作者感谢加拿大新前沿研究基金 （NFRF）、加拿大自然科学与工程研究委员会 （NSERC） 发现计划以及魁北克自然与技术研究基金会 （FRQNT） 硕士培训奖学金的资金支持。作者还要感谢McGill的Yaoyao Zhao教授团队对某些组件的3D打印提供的技术支持。

用于该方案的蚊子种类是未感染的成年雌性 埃及伊蚊 （A. aegypti），冷冻接收并储存在-20°C度的冰箱中。该物种由NIH/NIAID丝虫病研究试剂资源中心提供，通过BEI Resources，NIAID，NIH分发：未感染的 埃及伊蚊，菌株黑眼利物浦（冷冻），NR-48920。用于研究的试剂和设备列在 材料表中。
1. 从长鼻中解剖唇
<ol><li>使用镊子，将死蚊子放在显微镜下的载玻片上，并确保在长鼻的末端有一个锥形的尖端（图2A）。</li>
	<li>在将蚊子保持在载玻片上的同时，将手术刀刀片轻轻地放在蚊子头部附近的阴唇上（图2B）。</li>
	<li>继续在阴唇的整个上半部分（约 80 μm 的切口）做一个切口，在阴唇厚度上有一个较浅的穿透深度。确保对刀片施加轻微的压力，以便只切开阴唇，而不切开位于下方的束。</li>
	<li>用一把镊子牢牢握住蚊子的头部，然后用另一把精密镊子轻轻捏住锥形尖端和切口位置之间的任何位置的阴唇（图2B）。<ol><li>将夹住唇的镊子拉向锥形尖端的方向（图2C）。继续拉动镊子，直到阴唇脱离并完全从束中取出。</li>
	</ol></li>
	<li>将蚊子放在显微镜下，并确认盂唇的尖端是否存在。这可以通过束上存在锥形尖端来识别（图2D）。</li>
</ol>2. 将盂唇尖端与其他盂唇成员分开
<ol><li>夹住并闭合一组精密镊子的尖端，并将镊子的尖端放在靠近其尖端的盂唇旁边。</li>
	<li>使用镊子尖端在垂直于束长度的方向上对盂唇施加轻轻的力（图3A）。</li>
	<li>继续将盂唇推过载玻片，直到盂唇与其他束成员分离。</li>
	<li>在显微镜下检查样品，以验证盂唇和其他束成员之间已实现正确分离（图3，左）。如果分离不成功，请返回步骤 2.1。</li>
</ol>3. 切开盂唇尖端
<ol><li>当盂唇仍在载玻片上时，将手术刀刀片放在盂唇上，距离盂唇尖端约~200μm（图4A）。轻轻地施加足够的压力，将盂唇的尖端完全切开。虽然理想情况下，盂唇尖端应尽可能短，但 ~200 μm 是当前方法可以处理的最佳方法。</li>
	<li>使用任何数字测量软件测量切割盂唇的长度，以确保其长度不超过300μm（图4B）。在该协议中，使用ImageJ14。</li>
</ol>4.抓住盂唇的尖端
<ol><li>用一把精密镊子在载玻片上找到并隔离盂唇的尖端。丢弃载玻片上剩余的所有部分，但盂唇尖端除外。</li>
	<li>用同样精密的镊子，缓慢而轻微地捏住盂唇，使切割端自由且不受镊子的阻碍。此外，确保盂唇的方向平行于镊子长度的方向，并且盂唇的切割端远离镊子的身体。</li>
