这个项目是由土地巴登 - 符腾堡州，以及欧洲联盟，的Europäischer全宗资助献给死去大区ENTWICKLUNG。作者感谢赖维蒂希（ILM乌尔姆）提供的U251-MG-L106-Grx1-roGFP2细胞系和克劳迪娅·欣策的熟练的技术援助。

1，细胞球体生长，育成<ol><li>实验1：细胞球体培养与化疗药物<ol><li>通过添加0.45克琼脂糖至30ml培养基中（足够用于6孔板）制备琼脂糖1.5％的培养基。 </li><li>加热该混合物从步骤1.1.1至至少80℃，同时从时间搅拌时间。 </li><li>补加入50μl从步骤1.1.2加热混合物到96孔板的每个孔中，并让在1-2小时之固化。储存关闭和覆盖在冰箱中于5℃下直到使用的板。 </li><li>种子约150 MCF-7乳腺癌细胞中所制备的96孔板，在Dulbecco改良的Eagle培养基（DMEM）和火腿的F-12培养基中补充有10％胎牛血清（FCS）和1％青霉素/链霉素的每个孔。 </li><li>在5％CO 2和37在培养箱中生长球状5-7天达到直径约为200-300微米76℃。 </li><li>孵育的细胞球粒用蒽环类抗生素多柔比星盐酸盐为6小时，其浓度在5％CO 2和37℃下2微米和8微米（在培养基中）在培养箱之间的范围内。 </li><li>洗细胞球状体与培养基或Earle氏平衡盐溶液（EBSS）于显微镜之前。 </li></ol></li><li>实验2：在球体表达氧化还原传感器的氧化过程<ol><li>种子约96孔板中在DMEM 400 U251-MG-L106成胶质细胞瘤细胞中永久地转染了谷胱甘肽敏感的绿色荧光的氧化还原传感器Grx1-roGFP2在琼脂糖凝胶包被的孔（在步骤中描述的步骤1.1.1-1.1.3）培养基补充有10％FCS，1％青霉素/链霉素和潮霉素B（150微克/毫升）。 </li><li>在5％CO 2和37℃下生长的球体5-7天达到直径约为200-300微米的培养箱。 </li><li>添加10＃181;将在12小时用升的过氧化氢溶液（purum PA，≥30％），以990微升双蒸水来创建100mM的储备液。 </li><li>在实验前才从步骤稀释原液1.2.3到50μm的EBSS。 </li><li>测量与过氧化氢50μm的EBSS为氧化还原传感器的氧化过程中孵育通过微流体系统的细胞球状体。 </li></ol></li><li>实验3：启动和球体内部细胞坏死的标签<ol><li> 96孔板的DMEM培养基中补充有种子约400个细胞（ 如 U251-MG-L106-Grx1-roGFP2胶质母细胞瘤细 ​​胞），在琼脂糖凝胶包被的孔（在步骤中描述的步骤1.1.1-1.1.3） 10％FCS，1％青霉素/链霉素和潮霉素B（150毫克/毫升）。 </li><li>在5％CO 2和37℃下生长的球体5-7天达到直径约为200-300微米的培养箱。 </li><li>孵育细胞球体与在1μM的在培养基中的浓度的线粒体电子传递抑制剂鱼藤酮3小时以诱导细胞坏死。另外，以1微升的1毫升培养基中的浓度共同孵育带有绿色的细胞毒性染料3小时以标记坏死细胞。 </li><li>用培养基或EBSS之前镜检洗细胞球体。 </li></ol></li></ol> 2，光表调整注意：为了达到最好的结果是，必须确保该光板的光束腰是在物镜的聚焦平面上。束腰的位置可以仅通过观察光片在垂直位置相对于毛细管对齐（参见步骤2.5 - 2.8）。 <ol><li>用倒置显微镜和安装SPIM激励模块，在参考文献中描述。 16，在定位表的底板（ 见图1A）。 </li><li>装备显微镜用10X / 0.3或20X / 0.5的显微镜物镜和一个适当的长通滤波器（ 例如，λ≥515纳米），在显微镜的检测路径。 </li><li>安装积分照相机的显微镜的检测端口。 </li><li>使用激光或激光二极管的并联的准直光束用的优选470nm处的激发波长和其应用到SPIM模块。 </li><li>通过90度时，其中光片传送到用于光片的光束腰的轴向调整垂直位置旋转的圆柱形透镜。 </li><li>将包含在样品架荧光染料液体的毛细管。 </li><li>附加的样本保持器与毛细管到显微镜的定位表和对准毛细管，使得它的中心和光板的光腰是在焦点的位置。 </li><li>通过柱面透镜本身的轴向位置的变化调整束腰。回头的广告后，镜头校正配料。 </li></ol> 3，细胞球体的应用和显微镜饲料同步<ol><li>单独或在长方形的硼硅玻璃毛细管16内组将细胞球粒用的600微米×600微米的内横截面和120微米的壁厚。