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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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><tbody><tr><td>&lt;strong&gt;1. Glass Coverslip Cleaning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>32201</td><td>1 L</td></tr><tr><td>Coplin glass staining jar</td><td>Fisher Scientific</td><td>08-817</td><td>Staining jar with 8 grooves and molded glass cover</td></tr><tr><td>Coverslips</td><td>VWR</td><td>48404-467</td><td>24 mm x 30 mm (No.1&amp;frac12;, Rectangular)</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>E7023</td><td>1 L</td></tr><tr><td>KOH</td><td>Sigma</td><td>30603</td><td>Potassium hydroxide</td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>R80115TS</td><td>100 mm diameter, 15 mm deep</td></tr><tr><td>Sonicator</td><td>Branson</td><td>Z245143</td><td>Tabletop ultrasonic cleaner, 5510</td></tr><tr><td>&lt;strong&gt;2. Evaporative Deposition of Polystyrene Beads&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Clear storage container</td><td>Fisher Scientific</td><td>50-110-8222</td><td>26 x 18 x 15 in.</td></tr><tr><td>Desk fan</td><td>O2Cool</td><td>FD05001A</td><td>Any small desk (~5 in.) fan will work</td></tr><tr><td>Glass beaker</td><td>Fisher Scientific</td><td>02-555-25B</td><td>250 mL</td></tr><tr><td>Humidity meter</td><td>Fisher Scientific</td><td>11-661-19</td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>Fisher Scientific</td><td>21-402-903</td><td>1.5 mL</td></tr><tr><td>Polystyrene microspheres</td><td>Polysciences</td><td>18602-15</td><td>1.00 &amp;micro;m diameter, non-functionalized</td></tr><tr><td>Triton X-100 deturgent</td><td>Sigma</td><td>X100</td><td>100 mL</td></tr><tr><td>&lt;strong&gt;3. Bead Annealing for Reducing Pore Size in the Colloidal Crystal Template&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aluminum plate</td><td>Fisher Scientific</td><td>AA11062RY</td><td>Customized in-house to 14 cm x 14 cm</td></tr><tr><td>Ceramic hotplate</td><td>Fisher Scientific</td><td>HP88857100</td><td>13 x 8.2 x 3.8 in.</td></tr><tr><td>Temperature controller</td><td>McMaster-Carr</td><td>38615K71</td><td>Read temperature with thermocouple probe</td></tr><tr><td>Thermocouple probe</td><td>McMaster-Carr</td><td>9251T93</td><td>Type K, surface probe</td></tr><tr><td>&lt;strong&gt;4/5. Nanofabrication of Zero Mode Waveguides Using the Colloidal Crystal Template&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aluminum etchant</td><td>Transene</td><td>Type A</td><td></td></tr><tr><td>Aluminum pellets</td><td>Kurt J. Lesker</td><td>EVMAL40QXHB</td><td>For electron beam evaporation</td></tr><tr><td>Chloroform</td><td>Sigma</td><td>288306</td><td>1 L</td></tr><tr><td>Copper etchant</td><td>Transene</td><td>49-1</td><td></td></tr><tr><td>Copper pellets</td><td>Kurt J. Lesker</td><td>EVMCU40QXQA</td><td>For electron beam evaporation</td></tr><tr><td>Gold pellets</td><td>Kurt J. Lesker</td><td>EVMAUXX40G</td><td>For electron beam evaporation</td></tr><tr><td>Lens paper</td><td>Thorlabs</td><td>MC-5</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Scotch tape</td><td>Staples</td><td>MMM119</td><td></td></tr><tr><td>Thin film deposition system</td><td>Kurt J. Lesker</td><td>PVD-75</td><td>Tabletop thermal evaporation system will also work</td></tr><tr><td>Titanium pellets</td><td>Kurt J. Lesker</td><td>EVMTI45QXQA</td><td>For electron beam evaporation</td></tr><tr><td>Toluene</td><td>Sigma</td><td>244511</td><td>1 L</td></tr><tr><td>&lt;strong&gt;Representative Results&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>COMSOL Multiphysics Modeling Software</td><td>COMSOL, Inc.</td><td></td><td></td></tr><tr><td>Dual View spectral splitter</td><td>Photometrics, Inc.</td><td></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