	<li>一旦试样被牢固地夹住，就移除将镊子尖端固定在一起的夹紧力。盂唇的尖端会粘在镊子的一个尖端上。</li>
	<li>在显微镜下，检查镊子的尖端，并确保盂唇的尖端位于镊子的一个尖端上（图5）。如果盂唇的尖端不在镊子上，请返回步骤 4.2，如果盂唇的尖端不在镊子上也不在载玻片上，请返回步骤 1。</li>
</ol>5. 在无尖悬臂梁上涂上环氧树脂
<ol><li>将一滴 （~0.05 mL） 环氧树脂滴在新载玻片的边缘，方法是直接从原来的瓶子/容器中滴铸粘合剂。将含环氧树脂的载玻片放在探针台下方并聚焦在其上。</li>
	<li>通过固定底座（即较大的端），将无尖 AFM 悬臂安装到夹具组件上，使悬臂端保持自由并悬挂在空间中。确保悬臂梁的底部朝下。</li>
	<li>将机械手安装到探针台上。</li>
	<li>将机械手的 Z 轴升高到无尖悬臂位于含环氧树脂玻璃载玻片上方几毫米的位置。</li>
	<li>手动移动机械手，使无尖端悬臂在探针台上的摄像机的视野视图中可见。</li>
	<li>使用机械手，沿 X 和 Y 方向移动悬臂梁，直到悬臂的尖端直接位于载玻片边缘的环氧树脂上方。</li>
	<li>再次使用机械手，将无尖悬臂沿 Z 方向缓慢降低到载玻片边缘上方。</li>
	<li>当悬臂降低并接近载玻片时，请继续非常缓慢地降低悬臂，直到它第一次接触环氧树脂。不要再降低悬臂。</li>
	<li>小心地接合机械手，在 X 或 Y 方向上缓慢移动悬臂，并通过沿选定方向连续移动悬臂，将悬臂从环氧树脂池中取出，直到悬臂与载玻片上的环氧树脂完全分离。无尖悬臂的尖端应有一个微型环氧树脂气泡，在探针台下方可见。</li>
	<li>使用机械手向 Z 方向升高悬臂。</li>
</ol>6. 将盂唇的尖端粘合到无尖端悬臂梁上
<ol><li>将机械手绕悬臂的长轴旋转 90 度，然后将机械手侧放在探针台上。在这种配置中，沿悬臂梁长度的厚度在垂直方向上。</li>
	<li>将包含盂唇尖端的精密镊子放在探针台摄像头下方，使盂唇尖端的整个长度在计算机显示器上可见。</li>
	<li>将握住夹具和无尖端悬臂的机械手组件放置在探针台摄像头下方，使无尖端悬臂的整个长度在计算机显示器上可见。</li>
	<li>将探针台显微镜聚焦在盂唇的尖端和无尖端悬臂上。</li>
	<li>通过小心和手动旋转操纵器，将悬臂垂直于盂唇的尖端（图6A）。</li>
	<li>利用机械手的自由度，沿XY方向缓慢移动无尖悬臂，使悬臂上的胶水接触盂唇尖端的切割端（图6B）。</li>
	<li>固化环氧树脂，固化悬臂和蚊子盂唇之间的交叉点。</li>
	<li>环氧树脂固化后，轻轻地在XY方向上接合机械手，并将悬臂从镊子上移开，确认盂唇的尖端现在位于无尖端悬臂梁上（图6C）。</li>
</ol>