两种技术的细胞球状体的应用已经被证明是成功的。 <ol><li>以空毛细管直立用拇指和中指，然后封好上口与你的食指。使下部开口附近的细胞球状体在其周围的液体（EBSS或培养基）。从上部开口松开食指。液体，在它的细胞球体将被毛细力立刻浸泡。调整填充的毛细管内的旋转椭圆体通过重力在毛细管的各直立位置的位置。 </li><li>备选地，通过移液应用细胞球体到毛细管。小心塔吨移液器的尖端的开口，一方面，对于球体的尺寸足够大，并且在另一方面，小于或等于在尺寸上与毛细管的内径。 </li></ol></li><li>将毛细管与球体它在显微镜特殊的样品架。 </li><li>附加的样本保持器与毛细管到显微镜的定位表和对齐细胞球状体，使得它的中心和聚焦的位置。 </li><li>集光片和显微镜物镜，在检测路径中，通过调整反射镜的位置和柱面透镜的聚焦平面之间的匹配（参见图1B中的调整螺钉）。 </li><li>调节测微螺钉（参见图1B）根据折射指数和物镜的数值孔径，以补偿鱼缸效果。 注：样品和周围吨媒体不同的折射指数他物镜引到物镜的移位和焦平面的移动之间的差异。由于光片被物镜转台移动时，光片（对应于所述物镜的位移）和焦平面的换档的换档之间的不匹配是通过一杆臂补偿（参见图1B）。测微螺钉用来调整上部和下部销之间的距离来调整光片的根据物镜的数值孔径和折射率的转变。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig1highres.jpg" width="500" /> 单平面照明的图1（A）的照片的单一平面照明模块安装于倒置显微镜的定位表的底板，（B）的设置模块和显微镜进料同步补偿焦平面和照明平面之间的升降失配（鱼缸效应）。 ΔZ○表示物镜和Δ狕的变速F表示焦平面的偏移和光片。嵌体显示毛细血管含有球状体的横截面。左：矩形毛细管600微米（墙120微米）的内径。右：用于旋转设置。的内圆毛细管（内径为400μm，外径为550微米）的外侧的矩形毛细管内转动。两个毛细管之间的空间填充有浸没流体。 4，测量的动态液体环境注：先前的协议之前，测量其中的细胞球状体已预先温育，然后保持在适当位置中的毛细管通过简单的重力用于静态孵育。没有必要进行进一步fixati上。然而，对于在流动介质，其中的动态孵化，因此，在动态环境时，优选测量吸收动力学的测量，就可以做到这一点的子协议。步骤4.5-4.9是关键避免气泡到达样品。 <ol><li>填充FCS的毛细管30分钟，以支​​持细胞球状体的存货细胞粘附到内玻璃表面上。 </li><li>让剩余的FCS涂层在贫毛细管干燥至少12小时。 </li><li>按照步骤3.2中所述引入细胞球体的FCS涂层毛细管。 </li><li>留在培养箱中的毛细管，在5％CO 2和37℃下进行另外的2-4小时，以使球体的细胞粘连。 </li><li>设置了微流体系统的汇流部（参见图2）。使用蠕动泵以填充管的汇流（内径：0.89毫米），含有荧光染料，药物或试剂的培养基中。 </li><li>连接到打印机克拉微流体系统来的气泡捕捉器的汇流部（参见图2）。 </li><li>第一夹紧毛细管无气泡的汇流导管，然后将毛细管的另一端夹在排液管。 </li><li>调的水浴中以所希望的值（ 例如，37℃）的液体的温度。 </li><li>调节蠕动泵以期望的泵速度（ 例如，流速：9微升/分钟;泵速度在毛细管：25毫米/分钟）。 </li><li>收集泵送液体无论是在收件人（开环设置），反馈给源（闭环设置），或者直接将其送到蠕动泵（紧闭环设置）的管道。 </li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51993/51993fig2highres.jpg" width="500" /> 图2微流控设置（开环和紧闭环）;镶嵌：照明用的SPH使用连接到一个倒置显微镜光片以荧光显微镜微毛细管内eroid。 5，数据采集与分析<ol><li>设置激光功率和积分时间的图像采集。 </li><li>照顾，在步骤5.1中定义的参数不超过光的剂量值约为50-100焦耳/ cm 2的天然细胞或10-20焦耳/ cm 2的细胞培养用荧光染料或转染的荧光蛋白编码质粒来避免光毒性（详见参考文献12）。 </li><li>设置增量为Z堆叠到Δ狕= 5-10微米是优选的3维数据分析作为光片厚度为约10微米。 </li><li>由细胞球体内变化的焦平面的执行单个图像或Z-堆栈的测量。 </li></ol>

