美国国家科学基金会，MRI检查程序（CHE：1229103）被确认为购买新的电子控制装置，软件，液体电池，并装配了双AFM / STM所需的其他设备提供资金。我们承认在芝加哥NSF-MRSEC计划大学（DMR-0820054）与原子力显微镜仪器，培训和成像援助共用设施和仪器时提供的材料研究设施网络（DMR-0820054）。我们特别感谢泣涕郭医生，贾斯汀Jureller博士和嘉怡Lee教授为美国国家科学基金会，核磁共振建议，带来了必要的仪器，我们的校园资助之前，欢迎我们的学生。 （：P031C110157号）颁发给东北伊利诺伊大学所提供的暑期研究奖学金的学生和教师，以及支持消耗品我们承认，从第三章干补助资金。最后，我们感谢布鲁克纳公司的持续性支持和许可于reproduce剧情呈现PeakForce QNM的机制，用这个词ScanAsyst来形容自动化软件。

1，电脑和显微镜设置<ol><li>打开氮气气缸的阀和调节旋钮，以确保空气表是浮动电平。 </li><li>打开计算机，控制器和光纤在该顺序的光。 </li><li>将扫描仪的AFM / LFM模式和中心相机在原子力显微镜头。 </li><li>打开软件。选择实验类“纳米力学性能”，“定量纳米机械制图”下的实验组，与“PeakForce QNM流体力学”下的实验。点击载试验，然后安装。 </li><li>使用Z客观看舞台的表面对焦。使用粗上/下压电体移动大约1mm的原子力显微镜头孔的表面之下的阶段。 </li></ol> 2，准备云母表面的<ol><li>按粘合剂贴到一个干净的样品支撑盘（直径15毫米，厚0.8毫米）的脸。推云母V-4级盘（25×25×26毫米）上的粘合剂，直到牢固地附着。 </li><li>擦了一块透明胶带的到云母确保其完全遵守，没有气泡。从一个侧面拉出带了云母均匀地切割它。如果有必要，以确保该云母是平坦重复。 </li><li>抓地力的支持下盘，清洁云母面朝上，有一对圆形的镊子（磁盘夹子），并放置到扫描仪。云母应低于AFM头孔的水平。 </li></ol> 3，流体细胞大会和云母表面成像<ol><li>冲洗液细胞，O形环，注射器接头，软管接头，并在100％乙醇中的喷射软管。在冲洗去离子水的液体电池。让所有部件的空气完全干燥。 </li><li>将软管适配器插入干净的流体单元的端口。轻轻按下O型圈进用平头镊子流体单元的中心压痕。 </li><li>按春风等闲探头固定夹，并把它远离groovE对于小费。 </li><li>用平头镊子抓住探头上的长端离一角。把探头放入朝向小区的中心的末端的槽。推动在春季和调整固定夹在探头固定（ 图1B）。 </li><li>签上的原子力显微镜的夹紧，以确保它完全缩回（ 图1C）。 </li><li>将流体细胞，O型​​环和探针朝下，在云母样品的顶部。允许流体细胞休息到原子力显微镜的螺栓。放下夹具，以稳定在舞台上的流体单元。按下显微镜下开关，直到视力检查显示O型圈支撑盘和流体细胞之间紧紧压缩。 </li><li>一边看着监视器，聚焦粗调旋钮来可视化屏幕上的流体池的顶部。 </li><li>移动显微镜，直到有尖端的使用X / Y轴调节旋钮以及Z客观清晰的图像。该提示将APP耳朵像金属，金三角。移动的前端更接近使用在显微镜的下压电杆的样品表面。专注于尖端的反映。保持关系，它的反射轨迹的实际悬臂。在此阶段，应紧密配合，但不完全重叠。 </li><li>附上注射器接头到无菌1ml注射器。附加的喷射软管的注射器适配器的一端，并放置在缓冲溶液的容器，软管的另一端。充分延伸注射器的柱塞。 </li><li>连接软管上的流体单元的软管接头。轻轻按压活塞，直到液体完全充满中心室。检查细胞以确保没有气泡。检查，看看有没有流体从流体细胞溅出到显微镜。放置在一个凸起的固相支持物的注射器。 </li><li>使用相机饲料和X / Y方向平移旋钮来定位屏幕上的激光（漫红点）。使用激光MOVEM耳鼻喉科控制旋钮，将激光上一角。 </li><li>精细地调整激光及反射镜控制，直到在显微镜下显示的和信号被最大化（值= 4 - 6）。 </li><li>调整用的左后侧上的旋钮和扫描头的左上侧，以降低在显微镜接近零越好（±0.1）所显示的垂直和水平偏转的象限二极管的角度。 </li><li>在软件中，选择收购方案。至少，选择身高（跟踪和回扫），峰值​​力误差和变形。其他选项是依赖于用户的，并且可以根据期望的实验者的特定信息进行定制。 </li><li>选择“行”的RT飞机适合在每个采集窗口。选择“无”旧约飞机适合在高窗。设置的X和Y偏移量和扫描角为零时，如果尚未完成。 </li><li>设置扫描速率为1赫兹，每行的样品256中，ScanAsyst自动控制个别和ScanAsyst Auto Z技术限制为关闭。改变Z轴限制到500纳米。设定扫描尺寸为1微米。 </li><li>点击参与。在这一点上，末端接近的表面上。而尖端临近，观察屏幕的左下角Ž压电电压变化。如果针尖进入的范围之外，撤回尖和重新的做法。如果这是不成功的，尖端可能需要为了有一个成功的方法要改变。一旦该尖端部接合，该线将开始出现在每一个采集窗口，这将构成一个图像。 </li><li>取云母的图像以1×1微米和放大慢慢以确保该云母表面是干净的和平坦的。点击连续捕捉图像保存，因为它们出现。放大和周围的表面根据需要移动。 </li></ol> 4，蛋白沉积和样本进行成像的制备<ol><li>得到纯化的蛋白样品38并保持在冰上。如果存储在冷冻箱中，在解冻的样品冰浴5分钟。 </li><li>稀释蛋白质到0.0010毫克/毫升用冷藏成像缓冲液。这里所使用的成像缓冲器是15毫摩尔Tris-盐酸（pH值= 8.0），150mM的NaCl和15mM的MgCl 2的。 </li><li>补4室（100×15毫米）培养皿冷藏影像缓冲溶液。填充每个腔室具​​有至少5毫升溶液。轻轻混匀与连接到微量移液器尖端稀释的蛋白溶液。 </li><li>申请加入200μl稀释的蛋白溶液，以云母的已制备的表面。 30秒后，拿起蛋白/云母表面与圆形镊子（磁盘夹持器）。保持平行实验台上的样品。 </li><li>同时保持样品，立即浸入冷水中含有的培养皿中（ 步骤4.2）的第一象限中的缓冲溶液中的样品，同时确保该样品是平行于培养皿的盆地。经过1秒，取出样品。 </li><li>继续保持平行的试样的盆在培养皿中，重复步骤4.5其余三个象限。 </li><li>放置在干燥的无尘布的盘，以吸收多余的水分和盘转移到显微镜的阶段。请勿触摸实际表面用布。不使样品干燥。 </li><li>重复步骤3.6 - 3.16。 </li></ol>在云母5成像蛋白质<ol><li>点击检查参数框在屏幕的左侧。弹簧大小和针尖半径应根据所使用的尖端被设置。 </li><li>点击参与。类似于云母成像过程（ 步骤3.17），观察到的z压电窗口的前端的方法。 </li><li>允许扫描仪图像的相同区域的至少两个连续的通行证。点击连续拍摄，保存收集的每个图像。 </li><li>将扫描仪到一个新的区域和/或根据需要改变图像的大小。 </li><li>之后，自动化软件已经完成调整扫描参数为特定的图像大小，把软件关闭。 </li><li>为了手动调节扫描速率和z限制，行数，和电压微调的图像的焦点。如果图像变得太远失焦，打开软件，重新回到原来的起始位置。以增加，而对蛋白质放大的图像的分辨率，降低扫描速率和增加的行数成比例。 </li><li>在实验结束后，清洗液细胞用100％乙醇和冲洗用DI H 2 O。使细胞液充分干燥。 </li><li>打开软件的数据处理原始数据图像。首先，通过点击平铺图标拼合图像。选择零阶 ，然后点击执行。根据图像的不同，平面拟合可能是必要的，以获得适当的平面图像。在这种情况下，选择合适的飞机图标和借鉴使用光标图像的平坦区域的平面。点击执行。根据需要重复</li><li> SEL等截面标签来测量蛋白质的尺寸。绘制光标线移动在分子或区域进行分析。重复用于多个领域，该软件会产生叠加。导出照片或​​用于生成图像兼容其他软件和绘图软件中的格式的数据。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Near-Infared+Fluorescent+Protein+for+in+vivo+Imaging.">Stable Near-Infared Fluorescent Protein for in vivo Imaging.</a> Nature Biotech. 29, 757-761 (2011).</li><li>Samma, A. A., Johnson, C. K., Song, S., Alvarez, S., Zimmer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Origin+of+Fluorescence+in+Bacteriophytochrome+Infrared+Fluorescent+Proteins.">On the Origin of Fluorescence in Bacteriophytochrome Infrared Fluorescent Proteins.</a> J. Phys. Chem. B. 114, 15362-15369 (2011).</li><li>Lehtivuori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Properties+of+the+Chromophore-Binding+Domain+of+Bacteriophytochrome+from+Deinococcus+radiodurans.">Fluorescence Properties of the Chromophore-Binding Domain of Bacteriophytochrome from Deinococcus radiodurans.</a> J. Phys. Chem. B. 117, 11049-11057 (2013).</li><li>Toh, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Reactions+of+Bacteriophytochrome+Observed+with+Ultrafast+Mid-Infrared+Spectroscopy.">Primary Reactions of Bacteriophytochrome Observed with Ultrafast Mid-Infrared Spectroscopy.</a> J. Phys. Chem. A. 115, 3778-3786 (2011).</li><li>Auldridge, M. E., Satyshur, K. A., Anstrom, D. M., Forest, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-guided+engineering+enhances+a+phytochrome-based+infrared+fluorescent+protein.">Structure-guided engineering enhances a phytochrome-based infrared fluorescent protein.