带有蚊唇的生物杂交 AFM 探针，用于研究叮咬行为

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+vector+control+response+2017&#8211;2030.">Global vector control response 2017&#8211;2030.</a> World Health Organization. , (2017).</li><li>Gurera, D., Bhushan, B., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+mosquitoes&#8217;+painless+piercing.">Lessons from mosquitoes&#8217; painless piercing.</a> J Mech Behav Biomed Mater. 84, 178-187 (2018).</li><li>Ma, G., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle,+bio-microneedle+and+bio-inspired+microneedle:+A+review.">Microneedle, bio-microneedle and bio-inspired microneedle: A review.</a> Journ of Contr Relea. 251, 11-23 (2017).</li><li>Kong, X., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micronano+structure+and+mechanics+behavior+of+mosquito&#8217;s+proboscis+biomaterials+with+applications+to+microneedle+design.">Micronano structure and mechanics behavior of mosquito&#8217;s proboscis biomaterials with applications to microneedle design.</a> Advan Mater Res. 299-300, 376-379 (2011).</li><li>Li, A. D. R., Putra, K. B., Chen, L., Montgomery, J. S., Shih, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+proboscis-inspired+needle+insertion+to+reduce+tissue+deformation+and+organ+displacement.">Mosquito proboscis-inspired needle insertion to reduce tissue deformation and organ displacement.</a> Sci Rep. 10 (1), 12248 (2020).</li><li>Meyer, E., Hug, H. J., Bennewitz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+Scanning+Probe+Microscopy.">Introduction to Scanning Probe Microscopy.</a> Scanning Probe Microscopy. 1, 1-13 (2004).</li><li>Garc&#237;a, R., Per&#233;z, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+atomic+force+microscopy+methods.">Dynamic atomic force microscopy methods.</a> Surf Sci Rep. 47 (6), 197-301 (2002).</li><li>Thurner, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+and+indentation+force+measurement+of+bone.">Atomic force microscopy and indentation force measurement of bone.</a> WIREs Nanomed and Nanobio. 1 (6), 624-649 (2009).</li><li>M&#252;ller, D. J., Dufr&#234;ne, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+as+a+multifunctional+molecular+toolbox+in+nanobiotechnology.">Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology.</a> Nat Nanotech. 3 (5), 261-269 (2008).</li><li>Hussain, D., Ahmad, K., Song, J., Xie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+atomic+force+microscopy+for+-critical+dimension+metrology.">Advances in the atomic force microscopy for -critical dimension metrology.</a> Meas Sci Technol. 28 (1), 012001 (2017).</li><li>Kong, X. Q., Wu, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+proboscis:+An+elegant+biomicroelectromechanical+system.">Mosquito proboscis: An elegant biomicroelectromechanical system.</a> Phys Rev E. 82 (1), 011910 (2010).</li><li>Kong, X. Q., Wu, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+and+prediction+of+insertion+force+for+the+mosquito.">Measurement and prediction of insertion force for the mosquito.</a> J Bionic Eng. 6 (2), 143-152 (2009).</li><li>Ramasubramanian, M. K., Barham, O. M., Swaminathan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+a+mosquito+bite+with+applications+to+microneedle+design.">Mechanics of a mosquito bite with applications to microneedle design.</a> Bioinspir Biomim. 3 (4), 046001 (2008).</li><li>Dai, G., 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=Nanoscale+surface+measurements+at+sidewalls+of+nano-+and+micro-structures.">Nanoscale surface measurements at sidewalls of nano- and micro-structures.</a> Measur Sci and Technol. 18 (2), 334-341 (2007).</li></ol>

作者无需声明任何利益冲突。

该协议的第 1 步旨在清洁不需要的阴唇的生物样本。为了实现这一点，在阴唇上做一个切口，但不在位于阴唇正下方的束上做一个切口（图1）。由于束和唇在其界面处没有连接在一起（即，唇可以沿着束自由滑动，并且仅通过其附着在蚊子头上保持到位），因此进行的切口旨在将唇的一部分与蚊子的头部分开，从而促进外罩的移除。当唇形物从样品中拉出时，技术人员应该...

