Name: fabrication of zero mode waveguides for high concentration single molecule microscopy
Uploaded: 2020-05-12T13:00:00
Description: Watch this Scientific Journal Video about Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy at JoVE.com

这项工作得到了国家卫生研究院授予R01GM080376、R35GM118139和NSF工程机械生物学中心CMMI：15-48571至Y.E.G.的支持，以及国家艾滋病规划署博士前NRSA研究金F30AI114187对R.M.J.的支持。

注：所有步骤都可以在一般实验室空间中完成。
1. 玻璃盖唇清洁
<ol><li>为了提供胶体颗粒蒸发沉积的清洁表面，将 24 x 30 mm 光学玻色硅酸盐玻璃盖片（0.16×0.19 毫米厚度）放置在 coplin 玻璃染色罐的凹槽插入件内进行清洁。 注意：确保盖唇直立且分离良好，以便在清洁过程中所有表面都清晰暴露。</li>
	<li>将足够的丙酮倒入染色罐中，以覆盖盖片、盖上盖子，并在 40 °C 下...

对于胶体自组装（协议第2节），使用乙醇而不是水作为悬浮溶剂加速蒸发过程，使模板在沉积后2~3分钟准备好，而不是像以前的方法48，49那样在1~2小时。这里提出的蒸发沉降协议也比以前的沉降协议简单，需要控制悬浮层49、50、51以上的表面倾斜、温度和空气体积。本协议中使用?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61154">Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy</a>. A figure was updated.
Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61154/61154fig3.jpg" /> 
Figure 3: Representative results from evaporative deposition of colloids. (A) Example of optimal colloid deposition. (B) Example of an acceptable colloid deposition in which conditions were more humid (80% RH) than ideal. Holes in the crystal monolayer are apparent. (C) Example of an acceptable colloid deposition in which conditions were drier (65% RH) than optimal. The monolayer regions are slightly translucent while multilayered areas are white and opaque (perimeter and streaks inward). (D) A colloidal crystal illuminated with white light to highlight the rainbow diffraction from the crystals. (E) AFM image (tapping probe AFM in air) of a monolayer of hexagonally packed polystyrene beads from a successful colloid deposition (scale bar = 10 &#181;m). (F) Expanded AFM image of packed beads (scale bar = 2 &#181;m). <a href="https://www.jove.com/files/ftp_upload/61154/61154fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61154/61154fig3v2.jpg" /> 
Figure 3: Representative results from evaporative deposition of colloids. (A) Example of optimal colloid deposition. (B) Example of an acceptable colloid deposition in which conditions were more humid (80% RH) than ideal. Holes in the crystal monolayer are apparent. (C) Example of an acceptable colloid deposition in which conditions were drier (65% RH) than optimal. The monolayer regions are slightly translucent while multilayered areas are white and opaque (perimeter and streaks inward). (D) A colloidal crystal illuminated with white light to highlight the rainbow diffraction from the crystals. (E) AFM image (tapping probe AFM in air) of a monolayer of hexagonally packed polystyrene beads from a successful colloid deposition (scale bar = 10 &#181;m). (F) Expanded AFM image of packed beads (scale bar = 2 &#181;m). <a href="https://www.jove.com/files/ftp_upload/61154/61154fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61154">Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy</a>. A figure was updated.