Universidad de Cartagena UDEC

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

Fabrication of Silica Ultra High Quality Factor Microresonators

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the quality factor (Q) of the cavity, can be in excess of 500ns for cavities with Q factors above 100 million. As a result of their low material losses, silica microcavities have demonstrated some of the longest photon lifetimes to date1-2. Since a portion of the circulating light extends outside the resonator, these devices can also be used to probe the surroundings. This interaction has enabled numerous experiments in biology, such as single molecule biodetection and antibody-antigen kinetics, as well as discoveries in other fields, such as development of ultra-low-threshold microlasers, characterization of thin films, and cavity quantum electrodynamics studies.3-7
The two primary silica resonant cavity geometries are the microsphere and the microtoroid. Both devices rely on a carbon dioxide laser reflow step to achieve their ultra-high-Q factors (Q&gt;100 million).1-2,8-9 However, there are several notable differences between the two structures. Silica microspheres are free-standing, supported by a single optical fiber, whereas silica microtoroids can be fabricated on a silicon wafer in large arrays using a combination of lithography and etching steps. These differences influence which device is optimal for a given experiment.
Here, we present detailed fabrication protocols for both types of resonant cavities. While the fabrication of microsphere resonant cavities is fairly straightforward, the fabrication of microtoroid resonant cavities requires additional specialized equipment and facilities (cleanroom). Therefore, this additional requirement may also influence which device is selected for a given experiment.
Introduction
An optical resonator efficiently confines light at specific wavelengths, known as the resonant wavelengths of the device. 1-2 The common figure of merit for these optical resonators is the quality factor or Q. This term describes the photon lifetime (&tau;o) within the resonator, which is directly related to the resonator's optical losses. Therefore, an optical resonator with a high Q factor has low optical losses, long photon lifetimes, and very low photon decay rates (1/&tau;o). As a result of the long photon lifetimes, it is possible to build-up extremely large circulating optical field intensities in these devices. This very unique property has allowed these devices to be used as laser sources and integrated biosensors.10
A unique sub-class of resonators is the whispering gallery mode optical microcavity. In these devices, the light is confined in circular orbits at the periphery. Therefore, the field is not completely confined within the device, but evanesces into the environment. Whispering gallery mode optical cavities have demonstrated some of the highest quality factors of any optical resonant cavity to date.9,11 Therefore, these devices are used throughout science and engineering, including in fundamental physics studies and in telecommunications as well as in biodetection experiments. 3-7,12
Optical microcavities can be fabricated from a wide range of materials and in a wide variety of geometries. A few examples include silica and silicon microtoroids, silicon, silicon nitride, and silica microdisks, micropillars, and silica and polymer microrings.13-17 The range in quality factor (Q) varies as dramatically as the geometry. Although both geometry and high Q are important considerations in any field, in many applications, there is far greater leverage in boosting device performance through Q enhancement. Among the numerous options detailed previously, the silica microsphere and the silica microtoroid resonator have achieved some of the highest Q factors to date.1,9 Additionally, as a result of the extremely low optical loss of silica from the visible through the near-IR, both microspheres and microtoroids are able to maintain their Q factors over a wide range of testing wavelengths.18 Finally, because silica is inherently biocompatible, it is routinely used in biodetection experiments.
In addition to high material absorption, there are several other potential loss mechanisms, including surface roughness, radiation loss, and contamination loss.2 Through an optimization of the device size, it is possible to eliminate radiation losses, which arise from poor optical field confinement within the device. Similarly, by storing a device in an appropriately clean environment, contamination of the surface can be minimized. Therefore, in addition to material loss, surface scattering is the primary loss mechanism of concern.2,8
In silica devices, surface scattering is minimized by using a laser reflow technique, which melts the silica through surface tension induced reflow. While spherical optical resonators have been studied for many years, it is only with recent advances in fabrication technologies that researchers been able to fabricate high quality silica optical toroidal microresonators (Q&gt;100 million) on a silicon substrate, thus paving the way for integration with microfluidics.1
The present series of protocols details how to fabricate both silica microsphere and microtoroid resonant cavities. While silica microsphere resonant cavities are well-established, microtoroid resonant cavities were only recently invented.1 As many of the fundamental methods used to fabricate the microsphere are also used in the more complex microtoroid fabrication procedure, by including both in a single protocol it will enable researchers to more easily trouble-shoot their experiments.

We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids. The reflow method removes surface imperfections, allowing long photon lifetimes within both devices. The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to biodetection.

硅胶超高品质因子微谐振器的制造

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the ...