世界卫生组织 （WHO） 报告说，病媒传播疾病 （VBD） 占所有传染病的 17% 以上，每年导致全球超过 7,00,000 人死亡。例如，作为世界上最致命的动物，蚊子通过吸血节肢动物传播登革热、疟疾和寨卡病毒等多种病原体，每年导致 7 亿例感染1。探索制定有效措施预防VBDs至关重要，包括模仿蚊子的侵入行为以研究其叮咬机制，以及研究潜在障碍以证明其预防侵入的有效性。一个关键的挑战是制定适当的方法来进行这种调查。在文献中已经做出了努力，包括开发类似于蚊子毒刺几何形状的微尺度针头;然而，许多用于制造这些微针的材料（即粘弹性材料2、硅 （Si）、玻璃、陶瓷3 等）与蚊子长鼻的生物材料具有不同的机械性能。工程材料可能很脆，容易断裂和屈曲 3,4，而蚊子的长鼻可以更好地承受断裂或屈曲4.使用蚊子的盂唇而不是工程材料的生物混合探针的好处是，它可以更准确地表示蚊子的穿刺机制。此外，必须将专用工具与微针集成以执行定量研究，例如力5 的准确测量，这对于使用工程微针的定制设置来说不容易实现。
基于原子力显微镜 （AFM） 的方法很有前途的，因为它通过使用带有超细尖端的悬臂来操作，该悬臂被小心地放置在靠近样品表面的位置。尖端既可以扫描表面，也可以被压向/压入表面，由于其与样品的相互作用，会受到不同的吸引力或排斥力6.这些相互作用导致悬臂的偏转，通过激光束从悬臂顶部反射到光电探测器6 上来跟踪偏转。AFM 对系统运动的超强灵敏度使 AFM 能够进行各种测量，包括但不限于皮米精度的形态映射、从皮牛顿到微牛顿的力测量以及全面的多物理场研究7。例如，可以执行 AFM 压痕以精确评估对样品施加力的响应，并通过与适当的分析模型耦合来测量样品的硬度、弹性和其他机械性能8。AFM 的探针通常由硅 （Si） 或氮化硅 （Si3N4）8 制成，长度为 20-300 μm9 ，尖端半径约为几到几十纳米10。纳米级针尖半径是高分辨率成像等应用的理想选择;然而，对于试图在刚度、半径、形状和纵横比方面模仿穿透行为的研究，它不具备生物毒刺的特征。例如，蚊子的微针结构是束，其纵横比为 ~6011 （长度 ~1.5 毫米至 2 毫米;直径 ~30 μm）12。虽然可以假设传统的AFM探针类似于盂唇等生物毒刺，但其独特的材料特性和尺寸并不能反映咬合过程中的真实情况。
为了能够对模仿昆虫或其他动物用毒刺的生物叮咬的穿透行为进行定量研究，这里开发了一种制造生物混合AFM悬臂的工艺，该工艺以生物毒刺为尖端。作为一个案例研究，成功地展示了一种带有蚊子盂唇尖端的AFM悬臂。利用文献中关于蚊子用来刺穿受害者皮肤的典型插入力的现有信息12,13，这种生物混合AFM悬臂有可能在常规AFM下实现蚊子叮咬的近乎真实的模拟。利用微生物毒刺制造生物混合 AFM 悬臂的方案也可应用于开发其他基于 Sharp Stinger 的生物混合 AFM 悬臂，用于对各种咬合机制进行定量研究。
术语 长鼻及其相关组件的示意图如图 1 所示，其定义是 （1） 长鼻：蚊子口中的身体部位，允许蚊子自我进食，其核壳结构由束（核心）和唇（壳）组成，（2） 唇：长鼻的深色和钝的外壳2，（3）束：一组细长的针头包含在唇内，包括两个上颌骨、两个下颌骨、一个下咽部和一个盂唇2、（4）下咽部：负责将唾液分泌到宿主的血液中2、（5）上颌骨：协助摄食机制的锯齿状构件2、（5）下颌骨：类似于上颌骨，它们帮助蚊子摄食机构，并有一个锋利的尖端2，（6）盂唇：穿透受害人皮肤的主要构件，比上颌骨、下颌骨和下咽部大得多。它还具有感觉结构，使其能够在皮肤下找到血管和内部通道2，（7） 操纵器：具有三个自由度和微米级定位精度的组件，允许在 XYZ 方向上移动，（8） 夹具组件：安装在机械手上的定制 2 部分夹具，用于在实验过程中夹紧无尖端 AFM 悬臂。

<table><tbody><tr><td>&amp;nbsp;5-SA-SE Straight Tapered Ultra Fine-Pointed Tweezers</td><td>Excelta</td><td>N/A</td><td>For manipulating/dissecting the proboscis.</td></tr><tr><td>C-4D Probe station</td><td>Everbeing Int&amp;rsquo;l Corp&amp;nbsp;</td><td>N/A</td><td>Used for AFM assembly.</td></tr><tr><td>Tipless Tapping Mode Cantilever</td><td>NanoAndMore USA</td><td>TL-NCH</td><td>AFM cantilever used for mounting the labrum.&lt;br/&gt; Specs are shown here:&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Shape&lt;/strong&gt;: Beam&lt;br/&gt; &lt;strong&gt;Force Constant&lt;/strong&gt;: 42 N/m (10 - 130 N/m)&lt;br/&gt; &lt;strong&gt;Resonance Frequency&lt;/strong&gt;: 330 kHz (204 - 497 kHz)&lt;br/&gt; &lt;strong&gt;Length&lt;/strong&gt;: 125 &amp;micro;m (115 - 135 &amp;micro;m)&lt;br/&gt; &lt;strong&gt;Width&lt;/strong&gt;: 30 &amp;micro;m (22.5 - 37.5 &amp;micro;m)&lt;br/&gt; &lt;strong&gt;Thickness&lt;/strong&gt;: 4 &amp;micro;m ( 3 - 5 &amp;micro;m)</td></tr><tr><td>UV Expoxy</td><td>Let&#039;s resin</td><td>ALR00146</td><td>For stinger attachment.</td></tr></tbody></table>