Erratum: Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

单分子荧光共振能量转移（smFRET）或单分子荧光相关光谱（FCS）等单分子技术是分子生物物理学的有力工具，能够研究单个生物分子在转录1、2、3、翻译4、5、6等过程中的动态运动、构象和相互作用。对于 smFRET 来说，总内部反射荧光 （TIRF） 显微镜是一种常见方法，因为随着时间的推移，许多系绳分子可能会被跟踪，而 TIR 产生的消散波仅限于与盖唇8相邻的 100 至 200 nm 区域。然而，即使这种对激发体积的限制，感兴趣的荧光仍然需要稀释到pM或nM范围，以检测单分子信号以上的背景荧光9。由于细胞酶的Mi michaelis-Menten常数通常在μM到mM范围10之间，单分子研究中的生化反应通常比细胞中的生化反应慢得多。例如，蛋白质合成发生在大肠杆菌11，12中每秒15-20氨基酸，而大多数蛋白核糖体在smFRET实验中翻译为每秒0.1+1氨基酸每秒13。在蛋白质合成中，停滞核糖体上的晶体结构和smFRET显示，在tRNA-mRNA转移步骤14、15之前，转移RNA（tRNA）在"混合"和"经典"状态之间波动。然而，当迁移GTPase因子（EF-G）的生理浓度存在时，在smFRET6中观察到了混合状态和经典状态之间的不同构象。以与细胞中相似的速度和浓度研究动态分子过程很重要，但仍然是一项技术挑战。
提高荧光基板浓度的策略是使用金属基亚可见波长光圈（称为零模式波引（ZMWs））来生成密闭激发场，选择性地激发孔径内局部化的生物分子（图1）。光圈的直径一般为100~200纳米，深度为100~150纳米。在与井的大小和形状相关的截止波长以上（≈以水为介电介质18的圆形波导直径为 2.3 倍），波导不允许传播模式，因此称为"零模式波导"。然而，一个振荡的电磁场，称为消逝波，呈指数级衰减的强度仍然隧道短距离进入波导18，19。虽然与 TIR 消逝波类似，但 ZMW 消逝波的衰变常数较短，导致波导内的有效激发区域为 10~30 nm。在荧光标记配体的微摩尔浓度下，激发区域内同时存在一个或几个分子。这种对激发体积的限制以及随之而来的背景荧光减少，使单分子在生物学上相关的浓度下能够进行荧光成像。这已应用于许多系统20，包括FCS测量单一蛋白质扩散21，单分子FRET测量低亲和力配体蛋白22和蛋白质-蛋白质相互作用23，光谱电化学测量单分子周转事件24。
ZMW是使用离子束铣削25、26或电子束光刻（EBL）直接对金属层进行图案化而生产的，然后是等离子体蚀刻16、27。这些无面具光刻方法可创建系列波导，通常需要访问专门的纳米制造设施，从而阻止 ZMW 技术的广泛采用。另一种方法，紫外线纳米印花平版印刷升空28，使用石英滑动模具按反ZMW模板到电阻膜像邮票。虽然这种方法更精简，它仍然需要EBL来制造石英模具。本文介绍了一种简单且廉价的模板制造方法的协议，该方法不需要 EBL 或离子束铣削，并且基于纳米球的紧密包装，以形成平版面罩。
纳米球或"自然"光刻学于1982年由德克曼和邓斯缪尔29，30首次提出，使用单分散胶体粒子的自组装，从几十纳米到几十微米31，通过蚀刻和/或材料沉积创建表面图案的模板。二维（2D）或三维（3D）胶体粒子的扩展周期阵列，称为胶体晶体，其特点是散射和衍射32的明亮虹彩。虽然比电子束或光刻技术应用较少，但这种遮盖方法简单、成本低，而且很容易缩小，以创建低于 100 nm 的功能大小。
指导胶体粒子的自组装决定了使用胶体晶体作为表面图案的面具的成功。如果粒子的大小和形状是同质的，胶体粒子可以很容易地用六边形包装自行组装，由热带耗竭33驱动。滴涂后水蒸发是沉积胶体颗粒的有效途径，但其他方法包括浸涂34、旋转涂层35、电泳沉积36和空气-水界面37的巩固。下面提出的协议基于蒸发沉积方法，这是最简单的实现方法。紧密包装的聚苯乙烯珠之间的三角形间歇形成开口，在开口中镀一个牺牲金属，形成柱子（图2 和 补充图1）。在此步骤调整这些柱子的形状和直径之前，珠子的简短退火。删除珠子，在柱子周围沉积最后一层金属层，然后移除柱子。在两个金属沉积步骤进入胶体纳米瘤、移除中间柱子以及用于钝化和系绳的表面化学修改后，ZMW 阵列已准备好用于单分子成像。在制造后对ZMW光学特性的更广泛描述可以在附下的第38条中找到。除了用于金属蒸汽沉积的热蒸发器外，不需要专门的工具。

