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 下声波 10 分钟。</li>
	<li>倒出丙酮，冲洗盖片，用蒸馏的H2O填充染色罐，倒出水。再重复 2 次。</li>
	<li>再次重复丙酮声波（步骤 1.2 和 1.3）。</li>
	<li>将足够的 200 mM KOH 倒入罐中，覆盖覆盖的覆盖唇和声波，在 40 °C 下浇灌 20 分钟。 注:KOH 稍微蚀刻玻璃。</li>
	<li>用蒸馏的 H2O 冲洗盖片 6 次。</li>
	<li>添加乙醇覆盖盖片，添加盖子，并在40°C下声波10分钟。</li>
	<li>用蒸馏的 H2O 冲洗盖片 3 次。</li>
	<li>使用柔和的钳子在边缘拾取每个盖片，然后用 N2 气体干燥盖唇。只触摸盖片的边缘。将每个干燥、清洁的盖片放在单独清洁的 Petri 盘中。</li>
</ol>2. 聚苯乙烯珠的蒸发沉积
<ol><li>要为 ZMW 阵列创建胶体晶体面膜，离心机 50 μL 直径为 1 μm，非功能化聚苯乙烯珠（水中为 2.5% w/v），15，000 x g，25 °C 5 分钟。 注:在管道珠子之前，应短暂地将库存溶液旋涡，以防珠子沉入瓶底。</li>
	<li>丢弃超高超，留下尽可能少的水。 注:残留水可以改变乙醇再悬念39的蒸发特性，因此去除少量珠子以去除所有水是可以接受的。</li>
	<li>从步骤 2.2 中重新使用 2.2 步骤中的珠子，50 μL 为 1:400 TritonX-100:乙醇溶剂。派佩特上下几次，彻底混合珠子与溶剂。 注:TritonX-100:乙醇溶剂使用后应用石蜡薄膜密封，每月准备一次新鲜。珠子倾向于粘附在塑料容器的两侧，如微中心管，因此移液器沿两侧，以确保所有珠子被重新使用。</li>
	<li>要设置一个湿度室进行沉积，将6个Petri菜肴（每个菜都有一个盖片）放在长凳上，内侧的盖子稍稍偏开。在每个菜中，将盖唇移到开放区域，以便在下一步增加湿度时，盖唇暴露在环境中。</li>
	<li>放置一个卫生仪和一个小电风扇，以培养皿为中心。</li>
	<li>记录实验室中的起始相对湿度 （RH）。将 200 mL 烧饼装满 150 至 200 mL 的 +75 °C 水，并放在风扇后面。</li>
	<li>打开风扇，用翻倒的透明塑料存储容器（66 厘米 x 46 厘米 x 38 厘米）盖住 Petri 菜肴、风扇、烧甲和卫生表。</li>
	<li>让腔室中的 RH 上升到 70+75%，这通常需要 5~10 分钟。 注意:如果环境实验室 RH 较低（低于 +50%），请让腔室达到较高的 RH，但不超过 80%，以补偿沉积过程中的湿度损失（见下文）。</li>
	<li>当 RH 达到 70+75% 时，记录 RH 并稍微抬起塑料存储容器，以快速将盖子放在 Petri 盘上，从而防止盖片过度湿润。 注:由于加湿，室内温度将略高于室温，通常为 25°26 °C。如果在表盖上可见水分，则玻璃表面太湿。商业手套箱可能会简化协议的这一部分。</li>
	<li>让室内的 RH 继续上升到 85% 。此时，在湿度室中记录 RH，将珠子悬架的移液器 5 μL 记录到每个盖片的中心。</li>
	<li>每次沉积后关闭房间和培养皿，以尽量减少湿度损失。目标是在2分钟内完成所有6个证词。</li>
	<li>证词后在腔室中记录 RH。 注:沉积后的RH将有助于测量沉积过程中湿度损失的速度，这取决于环境实验室条件。对于典型的成功运行，腔室将在沉积前以 85% RH 开始，在沉积后以 70+75% RH 结束。</li>
	<li>让珠子飞沫扩散干燥5分钟。 注意:如果胶体晶体有许多孔或多层区域，则腔室可能分别太潮湿或干燥。调整相对湿度，以关闭培养皿并开始沉积（参见结果部分，以进一步讨论优化）。</li>
</ol>3. 用于减少胶体晶体模板孔隙大小的珠子退火
<ol><li>为降低聚苯乙烯珠的退火温度，缩小珠间插座，并圆闭盘间角落，在标准陶瓷热板顶部放置一个扁平的磨铝板。</li>
	<li>将热板温度设定为107°C，聚苯乙烯玻璃过渡温度为40°C。 注:为了获得稳定和准确的温度，在铝板的2~3毫米宽和4~5毫米深的孔中举行了热电偶探针。</li>
	<li>将包含珠子模板的盖片放在热铝板上，退火 20s（请参阅讨论部分，解释熔化时间）。</li>
	<li>加热后，从铝板上取下盖片，并及时将其放置在另一室温铝表面冷却。 注:将盖片稍微挂在板的边缘或将浅通道（参见所附视频）插入板中，以方便取回盖唇，这很有帮助。</li>
</ol>4. 使用胶体晶体模板对铝零模式波导进行纳米制造
<ol><li>利用热束或电子束蒸发沉积，在胶体晶体模板上以2~s沉积300纳米铜，在珠子之间的隔板中产生柱子。</li>
	<li>用胶带轻轻按压表面，去除珠子顶部多余的金属。慢慢剥去胶带，把金属拉下来。 注意:胶带拉动后，可能会保留一些微小的反射多余金属斑块，这些斑块通常可以通过 N2 气体流去除。如果胶带拉动后仍保留大量反射多余的金属，请尝试将模板浸泡在甲苯中 2 小时，以部分溶解聚苯乙烯珠。用蒸馏水清洗盖唇，用N2干燥，并重复胶带拉。额外的浸泡不应完全溶解珠子，因为珠子有助于保护柱子在胶带拉扯过程中免受损坏。</li>
	<li>要溶解聚苯乙烯珠，将珠子模板放入甲苯中，浸泡过夜。 警告:甲苯烟雾可能是有毒的。在通风良好的罩下使用甲苯，并佩戴个人防护设备，包括手套、安全眼镜和实验室外套。甲苯应储存在指定用于易燃液体的通风柜中。</li>
	<li>甲苯孵化后，用氯仿冲洗模板一次，用乙醇冲洗两次。此时小心处理盖片，因为精致的 200-300 nm 高金属柱现在已露出来。用 N2 干燥模板，在氧气等离子体清洁剂中去除残留聚合物和污染物 30 分钟。 警告:氯仿烟雾可能有毒。在通风良好的罩下使用氯仿，并佩戴个人防护设备，包括手套、安全眼镜和实验室外套。氯仿应储存在通风柜中，远离其他易燃溶剂。</li>
	<li>使用热束或电子束蒸发沉积，将钛粘附层的 3 纳米沉积在 1 +/s，然后以 4 +/s 左右和铜柱顶部的 100×150 nm 铝沉积。 注:人们可以使用更厚的包层来获得更深的指导和更好的背景荧光衰减，但这也会降低下一步暴露和溶解帖子后的产量（参见讨论部分）。</li>