科研

工程学

Terahertz Microfluidic Sensing Using a Parallel-plate Waveguide Sensor

Refractive index (RI) sensing is a powerful noninvasive and label-free sensing technique for the identification, detection and monitoring of microfluidic samples with a wide range of possible sensor designs such as interferometers and resonators 1,2. Most of the existing RI sensing applications focus on biological materials in aqueous solutions in visible and IR frequencies, such as DNA hybridization and genome sequencing. At terahertz frequencies, applications include quality control, monitoring of industrial processes and sensing and detection applications involving nonpolar materials.
Several potential designs for refractive index sensors in the terahertz regime exist, including photonic crystal waveguides 3, asymmetric split-ring resonators 4, and photonic band gap structures integrated into parallel-plate waveguides 5. Many of these designs are based on optical resonators such as rings or cavities. The resonant frequencies of these structures are dependent on the refractive index of the material in or around the resonator. By monitoring the shifts in resonant frequency the refractive index of a sample can be accurately measured and this in turn can be used to identify a material, monitor contamination or dilution, etc.
The sensor design we use here is based on a simple parallel-plate waveguide 6,7. A rectangular groove machined into one face acts as a resonant cavity (Figures 1 and 2). When terahertz radiation is coupled into the waveguide and propagates in the lowest-order transverse-electric (TE1) mode, the result is a single strong resonant feature with a tunable resonant frequency that is dependent on the geometry of the groove 6,8. This groove can be filled with nonpolar liquid microfluidic samples which cause a shift in the observed resonant frequency that depends on the amount of liquid in the groove and its refractive index 9.
Our technique has an advantage over other terahertz techniques in its simplicity, both in fabrication and implementation, since the procedure can be accomplished with standard laboratory equipment without the need for a clean room or any special fabrication or experimental techniques. It can also be easily expanded to multichannel operation by the incorporation of multiple grooves 10. In this video we will describe our complete experimental procedure, from the design of the sensor to the data analysis and determination of the sample refractive index.

The procedure for implementing a refractive index sensor for terahertz frequencies based on a grooved parallel-plate waveguide geometry is described here. The method yields a measurement of the refractive index of a small volume of liquid through monitoring of the shift in the resonant frequency of the waveguide structure

太赫兹微流控测量中的一个平行板波导传感器

Refractive index (RI) sensing is a powerful noninvasive and label-free sensing technique for the identification, detection and monitoring of microfluidic ...

Fabrication And Characterization Of Photonic Crystal Slow Light Waveguides And Cavities

Slow light has been one of the hot topics in the photonics community in the past decade, generating great interest both from a fundamental point of view and for its considerable potential for practical applications. Slow light photonic crystal waveguides, in particular, have played a major part and have been successfully employed for delaying optical signals1-4 and the enhancement of both linear5-7 and nonlinear devices.8-11
Photonic crystal cavities achieve similar effects to that of slow light waveguides, but over a reduced band-width. These cavities offer high Q-factor/volume ratio, for the realization of optically12 and electrically13 pumped ultra-low threshold lasers and the enhancement of nonlinear effects.14-16 Furthermore, passive filters17 and modulators18-19 have been demonstrated, exhibiting ultra-narrow line-width, high free-spectral range and record values of low energy consumption.
To attain these exciting results, a robust repeatable fabrication protocol must be developed. In this paper we take an in-depth look at our fabrication protocol which employs electron-beam lithography for the definition of photonic crystal patterns and uses wet and dry etching techniques. Our optimised fabrication recipe results in photonic crystals that do not suffer from vertical asymmetry and exhibit very good edge-wall roughness. We discuss the results of varying the etching parameters and the detrimental effects that they can have on a device, leading to a diagnostic route that can be taken to identify and eliminate similar issues.
The key to evaluating slow light waveguides is the passive characterization of transmission and group index spectra. Various methods have been reported, most notably resolving the Fabry-Perot fringes of the transmission spectrum20-21 and interferometric techniques.22-25 Here, we describe a direct, broadband measurement technique combining spectral interferometry with Fourier transform analysis.26 Our method stands out for its simplicity and power, as we can characterise a bare photonic crystal with access waveguides, without need for on-chip interference components, and the setup only consists of a Mach-Zehnder interferometer, with no need for moving parts and delay scans.
When characterising photonic crystal cavities, techniques involving internal sources21 or external waveguides directly coupled to the cavity27 impact on the performance of the cavity itself, thereby distorting the measurement. Here, we describe a novel and non-intrusive technique that makes use of a cross-polarised probe beam and is known as resonant scattering (RS), where the probe is coupled out-of plane into the cavity through an objective. The technique was first demonstrated by McCutcheon et al.28 and further developed by Galli et al.29

Use of photonic crystal slow light waveguides and cavities has been widely adopted by the photonics community in many differing applications. Therefore fabrication and characterization of these devices are of great interest. This paper outlines our fabrication technique and two optical characterization methods, namely: interferometric (waveguides) and resonant scattering (cavities).