</a> J. Biol. Chem. 287, 7000-7009 (2012).</li><li>Toh, K. C., Stojkovic, E. A., van Stokkum, I. H., Moffat, K., Kennis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-transfer+and+hydrogen-bond+interactions+determine+fluorescence+quantum+yield+and+photochemical+efficiency+of+bacteriophytochrome.">Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome.</a> Proc. Natl. Acad. Sci. U S A. 107, 9170-9175 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pymol+Molecular+Graphic+System+v.1.5.0.1.">The Pymol Molecular Graphic System v.1.5.0.1.</a> , .</li><li>Yang, X., Stojkovic, E. A., Kuk, J., Crystal Moffat, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+the+chromophore+binding+domain+of+an+unusual+bacteriophytochrome,+RpBphP3,+reveals+residues+that+modulate+photoconversion.">structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion.</a> Proc. Natl. Acad. Sci. USA. 104, 12571-12576 (2007).</li><li>Yang, X., Kuk, J., Moffat, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+Pseudomonas+aeruginosa+bacteriophytochrome:+photoconversion+and+signal+transduction.">Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: photoconversion and signal transduction.</a> Proc. Natl. Acad. Sci. USA. 105, 14715-14720 (2008).</li><li>Woitowich, N. K., Garrido, C., Ozarowski, W., Stojkovic, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+X-ray+Crystallographic+and+Structural+Analyses+of+a+Bacteriophytochrome+from+Stigmatella+aurantiaca.">Preliminary X-ray Crystallographic and Structural Analyses of a Bacteriophytochrome from Stigmatella aurantiaca.</a> The FASEB Journal. 25, 928 (2011).</li><li>Wagner, J. R., Zhang, J., Brunzelle, J. S., Vierstra, R. D., Forest, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+structure+of+Deinococcus+bacteriophytochrome+yields+new+insights+into+phytochrome+architecture+and+evolution.">High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution.</a> J. Biol. Chem. 282, 12298-12309 (2007).</li><li>Wagner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+Deinococcus+radiodurans+bacteriophytochrome+reveals+key+amino+acids+necessary+for+the+photochromicity+and+proton+exchange+cycle+of+phytochromes.">Mutational analysis of Deinococcus radiodurans bacteriophytochrome reveals key amino acids necessary for the photochromicity and proton exchange cycle of phytochromes.</a> J. Biol. Chem. 283, 12212-12226 (2008).</li></ol>