	<li>要溶解金属柱，将盖片浸泡在铜等（柠檬酸基: 材料表）2 小时。 警告:金属等损伤可导致皮肤灼伤。在通风良好的罩下处理等，并佩戴防护设备。处理后彻底洗手。金属等应储存在指定用于腐蚀性液体的通风柜中。</li>
	<li>用蒸馏水冲洗盖片，用N2干燥，用透镜纸轻轻擦去金属包层的表面，露出任何仍然覆盖在包层中的柱子。将盖片放回铜等，再放2小时，然后用蒸馏水再次冲洗，用N2干燥。 注意:ZMW 滑梯应存储在盖有盖、干净的培养皿中，以使其不含污染物。</li>
</ol>5. 使用胶体晶体模板对金零模式波导进行纳米制造
注:本节提供了制造黄金ZMW的方法（补充图1），它反映了制造铝ZMW的协议。
<ol><li>使用热束或电子束蒸发沉积，将 3 纳米的钛粘附层沉积在 1 é/s，然后以 4 é/s 沉积 300 nm 铝。</li>
	<li>用胶带轻轻按压表面，去除珠子顶部多余的金属。慢慢剥去胶带，把金属拉下来。</li>
	<li>要溶解聚苯乙烯珠，将珠子模板放入甲苯中，浸泡过夜。</li>
	<li>甲苯孵化后，用氯仿冲洗模板一次，用乙醇冲洗两次。用 N2 干燥模板，在氧气等离子体清洁剂中去除残留聚合物污染物 30 分钟。</li>
	<li>使用热束或电子束蒸发沉积，在铝柱周围和顶部以 5 +/s 沉积 100×150 nm 的黄金。</li>
	<li>要溶解金属柱，将盖片浸泡在铝等（磷酸基:磷酸基:磷酸基）中浸泡。 材料表）1 小时。</li>
	<li>用蒸馏水冲洗盖片，用N2干燥，用透镜纸轻轻擦去金属包层的表面，露出任何仍然覆盖在包层中的柱子。将盖片放回铝等1小时，然后用蒸馏水再次冲洗，用N2干燥。 注:ZMW 幻灯片应存储在盖有盖、干净的培养皿中。</li>
</ol>

作者没有什么可透露的。

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。

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			<td height="21" style="height:21px;width:215px;">1. Glass Coverslip Cleaning</td>
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			<td height="21" style="height:21px;width:215px;">Acetone</td>
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			<td style="width:119px;">32201</td>
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		</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 ...

fabrication-zero-mode-waveguides-for-high-concentration-single

Research

JoVE Journal

Bioengineering

7.9K 次观看.  University of Pennsylvania. 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。

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

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

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-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

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 ...

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

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

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 in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

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

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

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

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

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., ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

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 in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

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

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

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 ...