光子晶体慢光波导和腔体的制备与表征

Slow light has been one of the hot topics in the photonics community in the past decade, generating great interest both from a fundamental point of view ...

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

折光传感荧光芯微腔的合成和操作

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

制造，操作和表面声波驱动声逆流微流体流动可视化

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

Implementation of a Reference Interferometer for Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing its free spectral range (FSR), the module is put in parallel with a fiber taper-microcavity system and then calibrated through isolating and eliminating random shifts in the laser frequency (i.e. laser jitter noise). To realize the taper-microcavity junction and to maximize the optical power that is transferred to the resonator, a single-mode optical fiber waveguide is pulled. Solutions containing polystyrene nanobeads are then prepared and flown to the microcavity in order to demonstrate the system&#8217;s ability to sense binding to the surface of the microcavity. Data is post-processed via adaptive curve fitting, which allows for high-resolution measurements of the quality factor as well as the plotting of time-dependent parameters, such as resonant wavelength and split frequency shifts. By carefully inspecting steps in the time-domain response and shifting in the frequency-domain response, this instrument can quantify discrete binding events.

A reference interferometer technique, which is designed to remove undesirable laser jitter noise for nanodetection, is utilized for probing an ultra-high quality factor microcavity. Instructions for assembly, setup, and data acquisition are provided, alongside the measurement process for specifying the cavity quality factor.

一个参考干涉仪的Nanodetection实施

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing ...

Stimulated Stokes and Antistokes Raman Scattering in Microspherical Whispering Gallery Mode Resonators

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres can be thought as a store of energy with a huge variety of applications: compact laser sources, highly sensitive biochemical sensors and nonlinear phenomena. A protocol for the fabrication of both the microspheres and coupling system is given. The couplers described here are tapered fibers. Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS). A proof of the cavity-enhanced phenomenon is given by the lack of correlation among the pump, signal and idler: a resonant mode has to exist in order to obtain the pair of signal and idler. In the case of hyperparametric oscillations (four wave mixing and stimulated anti-stokes Raman scattering), the modes must fulfill the energy and momentum conservation and, last but not least, have a good spatial overlap.

Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS).

刺激斯托克斯和反斯托克斯拉曼散射在微球形回音壁模式谐振器

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres ...

Fabrication of 1-D Photonic Crystal Cavity on a Nanofiber Using Femtosecond Laser-induced Ablation

We present a protocol for fabricating 1-D Photonic Crystal (PhC) cavities on subwavelength-diameter tapered optical fibers, optical nanofibers, using femtosecond laser-induced ablation. We show that thousands of periodic nano-craters are fabricated on an optical nanofiber by irradiating with just a single femtosecond laser pulse. For a typical sample, periodic nano-craters with a period of 350 nm and with diameter gradually varying from 50 - 250 nm over a length of 1 mm are fabricated on a nanofiber with diameter around 450 - 550 nm. A key aspect of such a nanofabrication is that the nanofiber itself acts as a cylindrical lens and focuses the femtosecond laser beam on its shadow surface. Moreover, the single-shot fabrication makes it immune to mechanical instabilities and other fabrication imperfections. Such periodic nano-craters on nanofiber, act as a 1-D PhC and enable strong and broadband reflection while maintaining the high transmission out of the stopband. We also present a method to control the profile of the nano-crater array to fabricate apodized and defect-induced PhC cavities on the nanofiber. The strong confinement of the field, both transverse and longitudinal, in the nanofiber-based PhC cavities and the efficient integration to the fiber networks, may open new possibilities for nanophotonic applications and quantum information science.

We present a protocol for fabricating 1-D photonic crystal cavities on subwavelength diameter silica fibers (optical nanofibers) using femtosecond laser-induced ablation.

1-D光子晶体空穴对纳米纤维使用飞秒激光诱导消融制造

We present a protocol for fabricating 1-D Photonic Crystal (PhC) cavities on subwavelength-diameter tapered optical fibers, optical nanofibers, using ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

生物化学

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

生物医学微流体折射率匹配器件的研制

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...