There are no competing financial interests or conflicts of interest.

原子力显微镜是一种扫描探针显微镜方法完全能够成像的蛋白质和其他生物大分子的生理相关条件。相比于X射线晶体学和核磁共振，原子力显微镜中的一个限制是它不能达到相同的分辨率，特别是横向分辨率。当使用原子力显微镜来分析在任何表面上的分子，该表面的分子的图象上造成的影响和探针也必须当数据进行分析考虑。所获取的图像上的探测​​器的影响去卷积可以用适当的软件来完成...

原子力显微镜（AFM）已成为自1986年发明（ 图1），调查表面，薄膜，和单个分子的结构和机械性能的非常重要的工具。1-3使用的液体芯，该方法具有成为在生物大分子的研究，即使活细胞是特别有用的生理学相关的环境。4-10轻敲模式原子力显微镜在传统上被用于成像软材料或松散结合的分子的表面，由于接触模式AFM通常是不合适的，由于由悬臂引起的横向力的损害作用在样品11攻牙模式AFM基本上由具有前端间歇触摸表面，而不是在恒定接触而减少了这些力。在这种模式下，悬臂摆动达到或接近其共振频率与表面垂直。同样地，以非接触式原子力显微镜模式，地势肛门通过绘制Z-压电的运动为XY（距离）的函数yzed。 悬臂动力学可以是相当不稳定的或接近共振;因此，他们是非常具有挑战性的自动化的“稳定状态”的情况外。具体地，这些动力学依赖于两个样品的性质和扫描环境。对于软分子吸附到硬盘（ER）的表面，经过良好调优的反馈回路的分子可能会导致反馈振荡的表面。操作在流体进一步复杂悬臂的调谐。温度变化或流体水平要求设定点，收益和其他成像参数的不断调整。这些调整往往是非常耗时和对用户的挑战性。 峰力定量纳米机械性能映射（PF-QNM），如轻敲模式原子力显微镜，通过样品（ 图2）间歇性地接触，避免了横向的互动。12-15 &lt;/ SUP&gt;然而，PF-QNM工作在非谐振模式和频率比轻敲模式原子力显微镜低得多。这消除了轻敲模式原子力显微镜，特别是那些由流体的存在而加剧的调谐挑战。随着PF-QNM，图像通过采取武力响应曲线在每一个接触点收集。通过加入ScanAsyst软件，15调整扫描参数可被自动和高分辨率的图像由没有经验的用户获得在几分钟之内。一旦用户变得更加熟悉的原子力显微镜，任何自动或所有参数可以在任何时间，其允许实验者微调图像质量手动禁用。公司自成立以来，PF-QNM已经应用到地图细菌视紫红质，膜蛋白，以及其他天然蛋白在亚分子水平。16-18对细菌视紫红质，有弹性的蛋白质和X-射线晶体结构之间有直接的关系。12犯规-qnM的被利用来研究具有高分辨率的活细胞。19,20此外，PF-QNM数据已经阐明的红血球膜，这对细胞的完整性和功能是至关重要的内结构和力学之间的重要连接。21 我们已经采用扫描探针显微镜（SPM）的方法，22，包括原子力显微镜，23学习的红色光的光感受器的结构称为bacteriophytochromes（BphPs）。24,25它们由共价连接到信令执行器模块这样的光感测模块的因为组氨酸激酶（香港）26的光感测模组通常包含一个胆色素生色团，其在吸收光子发生结构转变，一系列深远的信号效应器模块，并导致整个蛋白质的全球转变的结构性变化，24,27-29基于这种转变，有两种截然不同的光吸收šBphPs，红色和远红外光吸收状态的美帝，表示为Pr和PFR。 Pr为热稳定的，暗适应状态下的最BphPs 28的PR / PFR光转化的分子基础是不完全是由于这些蛋白质的有限结构的知识的理解。随着从D除了一个结构球菌 ，这些蛋白质的30所有发表的X射线晶体结构是在暗适应状态下，缺乏效应器结构域。完整BphPs是太大而不能有效地通过核磁共振（NMR）研究，并且非常难以在其完整形式结晶（特别是在光适应状态下）的X射线晶体结构。 BphPs最近被改造为红外荧光蛋白标记（IFP的），这些蛋白31的结构特征可以进一步帮助有效IFP设计。32-36 本文的重点是介绍一个程序，影像通过PF-QNM使用液体电池的AFM BphPs的。该方法是从光合细菌R.表现在bacteriophytochrome RpBphP3（P3）的光适应状态下的学习沼泽 。这里介绍的AFM程序是用于蛋白质的成像方便和简单的方法，以及其他的生物大分子。用这种方法，单个分子的结构细节可以被收集的时间，类似于上位科学实验室课程会话短时间。通过测量断面，完成进一步的维分析，实验数据可以比较有效的计算模型。37-42