development of a bio-hybrid mosquito stinger-based atomic force microscopy probe

蚊子因其传播疾病的能力而臭名昭著，是对人类最致命的动物，对公共卫生构成持续挑战。目前使用的一级预防策略涉及化学驱虫剂，由于蚊子迅速产生抗药性，化学驱虫剂往往被证明是无效的。因此，发明新的预防方法至关重要。这种发展取决于对蚊子叮咬行为的透彻理解，因此需要一种实验装置，该实验装置能够通过可控的测试参数和定量测量准确复制实际叮咬场景。为了弥合这一差距，设计了一种生物混合原子力显微镜（AFM）探针，其尖端是生物毒刺 - 特别是蚊子盂唇。这种生物混合探针与标准AFM系统兼容，可以对蚊子的渗透行为进行近乎真实的模拟。这种方法标志着在叮咬机制的定量研究方面向前迈出了一步，有可能为对抗病媒传播疾病（VBDs）建立有效的屏障，并为对抗蚊媒传播疾病开辟新的途径。

制备的生物杂化AFM探针的扫描电子显微镜（SEM）图像如图 7所示。盂唇的末端成功地粘在无尖端悬臂梁上。由于蚊子毒刺的自然曲率和所提出协议的手动操作，因此获得具有完全垂直于悬臂的毒刺尖端的悬臂是极其困难的。毒刺与垂直于悬臂的假想中心线之间的偏心角通常为 ~10 度。虽然将探针安装到 AFM 悬臂15 上似乎是一个常见问题，但在执行力/应力分析...

Watch this Scientific Journal Video about Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms at JoVE.com

作者聚焦：用于定量分析蚊子叮咬机制的生物混合 AFM 悬臂的开发

对昆虫叮咬行为进行定量和受控调查对于制定有效的策略来对抗病媒传播疾病至关重要。在此背景下，介绍了一种制备生物杂化原子力显微镜（AFM）探针的方法。

基于生物杂化蚊虫叮咬的原子力显微镜探针的研制

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The ...

我们正在开发一种AFM悬臂，以潜在地定量研究蚊子的叮咬机制。我们的协议提供了一种新方法，用于创建准确、可控和可靠的 AFM 探针，以研究动物咬伤或刺痛过程。我们的协议旨在准确模拟动物咬伤过程，导致当前的方法要么不可控，无法以可靠的方式重现，要么在某些情况下完全不准确。我们工作的影响是双重的。首先，我们创建了一个协议，使其他研究人员能够制造他们自己的定制生物混合AFM悬臂。其次，我们的工作为创建其他协议铺平了道路，这些协议将测试系统与生物材料相结合，以创建更真实的测试场景。

development-bio-hybrid-mosquito-stinger-based-atomic-force-microscopy

Research

JoVE Journal

Engineering

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

708 次观看.  McGill University. 我们正在开发一种AFM悬臂，以潜在地定量研究蚊子的叮咬机制。我们的协议提供了一种新方法，用于创建准确、可控和可靠的 AFM 探针，以研究动物咬伤或刺痛过程。我们的协议旨在准确模拟动物咬伤过程，导致当前的方法要么不可控，无法以可靠的方式重现，要么在某些情况下完全不准确。我们工作的影响是双重的。首先，我们创建了一个协议，使其他研究人员能够?...

视频: 作者聚焦：用于定量分析蚊子叮咬机制的生物混合 AFM 悬臂的开发

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

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

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

重组DNA和蛋白质复合物，用原子力显微镜的可视化

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

科研

生物工程

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

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

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

免疫与感染

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 ...

工程学

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. ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

生物学

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

基于生物杂化蚊虫叮咬的原子力显微镜探针的研制

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重组DNA和蛋白质复合物，用原子力显微镜的可视化

警队复用单分子蛋白水解测量磁性镊子

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

荧

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

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

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

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

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

高速磁镊，用于力敏元件的纳米力学测量