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本手稿描述光片或单一平面照明显微镜（SPIM）装置，其3维电池系统进行了优化， 例如，多细胞肿瘤球体（MCTS）。三个示例性的应用包括：（1）吸收细胞生长抑制药物和其部分转化为降解产物（其对化疗疗效贡献仍有待评估）中，氧化还原状态的（2）通过采用遗传编码谷胱甘肽传感器在测量另外一种氧化剂，和（3）启动和细胞坏死时抑制线粒体呼吸链的标记。 本SPIM...

除了 ​​公认的方法（共焦或多光子激光扫描显微术1-4，结构照明显微镜5,6）光片或单个平面照明显微镜（SPIM）已被证明是三维成像7,8的一种有价值的方法， 9。特别令人感兴趣的是其应用到三维细胞培养物， 例如，多细胞肿瘤球体（MCTS），它们越来越多地用于药物发现研究10,11。再者，SPIM是当即使在长期暴露或重复测量低光剂量必须保持样品的可行性，因为用于样品仅此平面被曝光的每个平面的测定的优先方法。这是相对于其他显微技术，其中对于检测各焦平面的整个样品被照射，使记录的许多平面时的光量总和起来，并可能损坏样品12。 </P&gt; 光片显微镜或SPIM是根据与观察路径的样品在垂直方向上或者通过使用圆柱形透镜的或者通过扫描激动人心的激光束（用于见参考文献8评论）的照明。这通常需要特殊的样品室13,14或矩阵， 例如，琼脂糖7,15，在特殊的高性价比的显微镜来实现。作为替代那些系统中可比简单的照明装置，SPIM已经开发并适于常规的倒置显微镜16（参见图1）。它由激光束扩大到直径为8毫米，集中由一个圆柱形镜头（焦距：50毫米，数值孔径：0.08），以6-10微米厚的片状光通过景深约100微米。样品位于600至900微米内径的矩形毛细管放置在显微镜物镜的len的前S代表荧光检测。这些主要功能是目前完成，通过使用先进的微毛细管的方法，用于保持和转动用于荧光检测（相同光路中的样本，照射光片的同步调整（在轴向）和物镜的优化位移的长度，所需要的机械进给的校正），和一微流体系统的适配为应用荧光染料，药物制剂或药物，从而减少所需的数量和费用。

