Name: large-area scanning probe nanolithography facilitated by automated alignment and its application to substrate fabrication for cell culture studies
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Description: Watch this Scientific Journal Video about Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies at JoVE.com

作者承认包括英国工程和自然科学研究理事会在内的各种来源提供的财政支持 (赠款裁判。EP/K011685/1, EP/K024485/1) 和奖学金毕业生;Leverhulme 信托基金 (RPG-2014-292);威康信托机构战略支助基金 (105610/z/14/z);英国的理事会 (216196834);和曼彻斯特大学曼彻斯特大学研究所 (UMRI 泵启动基金) 和总统博士奖学金的 SW. 李博士 (Nanosurf AG) 的技术援助也得到了赞赏。

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对齐算法和软件由曼彻斯特大学开发, 并且是专有的。可在 http://www.click2go.umip.com/下载。

该协议为用户提供了一种方便的方法, 可以快速地实现高均匀性和可控制的功能大小 (cm2) 区域的 nanolithographic 模式。这些大面积 nanopatterns 的衬底可以为各种应用进一步阐述。这项技术的一个主要应用是在 nanofabricated 表面进行细胞表面相互作用研究。本报告展示了这些材料的细胞培养的一些例子, 展示了 nanofabricated 基质对 hMSC 形态的控制。
此协议的关键启用程序是对齐过程 (步骤 4) 的自动化, 它允许在表面上高度均匀和高通量的功能生产, 降低到纳米尺度分辨率, 从而实现细胞培养实验的快速周转。采用这种对准算法进行的聚合物笔光刻能够在大约30分钟内产生纳米尺度特征。自动对齐的再现性和准确性, 从而使图案化特征的一致性, 但严重依赖于所产生的探针阵列的质量 (步骤1和 2)。在准备过程中产生钝、折断或缺失探针的任何缺陷;如陷气气泡 (步骤 1.5) 或不正确的分离探针从主 (步骤 1.8) 可能导致不准确的对准和质量差光刻。
此报告方法与依赖于强制反馈的其他对齐方法共有一个限制。准确地确定探针与表面接触时, 由于需要考虑环境环境和样品阶段的运动引起的背景振动而受到限制。一般而言, 传感器在µN (本例中为2µN) 中具有强制灵敏度, 但对齐算法的设计目的是只注册至少490µN 的力作为探头和表面之间的最终接触, 以避免任何 "误报" 结果从背景噪音。13因此, 这种方法倾向于产生大的特征 (1-2 µm), 因为探头必须在z轴上扩展一个很大的距离 (随之而来的是更高的力), 以便注册接触。为了补偿, 更小的功能可以通过减少光刻步骤中的z轴距离来生成 (例如, 在步骤5.2.3.2 中输入 "黑色" 设置, 而不是5µm)。
尽管如此, 即使有了这一限制, 自动化算法也能够解决并行扫描探针光刻方法的应用中的一个关键方面, 因为对齐以前是最需要时间和不精确的步骤。这些技术的实施。这种自动化现在将制造过程的速率限制步骤从对齐到平版书写本身。虽然本协议演示了这种对齐程序在 PPL 中的应用, 但该框架可以应用于一些 SPL 技术, 如脂质 DPN26和矩阵辅助光刻27以及未来潜在的催化探头系统。28

