charging while achieving sufficient spatial resolution to image individual atomic dopants. The use of a capacitance technique enables observation of subsurface features, such as dopants buried many nanometers beneath the surface of a semiconductor material1,2,3. In principle, this technique can be applied to any system to resolve electron motion below an insulating surface.
As in other electric-field-sensitive scanned-probe techniques4, the lateral spatial resolution of the measurement depends in part on the radius of curvature of the probe tip. Using tips with a small radius of curvature can enable spatial resolution of a few tens of nanometers. This fine spatial resolution allows investigations of small numbers (down to one) of subsurface dopants1,2. The charge resolution depends greatly on the sensitivity of the charge detection circuitry; using high electron mobility transistors (HEMT) in such circuits at cryogenic temperatures enables a sensitivity of approximately 0.01 electrons/Hz&frac12; at 0.3 K 5.

Scanning-probe single-electron capacitance spectroscopy facilitates the study of single-electron motion in localized subsurface regions. A sensitive charge-detection circuit is incorporated into a cryogenic scanning probe microscope to investigate small systems of dopant atoms beneath the surface of semiconductor samples.

扫描探针单电子电容谱

The integration of low-temperature scanning-probe techniques and single-electron capacitance spectroscopy represents a powerful tool to study the ...

Quantum State Engineering of Light with Continuous-wave Optical Parametric Oscillators

Engineering non-classical states of the electromagnetic field is a central quest for quantum optics1,2. Beyond their fundamental significance, such states are indeed the resources for implementing various protocols, ranging from enhanced metrology to quantum communication and computing. A variety of devices can be used to generate non-classical states, such as single emitters, light-matter interfaces or non-linear systems3. We focus here on the use of a continuous-wave optical parametric oscillator3,4. This system is based on a non-linear &#967;2 crystal inserted inside an optical cavity and it is now well-known as a very efficient source of non-classical light, such as single-mode or two-mode squeezed vacuum depending on the crystal phase matching. 
Squeezed vacuum is a Gaussian state as its quadrature distributions follow a Gaussian statistics. However, it has been shown that number of protocols require non-Gaussian states5. Generating directly such states is a difficult task and would require strong &#967;3 non-linearities. Another procedure, probabilistic but heralded, consists in using a measurement-induced non-linearity via a conditional preparation technique operated on Gaussian states. Here, we detail this generation protocol for two non-Gaussian states, the single-photon state and a superposition of coherent states, using two differently phase-matched parametric oscillators as primary resources. This technique enables achievement of a high fidelity with the targeted state and generation of the state in a well-controlled spatiotemporal mode.

We describe the reliable generation of non-Gaussian states of traveling optical fields, including single-photon states and coherent state superpositions, using a conditional preparation method operated on the non-classical light emitted by optical parametric oscillators. Type-I and type-II phase-matched oscillators are considered and common procedures, such as the required frequency filtering or the high-efficiency quantum state characterization by homodyning, are detailed.

光与连续波光学参量振荡器的量子态工程

Engineering non-classical states of the electromagnetic field is a central quest for quantum optics1,2. Beyond their fundamental significance, such states ...

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, acceleration and forces involved in the locomotory behavior of nematodes suspended in a column of water in response to single wavelengths of light. We demonstrate how to evaluate the locomotion of a microscopic organism using Single Wavelength Shadow Imaging (SWSI) using two different examples.
The first example is a systematic and statistically viable study of the average descent of C. elegans in a column of water. For this study, we used living and dead wildtype C. elegans. When we compared the velocity and direction of nematode active movement with the passive descent of dead worms within the gravitational field, this study showed no difference in descent-times. The average descent was 1.5 mm/sec &#177; 0.1 mm/sec for both the live and dead worms using 633 nm coherent light.
The second example is a case study of select individual C. elegans changing direction during the descent in a vertical water column. Acceleration and force are analyzed in this example. This case study demonstrates the scope of other physical properties that can be evaluated using SWSI while evaluating the behavior using single wavelengths in an environment that is not accessible with traditional microscopes. Using this analysis we estimated an individual nematode is capable of thrusting with a force in excess of 28 nN.
Our findings indicate that living nematodes exert 28 nN when turning, or moving against the gravitational field. The findings further suggest that nematodes passively descend in a column of water, but can actively resist the force of gravity primarily by turning direction.

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

The technique presented here measures the path of freely swimming microscopic species using single wavelength exposure. C. elegans are used to demonstrate shadow imaging as an inexpensive alternative to costly microscopes. This technique can be adapted to accommodate various orientations, environments and species to measure direction, speed, acceleration and forces.

单波长阴影成像<em&gt;秀丽隐杆线虫</em&gt;步态包括武力在内的估算

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, ...

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

Making Record-efficiency SnS Solar Cells by Thermal Evaporation and Atomic Layer Deposition

Tin sulfide (SnS) is a candidate absorber material for Earth-abundant, non-toxic solar cells. SnS offers easy phase control and rapid growth by congruent thermal evaporation, and it absorbs visible light strongly. However, for a long time the record power conversion efficiency of SnS solar cells remained below 2%. Recently we demonstrated new certified record efficiencies of 4.36% using SnS deposited by atomic layer deposition, and 3.88% using thermal evaporation. Here the fabrication procedure for these record solar cells is described, and the statistical distribution of the fabrication process is reported. The standard deviation of efficiency measured on a single substrate is typically over 0.5%. All steps including substrate selection and cleaning, Mo sputtering for the rear contact (cathode), SnS deposition, annealing, surface passivation, Zn(O,S) buffer layer selection and deposition, transparent conductor (anode) deposition, and metallization are described. On each substrate we fabricate 11 individual devices, each with active area 0.25 cm2. Further, a system for high throughput measurements of current-voltage curves under simulated solar light, and external quantum efficiency measurement with variable light bias is described. With this system we are able to measure full data sets on all 11 devices in an automated manner and in minimal time. These results illustrate the value of studying large sample sets, rather than focusing narrowly on the highest performing devices. Large data sets help us to distinguish and remedy individual loss mechanisms affecting our devices.