<table border="0" cellpadding="0" cellspacing="0" width="944">
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			<td height="26" style="height:27px;width:243px;">Name of the Material/Equipment</td>
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			<td style="width:124px;">&#160;Catalog Number</td>
			<td style="width:376px;">Comments/Description(optional)</td>
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			<td height="26" style="height:27px;">microtiter plate</td>
			<td>Orange Scientific</td>
			<td>4430100</td>
			<td>for cell spheroid growing</td>
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		<tr height="26">
			<td height="26" style="height:27px;">agarose&#160;</td>
			<td>Carl Roth GmbH</td>
			<td>3810.1</td>
			<td>for cell spheroid growing</td>
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		<tr height="26">
			<td height="26" style="height:27px;">MCF-7 cell line</td>
			<td>CLS Cell Lines Service GmbH</td>
			<td>300273</td>
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		<tr height="26">
			<td height="26" style="height:27px;">U251-MG-L106 cell line</td>
			<td></td>
			<td></td>
			<td>cell line</td>
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			<td height="26" style="height:27px;">DMEM</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>FG0435</td>
			<td>culture medium</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">DMEM/Ham&#39;s F-12</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>FG4815</td>
			<td>culture medium</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">FCS</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>S0615</td>
			<td>cell culture supplement</td>
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		<tr height="26">
			<td height="26" style="height:27px;">penicillin/streptomycin</td>
			<td>Biochrom AG (Merck Millipore)</td>
			<td>A 2213</td>
			<td>antibiotics</td>
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		<tr height="26">
			<td height="26" style="height:26px;">hygromycin B</td>
			<td style="width:201px;">PAA Laboratories&#160;&#160;</td>
			<td>P02-015</td>
			<td>antibiotic</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">EBSS</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>E3024</td>
			<td>cell culture supplement</td>
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		<tr height="26">
			<td height="26" style="height:27px;">doxorubicin</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>D1515</td>
			<td>fluorescent dye (CAUTION: acute toxicity)</td>
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			<td height="26" style="height:27px;">green cytotoxicity dye</td>
			<td>Promega GmbH</td>
			<td>G8742</td>
			<td>CellTox - fluorescent cytotoxicity dye</td>
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		<tr height="26">
			<td height="26" style="height:27px;">rotenone</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>R8875</td>
			<td>cellular inhibitor (CAUTION: acute toxicity)</td>
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			<td height="26" style="height:27px;">hydrogen peroxide (H2O2)</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>95302</td>
			<td>reagent for oxidation (CAUTION: acute toxicity)</td>
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			<td height="26" style="height:27px;">capillary</td>
			<td>VitroCom&#160;</td>
			<td>8260-050</td>
			<td>sample preparation</td>
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		<tr height="26">
			<td height="26" style="height:27px;">microscope</td>
			<td>Carl Zeiss Jena</td>
			<td>Axiovert 200M</td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:27px;">AxioCam MRc CCD-camera</td>
			<td>Carl Zeiss MicroImaging GmbH</td>
			<td>426508-9901-000</td>
			<td>CCD-camera</td>
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		<tr height="26">
			<td height="26" style="height:27px;">AxioVision data aquisition software</td>
			<td>Carl Zeiss MicroImaging GmbH</td>
			<td>version 4.8.2.</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">laser diode</td>
			<td>Pico Quant GmbH</td>
			<td>LDH-P-C-470</td>
			<td>used with driver PDL800-B</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">perestaltic pump</td>
			<td>Ismatec Labortechnik</td>
			<td>MS-1 Reglo</td>
			<td>re-callibrated to reduce the minimum pump speed by 1/10&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">silicone tubes</td>
			<td>IDEX Health &#38; Science GmbH</td>
			<td>TYGON R3607</td>
			<td></td>
		</tr>
	</tbody>
</table>

single plane illumination module and micro-capillary approach for a wide-field microscope