纳米技术的进展依赖于能够高效、可靠地制造表面纳米尺度特征的技术的发展。1,2然而, 在大面积 (多 cm2) 上可靠地产生这样的特性, 而且成本相对较低, 这是一个非平凡的努力。大多数现有的技术, 从半导体工业, 依靠烧蚀光刻制造 ' 硬 ' 材料。最近, 从扫描探针显微镜 (SPM) 中获得的光刻技术已经成为纳米设计快速成型的一种方便、通用的方法。3基于 SPM 的技术能够方便快捷地 "编写" 任何用户定义的模式。最知名的这些是蘸笔光刻 (DPN), 率先由 Mirkin等人,4在那里扫描探针被用作 ' 钢笔 ' 转移分子 ' 墨水 ' 的表面产生的特点, 类似于写作。在环境条件下, 当探针在表面扫描时, "墨水" 分子通过在探针和表面之间形成的水半月板转移到表面 (图 1)。DPN 从而允许 nanolithographic 沉积广泛的材料, 包括 ' 软 ' 材料, 如聚合物和生物分子。nanopipettes 还报道了5项相关技术, 这些方法使用了用管道进行流体输送的探头, 不同的称为 ' ' ' ' 和 ' 纳米喷泉笔 '。6,7,8
更广泛地应用 SPM 衍生光刻的主要障碍是吞吐量, 因为它需要过长的时间, 以一个单一的探针模式厘米尺度区域。早期致力于解决这个问题的重点是基于悬臂式 DPN 的并行化, 同时报告了 "一维" 和 "二维" (2D) 探针阵列, 用于对厘米大小的区域进行光刻。5,9然而, 这些悬臂阵列是通过相对复杂的多步骤制造方法产生的, 而且相对脆弱。聚合物笔光刻 (PPL) 的发明解决了这个问题, 通过更换标准的 SPM 悬臂与2D 阵列软硅氧烷弹性探头粘结到玻璃滑梯。10这一简单的探头设置大大降低了阵列大面积的成本和复杂性, 为更广泛的应用开辟了光刻。这种无悬臂结构也被扩展到硬尖端软弹簧光刻,11提供了一个混合软弹性支承与硬硅小贴士, 使改进的分辨率相比, 使用软弹性体提示。
执行这些2D 阵列技术的一个关键因素是探针阵列必须与表面基底完全平行, 以便在使用光刻时, 所有探头同时与表面接触。即使是一个小的错位也会导致从阵列的一侧到另一个的特征大小有很大的差别, 因为一些探针会在阵列下降的时候与表面接触, 而另一些探头会在以后接触到或根本不出现。12精确对准是特别重要的, 由于软弹性体探头的变形性, 在那里接触表面的探头将被压缩, 留下更大的足迹在表面上。
早期的研究人员使用纯粹的视觉检查来指导对准过程, 利用在阵列上方的摄像头观察金字塔探针的变形, 因为它们接触到了表面。10对齐方式是通过观察探针的哪一侧接触到表面首先, 然后调整角度和重复该过程以迭代的方式来判断, 直到探头两侧接触的差异不区分的眼睛。由于该对准程序依赖于操作者主观视觉检查, 重现性较低。
随后, 开发了一种更客观的方法, 由安装在基板下的力传感器组成, 用来测量表面上探头接触时施加的作用力。12通过调整倾斜角度来实现对齐, 从而使施加的力最大化, 这表明所有探头同时处于接触中。该方法表明, 对0.004°内的表面平行是可能的。这项 "力反馈水准" 现已在两份独立报告中实施为全自动系统。13,14都使用在基板下方或阵列上方安装的力传感器的三重, 并测量探针阵列和曲面之间接触所施加的作用力量。这些系统给出了高精度, 报告≤0.001°的不协调度超过1厘米长,14或≤ 0.0003°over 1.4 厘米.13这些自动对齐系统还提供了大量节省操作员时间和总时间来完成光刻工艺。
这种技术所启用的高通量表面制造的一个主要应用是细胞培养基质的生成。现在已经确定了细胞表型可以通过控制细胞和表面特征之间的初始相互作用来操作, 并且可以在纳米尺度上增强。15具体地说, 扫描探针光刻方法已被证明是一种简便的方法来生产各种 nanofabricated 表面的这种细胞培养实验。16例如, 表面呈现的纳米结构的自组装膜和细胞外基质蛋白模板的 DPN 已被用来研究纳米改性材料在材料诱导分化的潜力干细胞。17
该协议描述了一种改进的原子力显微镜 (AFM) 系统的使用, 它能够实现大面积的 PPL。我们详细介绍了用多力传感器检测力的方法, 作为确定探针表面接触的手段, 以及自动化迭代对准过程的算法。对这些模式的后续功能化与细胞外基质蛋白纤维连接蛋白和人骨髓间充质干细胞 (hMSC) 的培养进行了描述, 作为一种用于细胞培养的人工表面的证明。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FlexAFM mounted on a motorised 5-axis (XYZ&amp;Theta;&amp;Phi;) translation and goniometer stage</td><td>NanoSurf</td><td>P40008</td><td></td></tr><tr><td>AFM control software</td><td>NanoSurf</td><td>C3000</td><td></td></tr><tr><td>Engraving pen</td><td>Sigma-Aldrich</td><td>Z225568</td><td></td></tr><tr><td>Plasma Cleaner</td><td>Harrick plasma</td><td>PDC-32G-2</td><td></td></tr><tr><td>PlasmaFlo</td><td>Harrick