<table border="0" cellpadding="0" cellspacing="0" style="width:667px;" width="668">
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			<td height="21" style="height:21px;width:215px;">1. Glass Coverslip Cleaning</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Acetone</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">32201</td>
			<td>1 L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Coplin glass staining jar</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">08-817</td>
			<td>Staining jar with 8 grooves and molded glass cover</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Coverslips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">48404-467</td>
			<td>24 mm x 30 mm (No.1&#189;, Rectangular)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">E7023</td>
			<td>1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">KOH</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">30603</td>
			<td>Potassium hydroxide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Petri dishes</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">R80115TS</td>
			<td>100 mm diameter, 15 mm deep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sonicator</td>
			<td style="width:103px;">Branson</td>
			<td style="width:119px;">Z245143</td>
			<td>Tabletop ultrasonic cleaner, 5510</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">2. Evaporative Deposition of Polystyrene Beads</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Clear storage container</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">50-110-8222</td>
			<td>26 x 18 x 15 in.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Desk fan</td>
			<td style="width:103px;">O2Cool</td>
			<td style="width:119px;">FD05001A</td>
			<td>Any small desk (~5 in.) fan will work</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Glass beaker</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">02-555-25B</td>
			<td>250 mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Humidity meter</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">11-661-19</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Microcentrifuge tubes</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">21-402-903</td>
			<td>1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polystyrene microspheres</td>
			<td style="width:103px;">Polysciences</td>
			<td style="width:119px;">18602-15</td>
			<td>1.00 &#181;m diameter, non-functionalized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Triton X-100 deturgent</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td>100 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">3. Bead Annealing for Reducing Pore Size in the Colloidal Crystal Template</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Aluminum plate</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">AA11062RY</td>
			<td>Customized in-house to 14 cm x 14 cm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Ceramic hotplate</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">HP88857100</td>
			<td>13 x 8.2 x 3.8 in.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Temperature controller</td>
			<td style="width:103px;">McMaster-Carr</td>
			<td style="width:119px;">38615K71</td>
			<td>Read temperature with thermocouple probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Thermocouple probe</td>
			<td style="width:103px;">McMaster-Carr</td>
			<td style="width:119px;">9251T93</td>
			<td>Type K, surface probe</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">4/5. Nanofabrication of Zero Mode Waveguides Using the Colloidal Crystal Template</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Aluminum etchant</td>
			<td style="width:103px;">Transene</td>
			<td style="width:119px;">Type A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Aluminum pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMAL40QXHB</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Chloroform</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">288306</td>
			<td>1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Copper etchant</td>
			<td style="width:103px;">Transene</td>
			<td style="width:119px;">49-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Copper pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMCU40QXQA</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Gold pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMAUXX40G</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Lens paper</td>
			<td style="width:103px;">Thorlabs</td>
			<td style="width:119px;">MC-5</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Plasma cleaner</td>
			<td style="width:103px;">Harrick Plasma</td>
			<td style="width:119px;">PDC-32G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Scotch tape</td>
			<td style="width:103px;">Staples</td>
			<td style="width:119px;">MMM119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Thin film deposition system</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">PVD-75</td>
			<td>Tabletop thermal evaporation system will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Titanium pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMTI45QXQA</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Toluene</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">244511</td>
			<td>1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Representative Results</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">COMSOL Multiphysics Modeling Software</td>
			<td>COMSOL, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dual View spectral splitter</td>
			<td style="width:103px;">Photometrics, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of zero mode waveguides for high concentration single molecule microscopy

在单分子荧光酶学中，溶液中标记基材的背景荧光通常将荧光浓度限制在皮科至纳米摩尔范围内，其数量级小于许多生理配体浓度。称为零模式波导 （ZMWs） 的光学纳米结构，直径为 100 至 200 nm，由铝或金等薄导金属制造，通过将可见光激发限制在沸点有效体积中，从而允许在氟叶微摩尔浓度下对单个分子进行成像。然而，对昂贵和专门的纳米制造设备的需求已经排除了ZMW的广泛使用。 通常，ZMW等纳米结构是通过直接书写获得的，电子束光刻是连续的和缓慢的。在这里，胶体或纳米球，光刻学被用作替代策略，以创建纳米级的面罩，用于波导制造。本报告详细描述了该方法，并考虑到每个阶段的实际情况。该方法允许数千个铝或金ZMW并行制造，最终波导直径和深度为100-200纳米。只需要普通的实验室设备和用于金属沉积的热蒸发器。通过使ZMW更容易进入生化社区，这种方法可以促进细胞浓度和速率分子过程的研究。

通过蒸发沉积（步骤 2.1+2.13）自组装聚苯乙烯胶体颗粒可以产生一系列结果，因为它需要控制溶剂蒸发率。但是，由于沉积速度很快（每轮 10-15 分钟），因此可以针对不同的环境实验室条件快速优化该过程。 图3A 显示沉积和蒸发后形成良好的胶体模板。从宏观上讲，珠子区域是圆形的，边界由不透明的多层珠环定义。图像中的半透明区域，但不是白色区域是所需的单层区?...

Watch this Scientific Journal Video about Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy at JoVE.com

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

这里描述的是一种纳米球平板成像方法，用于平行制造零模式波导，这是金属包玻璃显微镜覆盖带中的纳米阵列，用于在纳米到微摩尔浓度的荧光素下进行单分子成像。该方法利用胶体晶体自组装创建波导模板。

为高浓度单分子显微镜制造零模式波导

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

Research

JoVE Journal

Bioengineering

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

科研

生物工程

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

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 ...

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

制备碳纳米管高频纳电子生物传感器的传感高离子强度的解决方案

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

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

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

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

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

单分子荧光显微镜在平面支持双层膜

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

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

磁共振显微镜下切除活体脑组织的代谢支持

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...