这是很容易适应于一个倒置的宽视场显微镜和三维细胞培养物的优化，对于光片或单个平面照明显微镜（SPIM）中描述的模块， 例如，多细胞肿瘤球体（MCTS）。在SPIM激发模块的形状和偏转，使得样品被光片照射垂直于显微镜的检测路径的光。该系统的特点是利用一个矩形毛细管的用于保持（和在一个先进的版本也被用于旋转微毛细管的方法）的样品中，由照明光片的同步调整并用于荧光检测以及物镜通过微流体系统的应用的荧光染料，在少量的药物制剂或药物的适应性。给出的协议与该系统的工作，和一些技术细节的报告。代表性的结果包含的向上（1）的测量在添加氧化剂的需要抑制细胞生长的药物（阿霉素）和其部分转化为降解产物，（2）氧化还原测量通过使用遗传编码谷胱甘肽传感器，以及（3）启动和细胞坏死的经抑制的标签线粒体呼吸链。本SPIM模块与现有系统相比，差别和优点进行了讨论。

实验1：细胞球体培养与化疗药物 预先培养的MCF-7细胞球体（8μM多柔比星，6小时）的Z堆叠的扫描示于图3，它给出了关于细胞摄取和多柔比星17的分布和其降解产物18,19的详细信息。内的旋转椭圆体的红色荧光阿霉素的外细胞层主要定位于细胞核，而在内侧旋转椭球体的区域由降解产物发射绿色荧光变为主导的细胞膜19。 <p ...

Watch this Scientific Journal Video about Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope at JoVE.com

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

为单一平面照明显微镜的模块（SPIM）中描述了很容易适应的倒宽视场显微镜和3维细胞培养物进行了优化。样品位于一个矩形的毛细管，并通过微流体系统的荧光染料，药物制剂或药物可以以小批量应用。

单一平面照明模块和一个宽场显微镜微毛细管法

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and ...

single-plane-illumination-module-micro-capillary-approach-for-wide

Research

JoVE Journal

Engineering

9.6K 次观看.  Aalen University. 为单一平面照明显微镜的模块（SPIM）中描述了很容易适应的倒宽视场显微镜和3维细胞培养物进行了优化。样品位于一个矩形的毛细管，并通过微流体系统的荧光染料，药物制剂或药物可以以小批量应用。 

视频: Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities

High resolution microscope systems with optical traps allow for precise manipulation of various refractive objects, such as dielectric beads 1 or cellular organelles 2,3, as well as for high spatial and temporal resolution readout of their position relative to the center of the trap. The system described herein has one such "traditional" trap operating at 980 nm. It additionally provides a second optical trapping system that uses a commercially available holographic package to simultaneously create and manipulate complex trapping patterns in the field of view of the microscope 4,5 at a wavelength of 1,064 nm. The combination of the two systems allows for the manipulation of multiple refractive objects at the same time while simultaneously conducting high speed and high resolution measurements of motion and force production at nanometer and piconewton scale.

The system described herein employs a traditional optical trap as well as an independent holographic optical trapping line, capable of creating and manipulating multiple traps. This allows for the creation of complex geometric arrangements of refractive particles while also permitting simultaneous high-speed, high-resolution measurements of the activity of biological enzymes.

高分辨率显微镜与常规和全息光学捕获能力建设

High resolution microscope systems with optical traps allow for precise manipulation of various refractive objects, such as dielectric beads 1 or cellular ...