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>O4997-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Acros Organics</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Acros Organics</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimode 8 AFM</td>
			<td>Bruker-Nano</td>
			<td>492-008-011</td>
			<td>equipped with Nanoscope V controller and J scanner</td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>Bruker-Nano</td>
			<td>SNL-10, ScanAsyst-Fluid+</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapping Mode Fluid Cell</td>
			<td>Bruker-Nano</td>
			<td>MTFML</td>
			<td></td>
		</tr>
		<tr>
			<td>Mica V-4 Grade</td>
			<td>SPI supplies</td>
			<td>1150503</td>
			<td>25 x 25 x .26 mm</td>
		</tr>
		<tr>
			<td>Sample support disk</td>
			<td>nanoSurf</td>
			<td>BT02236</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Plasta-Medic, Inc.</td>
			<td></td>
			<td>100 mm x 15 mm&#160;</td>
		</tr>
		<tr>
			<td>micropipettors</td>
			<td>Denville Scientific</td>
			<td>XL 3000i</td>
			<td></td>
		</tr>
		<tr>
			<td>RpBphP3</td>
			<td></td>
			<td></td>
			<td>Prepared according to cited references</td>
		</tr>
		<tr>
			<td>Nanoscope software</td>
			<td>Bruker-Nano</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Light</td>
			<td>Digital Instruments Inc.</td>
			<td>F0-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Pelco AFM Disc Gripper</td>
			<td>Ted Pella Inc</td>
			<td>1668</td>
			<td>12 mm</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>McKesson</td>
			<td>102-ST1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Denville Scientific</td>
			<td>C2171</td>
			<td></td>
		</tr>
		<tr>
			<td>The Pymol Molecular Graphic System v.1.5.0.1</td>
			<td>Schrodinger, LLC</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

atomic force microscopy of red-light photoreceptors using peakforce quantitative nanomechanical property mapping

原子力显微镜（AFM）使用连接到悬臂探测表面的力响应金字塔尖。尖端的偏转可以被测量到〜10 PN通过激光和扇区检测器，它可以转换为图像的形貌。振幅调制或“轻敲模式”AFM涉及制备与表面间歇性接触，在其谐振频率而振荡，以产生图像的探测器。用于与流体细胞结合，轻敲模式原子力显微镜使生物大分子的影像，如在生理条件有关的蛋白质。轻敲模式原子力显微镜需要的探头和多种可挑战经验的用户扫描参数的频繁调整的手动调整。为了获得高质量的图像，这些调整是最耗时的。 PeakForce定量纳米机械性能映射（PF-QNM）通过测量力respon产生图像本身曲线的每个点与样品接触的。与ScanAsyst软件，PF-QNM可以自动化。这个软件调整设定点，驱动频率，扫描速度，增益和其他重要的扫描参数自动给定的样本。这不仅处理同时保护脆弱的探针和样品，它显著降低，得到高清晰度的图像所需的时间。 PF-QNM是原子力显微镜成像流体兼容;因此，它具有用于成像的生物相关物质的广泛应用。 本文提出的方法描述PF-QNM的应用程序来获取细菌红光感光，RpBphP3（P3）的图像，从光合河沼泽红假单胞菌在其光适应状态。使用这种方法，P3的个别蛋白二聚体和二聚体的聚集体已经观察到在云母表面在成像缓冲器的存在。以适当调整表面和/或溶液浓度，这种方法通常可应用于其它生物学相关的大分子和软质材料。

感光体蛋白，P3，在其光适应状态下的有代表性的AFM图像示于图3和图4。一个刚切割的云母衬底（ 图3A）是合适的，平坦的表面蛋白质的吸附。收集干净云母的图像作为阴性对照是非常重要的有以下几个原因。首先，它确保了液体电池的清洁，并从先前的实验无残留物质会污染地面。第二，它测试探针的质量。如果探头脏了或变形，这样会出现图像条纹或显示...