plasma</td><td>PDC-FMG-2</td><td></td></tr><tr><td>Economy Dry Oxygen Service Pump</td><td>Harrick plasma</td><td>PDC-OPE-2</td><td></td></tr><tr><td>Tube Rotator</td><td>Stuart</td><td>SB3</td><td></td></tr><tr><td>Vacuum Desiccator</td><td>Thermo Fisher Scientific</td><td>5311-0250</td><td></td></tr><tr><td>Milli-Q Water Purification System</td><td>Merck Millipore</td><td>ZRXQ015WW</td><td></td></tr><tr><td>Modular Humidity Generator</td><td>proUmid</td><td>MHG32</td><td></td></tr><tr><td>Proline Plus Pipette 100-1000 &amp;micro;L</td><td>Sartorius</td><td>728070</td><td></td></tr><tr><td>Silicon masters</td><td>NIL Technology</td><td>custom-made</td><td></td></tr><tr><td>Upright snapshot fluorescence microscope</td><td>Olympus</td><td>BX51</td><td></td></tr><tr><td>Microscope objectives</td><td>Olympus</td><td>10x and 60x UPlan FLN &amp;infin;/-/FN 26.5</td><td></td></tr><tr><td>Upright bright field microscope</td><td>Leica</td><td>DM 2500M</td><td></td></tr><tr><td>Ultrasonicator</td><td>Ultrawave Ltd.</td><td>U95</td><td></td></tr><tr><td>Spreadsheet for recording and intepreting automated alignment results</td><td>Microsoft</td><td>Excel</td><td></td></tr><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-propanol</td><td>Sigma-Aldrich</td><td>34863</td><td>FLAMMABLE</td></tr><tr><td>Microscope Sildes, Clear, Ground</td><td>Thermo Fisher Scientific</td><td>451000</td><td></td></tr><tr><td>(7&amp;ndash;8% vinylmethylsiloxane)-dimethylsiloxane copolymer, trimethylsiloxy-terminated</td><td>Gelest</td><td>VDT-731</td><td></td></tr><tr><td>1,3,5,7-tetramethyl-1,3,5,7-tetravinylcyclotetrasiloxane</td><td>Gelest</td><td>SIT7900.0</td><td></td></tr><tr><td>Platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution</td><td>Sigma-Aldrich</td><td>479527</td><td>HARMFUL, TOXIC</td></tr><tr><td>(25&amp;ndash;35% methylhydrosiloxane)-dimethylsiloxane copolymer, trimethylsiloxane-terminated</td><td>Gelest</td><td>HMS-301</td><td></td></tr><tr><td>Weigh Boat 100 mL</td><td>Scientific Laboratory Supplies</td><td>BALI828</td><td></td></tr><tr><td>Pasteur pipette</td><td>Appleton Woods</td><td>KS230</td><td></td></tr><tr><td>Petri dish</td><td>SARSTEDT</td><td>82.1473</td><td></td></tr><tr><td>Razor blade</td><td>Thermo Fisher