科研

工程学

Analysis of Contact Interfaces for Single GaN Nanowire Devices

Single GaN nanowire (NW) devices fabricated on SiO2 can exhibit a strong degradation after annealing due to the occurrence of void formation at the contact/SiO2 interface. This void formation can cause cracking and delamination of the metal film, which can increase the resistance or lead to a complete failure of the NW device. In order to address issues associated with void formation, a technique was developed that removes Ni/Au contact metal films from the substrates to allow for the examination and characterization of the contact/substrate and contact/NW interfaces of single GaN NW devices. This procedure determines the degree of adhesion of the contact films to the substrate and NWs and allows for the characterization of the morphology and composition of the contact interface with the substrate and nanowires. This technique is also useful for assessing the amount of residual contamination that remains from the NW suspension and from photolithographic processes on the NW-SiO2 surface prior to metal deposition. The detailed steps of this procedure are presented for the removal of annealed Ni/Au contacts to Mg-doped GaN NWs on a SiO2 substrate.

A technique was developed that removes Ni/Au contact metal films from their substrate to allow for the examination and characterization of the contact/substrate and contact/NW interfaces of single GaN nanowire devices.

单氮化镓纳米线设备分析接触界面

Single GaN nanowire (NW) devices fabricated on SiO2 can exhibit  a strong degradation after annealing due to the  occurrence of void formation at the ...

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

测量材料在微流使用1-2面流小角中子散射

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle&#039;s Mapping and Quantification in Organ Tissue

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

激光诱导击穿光谱：对于纳米粒子的映射和定量的器官组织的新方法

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

在透射电子显微镜原位时间相关的介质击穿：一种可能性，了解微电子器件的失效机理

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

紧凑的镜头少数字全息显微镜的MEMS检测与表征

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and ...

Hand Controlled Manipulation of Single Molecules via a Scanning Probe Microscope with a 3D Virtual Reality Interface

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building blocks in a bottom-up approach is still open. The scanning probe microscope (SPM) could be a tool of choice; however, SPM-based manipulation was until recently limited to two dimensions (2D). Binding the SPM tip to a molecule at a well-defined position opens an opportunity of controlled manipulation in 3D space. Unfortunately, 3D manipulation is largely incompatible with the typical 2D-paradigm of viewing and generating SPM data on a computer. For intuitive and efficient manipulation we therefore couple a low-temperature non-contact atomic force/scanning tunneling microscope (LT NC-AFM/STM) to a motion capture system and fully immersive virtual reality goggles. This setup permits &#34;hand controlled manipulation&#34; (HCM), in which the SPM tip is moved according to the motion of the experimenter&#39;s hand, while the tip trajectories as well as the response of the SPM junction are visualized in 3D. HCM paves the way to the development of complex manipulation protocols, potentially leading to a better fundamental understanding of nanoscale interactions acting between molecules on surfaces. Here we describe the setup and the steps needed to achieve successful hand-controlled molecular manipulation within the virtual reality environment.

We demonstrate the precise manipulation of individual organic molecules on a metal surface with the tip of a scanning probe microscope driven in 3D by the experimenter's hand using a motion capture system and fully immersive virtual reality goggles.

通过手扫描探针显微镜用三维虚拟现实接口控制单分子的操作

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building ...

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

Hyperlens 综合显微镜和超分辨率成像的演示

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics

The miniaturization of semiconductor devices to scales where small numbers of dopants can control device properties requires the development of new techniques capable of characterizing their dynamics. Investigating single dopants requires sub-nanometer spatial resolution, which motivates the use of scanning tunneling microscopy (STM). However, conventional STM is limited to millisecond temporal resolution. Several methods have been developed to overcome this shortcoming, including all-electronic time-resolved STM, which is used in this study to examine dopant dynamics in silicon with nanosecond resolution. The methods presented here are widely accessible and allow for local measurement of a wide variety of dynamics at the atomic scale. A novel time-resolved scanning tunneling spectroscopy technique is presented and used to efficiently search for dynamics.

We demonstrate an all-electronic method to observe nanosecond-resolved charge dynamics of dopant atoms in silicon with a scanning tunneling microscope.

全电子纳秒分辨扫描隧道显微镜: 促进单一掺杂剂电荷动力学的研究

The miniaturization of semiconductor devices to scales where small numbers of dopants can control device properties requires the development of new ...

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

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

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

用畸变校正扫描透射电镜进行单数字纳米电子束光刻

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