Watch this Scientific Journal Video about Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping at JoVE.com

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

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 ...

atomic-force-microscopy-red-light-photoreceptors-using-peakforce

Research

JoVE Journal

Engineering

11.6K 次观看.  Northeastern Illinois University. 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. 

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

Preparation and Friction Force Microscopy Measurements of Immiscible, Opposing Polymer Brushes

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low friction at the interface. Nevertheless, these systems can be sensitive to wear due to interdigitation of the opposing brushes. In a recent publication, we have shown via molecular dynamics simulations and atomic force microscopy experiments, that using an immiscible polymer brush system terminating the substrate and the slider surfaces, respectively, can eliminate such interdigitation. As a consequence, wear in the contacts is reduced. Moreover, the friction force is two orders of magnitude lower compared to traditional miscible polymer brush systems. This newly proposed system therefore holds great potential for application in industry. Here, the methodology to construct an immiscible polymer brush system of two different brushes each solvated by their own preferred solvent is presented. The procedure how to graft poly(N-isopropylacrylamide) (PNIPAM) from a flat surface and poly(methyl methacrylate) (PMMA) from an atomic force microscopy (AFM) colloidal probe is described. PNIPAM is solvated in water and PMMA in acetophenone. Via friction force AFM measurements, it is shown that the friction for this system is indeed reduced by two orders of magnitude compared to the miscible system of PMMA on PMMA solvated in acetophenone.

The methodology to perform friction force microscopy experiments for contacting brushes is presented: Two polymer brushes that are grafted from (a) substrates and (b) colloidal probes are slid to show that, by using two contacting immiscible brush systems, friction in sliding contacts is reduced compared to miscible brush systems.

互溶的测量准备和摩擦力显微镜，反对聚合物刷

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low ...

科研

工程学

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 ...

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

用同步快速反应层析成像动态孔隙规模油藏条件下成像碳酸盐

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

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 ...

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.

用金纳米粒子-嵌入式膜过滤法选择性脱盐放射性碘阴离子的有效方法

Here, we demonstrate a detail protocol for the preparation of nanomaterials-embedded composite membranes and its application to the efficient and ...

Surface Mapping of Earth-like Exoplanets using Single Point Light Curves

Spatially resolving exoplanet features from single-point observations is essential for evaluating the potential habitability of exoplanets. The ultimate goal of this protocol is to determine whether these planetary worlds harbor geological features and/or climate systems. We present a method of extracting information from multi-wavelength single-point light curves and retrieving surface maps. It uses singular value decomposition (SVD) to separate sources that contribute to light curve variations and infer the existence of partially cloudy climate systems. Through analysis of the time series obtained from SVD, physical attributions of principal components (PCs) could be inferred without assumptions of any spectral properties. Combining with viewing geometry, it is feasible to reconstruct surface maps if one of the PCs are found to contain surface information. Degeneracy originated from convolution of the pixel geometry and spectrum information determines the quality of reconstructed surface maps, which requires the introduction of regularization. For the purpose of demonstrating the protocol, multi-wavelength light curves of Earth, which serves as a proxy exoplanet, are analyzed. Comparison between the results and the ground truth is presented to show the performance and limitation of the protocol. This work provides a benchmark for future generalization of exoplanet applications.

The protocol extracts information from light curves of exoplanets and constructs their surface maps. It uses light curves of Earth, which serves as a proxy exoplanet, to demonstrate the approach.

使用单点光曲线绘制的地球样外行星的表面图

Spatially resolving exoplanet features from single-point observations is essential for evaluating the potential habitability of exoplanets. The ultimate ...