Scientific</td><td>ST10-031T</td><td></td></tr><tr><td>Adhesive Carbon Tape</td><td>Agar scientific</td><td>AGG3939</td><td></td></tr><tr><td>16-Mercaptohexadecanoic acid</td><td>Sigma-Aldrich</td><td>448303-1G</td><td>HARMFUL, TOXIC</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>34852</td><td>FLAMMABLE</td></tr><tr><td>Gold coated microscope slide</td><td>Sigma-Aldrich</td><td>643203</td><td>Once opened gold will remain reactive to thiols for at least 1 month</td></tr><tr><td>Thiourea</td><td>Sigma-Aldrich</td><td>T8656</td><td>HARMFUL, TOXIC</td></tr><tr><td>Iron(III) nitrate nonahydrate</td><td>Sigma-Aldrich</td><td>529303</td><td>HARMFUL, TOXIC</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>84415</td><td>HARMFUL, TOXIC</td></tr><tr><td>(11-Mercaptoundecyl)hexa(ethylene glycol)</td><td>Sigma-Aldrich</td><td>675105</td><td>HARMFUL, TOXIC</td></tr><tr><td>Fibronectin from human plasma</td><td>Sigma-Aldrich</td><td>F0895</td><td></td></tr><tr><td>Cobalt(II) nitrate hexahydrate</td><td>Sigma-Aldrich</td><td>203106</td><td>HARMFUL, TOXIC</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>MSCGM Mesenchymal Stem Cell Growth Medium</td><td>Lonza UK</td><td>PT-3001</td><td></td></tr><tr><td>Human Mesenchymal Stem Cells</td><td>Lonza UK</td><td>PT-2501</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>Heraeus Multifuge X1 Centrifuge</td><td>Thermo Fisher Scientific</td><td>75004210</td><td></td></tr><tr><td>CELLSTAR Centrifuge Tubes</td><td>Greiner Bio-One</td><td>188261</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>P/0840/53</td><td>HARMFUL, TOXIC</td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>&quot;Detergent&quot; in manuscript</td></tr><tr><td>VECTASHIELD Antifade Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Rabbit anti-fibronectin antibody</td><td>Abcam</td><td>ab2413</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594</td><td>Thermo Fisher Scientific</td><td>R37117</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A3912</td><td></td></tr><tr><td>12-well plate</td><td>Thermo Fisher Scientific</td><td>10253041</td><td></td></tr><tr><td>T75 tissue culture flask</td><td>Thermo Fisher Scientific</td><td>10790113</td><td></td></tr><tr><td>cantilever</td><td>BudgetSensor</td><td>ContAl-G</td><td></td></tr></tbody></table>