Tin sulfide (SnS) is a candidate material for Earth-abundant, non-toxic solar cells. Here, we demonstrate the fabrication procedure of the SnS solar cells employing atomic layer deposition, which yields 4.36% certified power conversion efficiency, and thermal evaporation which yields 3.88%.

通过热蒸发和原子层沉积使记录效率的SnS太阳能电池

Tin sulfide (SnS) is a candidate absorber material for Earth-abundant, non-toxic solar cells. SnS offers easy phase control and rapid growth by congruent ...

Free-form Light Actuators &#8212; Fabrication and Control of Actuation in Microscopic Scale

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted researchers&#39; attention in many fields. Most of the studies focused on macroscopic LCE structures (films, fibers) and their miniaturization is still in its infancy. Recently developed lithography techniques, e.g., mask exposure and replica molding, only allow for creating 2D structures on LCE thin films. Direct laser writing (DLW) opens access to truly 3D fabrication in the microscopic scale. However, controlling the actuation topology and dynamics at the same length scale remains a challenge.
In this paper we report on a method to control the liquid crystal (LC) molecular alignment in the LCE microstructures of arbitrary three-dimensional shape. This was made possible by a combination of direct laser writing for both the LCE structures as well as for micrograting patterns inducing local LC alignment. Several types of grating patterns were used to introduce different LC alignments, which can be subsequently patterned into the LCE structures. This protocol allows one to obtain LCE microstructures with engineered alignments able to perform multiple opto-mechanical actuation, thus being capable of multiple functionalities. Applications can be foreseen in the fields of tunable photonics, micro-robotics, lab-on-chip technology and others.

Here, we fabricate 3D polymeric micro/nano structures in which both the shape and the molecular alignment can be engineered with nanometer scale accuracy by the use of direct laser writing. Light induced deformation of several types of liquid crystalline elastomer microstructures can be controlled in the microscopic scale.

自由形式的光驱动器 - 制作和开动控制在微观尺度

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted ...

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 ...

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves several steps, include drawing samples, cooling, cutting, transport to the laboratory, and analysis. The whole analysis process requires more than 30 minutes, which hinders on-line process control. Laser-induced breakdown spectroscopy is an excellent on-line analysis method that can satisfy the requirements of vacuum induction melting because it is fast and noncontact and does not require sample preparation. The experimental facility uses a lamp-pumped Q-switched laser to ablate melted liquid steel with an output energy of 80 mJ, a frequency of 5 Hz, a FWHM pulse width of 20 ns, and a working wavelength of 1,064 nm. A multi-channel linear charge coupled device (CCD) spectrometer is used to measure the emission spectrum in real time, with a spectral range from 190 to 600 nm and a resolution of 0.06 nm at a wavelength of 200 nm. The protocol includes several steps: standard alloy sample preparation and an ingredient test, smelting of standard samples and determination of the laser breakdown spectrum, and construction of the elements concentration quantitative analysis curve of each element. To realize the concentration analysis of unknown samples, the spectrum of a sample also needs to be measured and disposed with the same process. The composition of all main elements in the melted alloy can be quantitatively analyzed with an internal standard method. The calibration curve shows that the limit of detection of most metal elements ranges from 20-250 ppm. The concentration of elements, such as Ti, Mo, Nb, V, and Cu, can be lower than 100 ppm, and the concentrations of Cr, Al, Co, Fe, Mn, C, and Si range from 100-200 ppm. The R2 of some calibration curves can exceed 0.94.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

激光击穿光谱对真空感应熔融的定量分析

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves ...

An Efficient Method for Selective Desalination of Radioactive Iodine Anions by Using Gold Nanoparticles-Embedded Membrane Filter

Here, we demonstrate a detail protocol for the preparation of nanomaterials-embedded composite membranes and its application to the efficient and ion-selective removal of radioactive iodines. By using citrate-stabilized gold nanoparticles (mean diameter: 13 nm) and cellulose acetate membranes, gold nanoparticle-embedded cellulose acetate membranes (Au-CAM) have easily been fabricated. The nano-adsorbents on Au-CAM were highly stable in the presence of high concentration of inorganic salts and organic molecules. The iodide ions in aqueous solutions could rapidly be captured by this engineered membrane. Through a filtration process using an Au-CAM containing filter unit, excellent removal efficiency (&gt;99%) as well as ion-selective desalination result was achieved in a short time. Moreover, Au-CAM provided good reusability without significant decrease of its performances. These results suggested that the present technology using the engineered hybrid membrane will be a promising process for the large-scale decontamination of radioactive iodine from liquid wastes.

An efficient method for the rapid and ion-selective desalination of radioactive iodine in several aqueous solutions is described by using gold nanoparticles-immobilized cellulose acetate membrane filters.

调查单分子粘附的原子力谱

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