Optimizing Magnetic Force Microscopy Resolution and Sensitivity to Visualize Nanoscale Magnetic Domains

Magnetic force microscopy (MFM) enables mapping local magnetic fields across a sample surface with nanoscale resolution. To perform MFM, an atomic force microscopy (AFM) probe whose tip has been magnetized vertically (i.e., perpendicular to the probe cantilever) is oscillated at a fixed height above the sample surface. The resultant shifts in the oscillation phase or frequency, which are proportional to the magnitude and sign of the vertical magnetic force gradient at each pixel location, are then tracked and mapped. Although the spatial resolution and sensitivity of the technique increases with decreasing lift height above the surface, this seemingly straightforward path to improved MFM images is complicated by considerations such as minimizing topographical artifacts due to shorter range van der Waals forces, increasing the oscillation amplitude to further improve sensitivity, and the presence of surface contaminants (in particular water due to humidity under ambient conditions). In addition, due to the orientation of the probe's magnetic dipole moment, MFM is intrinsically more sensitive to samples with an out-of-plane magnetization vector. Here, high-resolution topographical and magnetic phase images of single and bicomponent nanomagnet artificial spin-ice (ASI) arrays obtained in an inert (argon) atmosphere glovebox with &lt;0.1 ppm O2 and H2O are reported. Optimization of lift height and drive amplitude for high resolution and sensitivity while simultaneously avoiding the introduction of topographical artifacts is discussed, and detection of the stray magnetic fields emanating from either end of the nanoscale bar magnets (~250 nm long and &lt;100 nm wide) aligned in the plane of the ASI sample surface is shown. Likewise, using the example of a Ni-Mn-Ga magnetic shape memory alloy (MSMA), MFM is demonstrated in an inert atmosphere with magnetic phase sensitivity capable of resolving a series of adjacent magnetic domains each ~200 nm wide.

Magnetic force microscopy (MFM) employs a vertically magnetized atomic force microscopy probe to measure sample topography and local magnetic field strength with nanoscale resolution. Optimizing MFM spatial resolution and sensitivity requires balancing decreasing lift height against increasing drive (oscillation) amplitude, and benefits from operating in an inert atmosphere glovebox.

优化磁力显微镜分辨率和灵敏度，以可视化纳米级磁畴

Magnetic force microscopy (MFM) enables mapping local magnetic fields across a sample surface with nanoscale resolution. To perform MFM, an atomic force ...

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 ...

AFMS 中的并联有源悬臂阵列可实现高通量检测

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. However, due to their limited imaging throughput, AFMs have not been widely adopted for large-scale inspection purposes. Researchers have developed high-speed AFM systems to record dynamic process videos in chemical and biological reactions at tens of frames per second, at the cost of a small imaging area of up to several square micrometers. In contrast, inspecting large-scale nanofabricated structures, such as semiconductor wafers, requires nanoscale spatial resolution imaging of a static sample over hundreds of square centimeters with high productivity. Conventional AFMs use a single passive cantilever probe with an optical beam deflection system, which can only collect one pixel at a time during AFM imaging, resulting in low imaging throughput. This work utilizes an array of active cantilevers with embedded piezoresistive sensors and thermomechanical actuators, which allows simultaneous multi-cantilever operation in parallel operation for increased imaging throughput. When combined with large-range nano-positioners and proper control algorithms, each cantilever can be individually controlled to capture multiple AFM images. With data-driven post-processing algorithms, the images can be stitched together, and defect detection can be performed by comparing them to the desired geometry. This paper introduces principles of the custom AFM using the active cantilever arrays, followed by a discussion on practical experiment considerations for inspection applications. Selected example images of silicon calibration grating, highly-oriented pyrolytic graphite, and extreme ultraviolet lithography masks are captured using an array of four active cantilevers (&#34;Quattro&#34;) with a 125 &#181;m tip separation distance. With more engineering integration, this high-throughput, large-scale imaging tool can provide 3D metrological data for extreme ultraviolet (EUV) masks, chemical mechanical planarization (CMP) inspection, failure analysis, displays, thin-film step measurements, roughness measurement dies, and laser-engraved dry gas seal grooves.

作者聚焦：使用 Quattro-Parallel 悬臂阵列的主动探针原子力显微镜简介

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

使用Quattro-Parallel 悬臂阵列的主动探针原子力显微镜，用于高通量大规模样品检测

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. ...