large-area scanning probe nanolithography facilitated by automated alignment and its application to substrate fabrication for cell culture studies

扫描探针显微镜已使创建了各种方法的建设性 ("添加剂") 自上而下制造纳米尺度的特征。从历史上看, 扫描探针光刻的一个主要缺点是单探头系统的固有的低吞吐量。通过使用多个探测器阵列来解决这一问题, 从而提高了光刻吞吐量。为了实现这种并行光刻, 探针阵列与基体表面的精确对准是至关重要的, 因此当光刻模式开始时, 所有探针都能同时与表面接触。该协议描述了聚合物笔光刻在厘米大小的区域内产生纳米尺度特征的利用, 通过使用一种快速、精确和自动对准探针阵列的算法来促进。在这里, 光刻的硫醇的黄金基板显示的特点, 高均匀性的生成。这些模式, 然后功能性与纤连蛋白用于在表面定向细胞形态学研究的背景下使用。

为了检查自动对齐是否成功, 已检查从对齐数据 (在步骤4.8 中的电子表格中) 绘制的图表。如果对齐过程成功, 则两个地块对应于采样阶段沿θ和φ轴倾斜的角度, 显示了一系列上升和下降的数据点。在每个地块中, 两个数据点的线性拟合显示了一个定义良好的相交 "峰值", 表示最大z扩展和实现对齐的相应角度 (图 4A 和 4B)。此过程重复四次 (即每轴两次), 并绘制为一组四坐标。因此, 每对坐标的交集都显示出整体最佳角度 (图 4C)。13在对齐不成功的情况下, 可以观察到, 它们对应的θ和φ角图不提供高质量的线性匹配, 或者不相交 (图 5)。这种失败的对齐通常是由于阵列被不正确地修剪或装入探头持有者 (步骤1.7、1.8 和 2.2)。在这些情况下, 数组被丢弃, 一个新的准备和装载 (步骤1和 2), 并重复对齐过程 (步骤 4)。
成功地对齐和 MHA 后, 用侧向力显微镜对花纹金基片进行成像, 检查是否发生了沉积。对印刷表面进行更大面积的检查时, 还通过基体的光学显微镜对未被沉积的硫醇保护的金进行蚀刻 (图 6和图 7)。但是, 蚀刻图案不能用于进一步的功能化, 只应用于确认一批印刷表面基板的代表性样品的图案。如果蚀刻图案显示与单个钢笔对应的空白区域 (图 8), 则此结果指示探测阵列的生产未成功完成, 并且某些探头已损坏或丢失。探测器的这种不均匀性可能是由于使用了氟涂层已磨损的老主控形状 (步骤 1.3), 导致在阵列与主控形状分离时, 一些探头被撕掉。在这些情况下, 应使用新的主控形状。结果也可能是由于在玻璃衬底和主 (步骤 1.5) 之间存在气泡, 或者在固化后探头阵列与主控形状没有完全分离 (步骤 1.8)。
荧光灯显微图像的纤维连接蛋白功能性表面孵化 hMSCs 也收集 (图 9)。一般情况下, 所有基质在体外培养环境中都是稳定的, 细胞在被隔离的 20 x 20 组特征的情况下附着并适应了印刷图案的形态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56967/56967fig1.jpg"> 图1。聚合物笔光刻的示意图表示,通过探针尖端的水半月板显示分子墨水的传输.(a) 侧面视图和 (B) 聚合物笔阵列的顶部视图表明, 当探头阵列和表面基板完全对准时, 所有探头同时与表面接触, 导致并行光刻。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56967/56967fig2.jpg"> 图2。聚合物笔光刻原理图成立.(A) 扩大了实验装置的侧面视图, 准备好的探头阵列附着在探针支架上, 安装在 AFM 扫描仪上。基板放置在舞台上, 下面是三力传感器的位置。(B) 装配式仪器的表示, 显示 AFM 扫描头相对于样品阶段。(C) 显示力传感器位置的底部视图。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56967/56967fig3.jpg"> 图3。描述对齐过程的光谱学程序的示意图表示法.AFM 扫描仪被设置为在100毫秒内, 在250毫秒的位置进行10µm 的探针, 然后在100毫秒内收缩10µm, 然后在收回的位置为250毫秒举行。然后在整个对准过程中重复该运动。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56967/56967fig4.jpg"> 图4。图示成功对齐的图表.z 位置图对倾斜角度 (A) θ和 (B) φ为一个成功的对齐, 在那里表明测量的实际值和+表明最适合用最小二乘法。(C) φ与θ拟合角度的关系图, 达到最大 z 位置的四点。标记的交点是两个轴的最终最佳倾斜角度。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56967/56967fig5.jpg"> 图5。图示不成功的对齐方式.z 位置图对倾斜角度 (A) θ和 (B) φ为一个不成功的对齐, 在那里指示测量的实际值和+表明最适合用最小二乘法。(C) φ与θ拟合角度的关系图, 达到最大 z 位置的四点。没有明确的最优或相交点, 因此不解决最佳对准角。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56967/56967fig6.jpg"> 图6。说明光学显微学和原子力显微图像的黄金基质, 被排列的 MHA 阵列, 然后蚀刻.(a) 和 (B) 依次放大蚀刻图案的光学显微图像;(C) 是一个单一模式网格的 AFM 地形图像。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56967/56967fig7.jpg"> 图7。MHA 被排列的 PPL 阵列, 然后蚀刻的金色基底的光学显微图像.(a) 和 (B) 依次放大蚀刻图案的光学显微图像, (C) 是一种显示大面积均匀图案的低放大图像。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56967/56967fig8.jpg"> 图8。说明了一种金色基体的光学显微图像, 它的 MHA 不均匀, 然后蚀刻.预定的模式 (显示在嵌入) 是重复网格20点线排列在20行, 每两条线产生的z轴延长1µm (范围从5到-5 µm)。可以看到, 在某些区域, 由于在这些位置中缺少探测器, 因此不会生成任何模式。在仅产生两行点的区域中, 此结果表示探测器存在, 但它与完全运行的探头的高度不相同, 因此仅当阵列扩展到完全z轴距离时才会沉积特征。在这个图像中, 对比度被刻意改变, 以使观察部分印刷区域。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56967/56967fig9.jpg"> 图9。荧光显微图像的 hMSCs 培养的纤维粘连蛋白阵列模板的人.(a) 和 (B) 是显示单个细胞的高放大图像。(C) 在没有附着单元的情况下显示纤连阵列的示例模式, (D) 是在网格排列中培养的单元格的广域图像 (在镶嵌图中也显示了印刷图案的示意图)。细胞染色显示纤维粘连蛋白 (红色), F 肌动蛋白 (绿色) 和细胞核 (蓝色)。<a href="https://cloudflare.jove.com/files/ftp_upload/56967/56967fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies at JoVE.com

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

在这里, 我们提出了一个广域扫描探针光刻的协议, 由探针阵列的迭代对准所启用, 以及利用光刻模式进行细胞表面相互作用研究。

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

large-area-scanning-probe-nanolithography-facilitated-automated

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.6K 次观看.  University of Manchester. 在这里, 我们提出了一个广域扫描探针光刻的协议, 由探针阵列的迭代对准所启用, 以及利用光刻模式进行细胞表面相互作用研究。

视频: Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

化学

Atomically Traceable Nanostructure Fabrication

Reducing the scale of etched nanostructures below the 10 nm range eventually will require an atomic scale understanding of the entire fabrication process being used in order to maintain exquisite control over both feature size and feature density. Here, we demonstrate a method for tracking atomically resolved and controlled structures from initial template definition through final nanostructure metrology, opening up a pathway for top-down atomic control over nanofabrication. Hydrogen depassivation lithography is the first step of the nanoscale fabrication process followed by selective atomic layer deposition of up to 2.8 nm of titania to make a nanoscale etch mask. Contrast with the background is shown, indicating different mechanisms for growth on the desired patterns and on the H passivated background. The patterns are then transferred into the bulk using reactive ion etching to form 20 nm tall nanostructures with linewidths down to ~6 nm. To illustrate the limitations of this process, arrays of holes and lines are fabricated. The various nanofabrication process steps are performed at disparate locations, so process integration is discussed. Related issues are discussed including using fiducial marks for finding nanostructures on a macroscopic sample and protecting the chemically reactive patterned Si(100)-H surface against degradation due to atmospheric exposure.

We report a protocol for combining the atomic metrology of the Scanning Tunneling Microscope for surface patterning with selective Atomic Layer Deposition and Reactive Ion Etching. Using a robust process involving numerous atmospheric exposures and transport, 3D nanostructures with atomic metrology are fabricated.

原子纳米结构可追溯制造

Reducing the scale of etched nanostructures below the 10 nm range eventually will require an atomic scale understanding of the entire fabrication process ...

工程学

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

用畸变校正扫描透射电镜进行单数字纳米电子束光刻

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

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 ...

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

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微机械动态细胞培养平台

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

原子纳米结构可追溯制造

用畸变校正扫描透射电镜进行单数字纳米电子束光刻

无标记技术的单细胞分泌物的时空成像

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

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