Name: single plane illumination module and micro-capillary approach for a wide-field microscope
Uploaded: 2014-08-15T19:00:00
Description: Watch this Scientific Journal Video about Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope at JoVE.com

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...

<ol>
	<li>Pawley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biological confocal microscopy. , (1990).</li><li>Webb, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+optical+microscopy.">Confocal optical microscopy.</a> Rep. Prog. Phys. 59, 427-471 (1996).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>K&#246;nig, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+in+life+sciences.">Multiphoton microscopy in life sciences.</a> J. Microsc. 200 (2), 83-104 (2000).</li><li>Neil, M. A., Juskaitis, R., Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+obtaining+optical+sectioning+by+using+structured+light+in+a+conventional+microscope.">Method of obtaining optical sectioning by using structured light in a conventional microscope.</a> Opt. Lett. 22 (24), 1905-1907 (1997).</li><li>Gustafsson, M. G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+resolution+doubling+in+wide-field+fluorescence+microscopy+by+structured+illumination.">Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.</a> Biophys. J. 94 (12), 4957-4970 (2008).</li><li>Huisken, J., Swoger, J., del Bene, F., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+SPIM.">Optical sectioning deep inside live embryos by SPIM.</a> Science. 305 (5686), 1007-1009 (2004).</li><li>Huisken, J., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+plane+illumination+microscopy+techniques+in+development+biology.">Selective plane illumination microscopy techniques in development biology.</a> Development. 136 (12), 1963-1975 (2009).</li><li>Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+fluorescence+microscopy:+a+review.">Light sheet fluorescence microscopy: a review.</a> J. Histochem. Cytochem. 59 (2), 129-138 (2011).</li><li>Kunz-Schughart, L. A., Freyer, J. P., Hofstaedter, F., Ebner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+3-D+cultures+for+high+throughput+screening:+the+multi-cellular+spheroid+model.">The use of 3-D cultures for high throughput screening: the multi-cellular spheroid model.</a> J. Biomol. Screen. 9 (4), 273-285 (2004).</li><li>Schneckenburger, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-dimensional+fluorescence+microscopy+of+living+cells.">Multi-dimensional fluorescence microscopy of living cells.</a> J. Biophotonics. 4 (3), 143-149 (2011).</li><li>Schneckenburger, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+exposure+and+cell+viability+in+fluorescence+microscopy.">Light exposure and cell viability in fluorescence microscopy.</a> J. Microsc. 245 (3), 311-318 (2012).</li><li>Ritter, J. G., Veith, R., Veenendaal, A., Siebrasse, J. P., Kubitschek, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+microscopy+for+single+molecule+tracking+in+living+tissue.">Light sheet microscopy for single molecule tracking in living tissue.</a> PLoS One. 5 (7), e11639:1-e11639:7 (2010).</li><li>Mertz, J., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+light-sheet+microscopy+in+the+whole+mouse+brain+with+HiLo+background+rejection.">Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection.</a> J. Biomed. Opt. 15 (1), 016027 (2010).</li><li>Fahrbach, F. O., Rohrbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+line-scanned+light-sheet+microscope+with+phase+shaped+self-reconstructing+beams.">A line-scanned light-sheet microscope with phase shaped self-reconstructing beams.</a> Opt. Express. 18 (23), 24229-24244 (2010).</li><li>Bruns, T., Schickinger, S., Wittig, R., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+strategy+and+illumination+of+3D+cell+cultures+in+light-sheet+based+fluorescence+microscopy.">Preparation strategy and illumination of 3D cell cultures in light-sheet based fluorescence microscopy.</a> J. Biomed. Opt. 17 (10), 101518 (2012).</li><li>Ma, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids+as+an+in+vivo-like+tumor+model+for+three-dimensional+imaging+of+chemotherapeutic+and+nano+material+cellular+penetration.">Multicellular tumor spheroids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration.</a> Mol. Imaging. 11 (6), 487-498 (2012).</li><li>Weber, P., Wagner, M., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+dependent+uptake+and+interaction+of+doxorubicin+in+MCF-7+breast+cancer+cells.">Cholesterol dependent uptake and interaction of doxorubicin in MCF-7 breast cancer cells.</a> Int. J. Mol. Sci. 14 (4), 8358-8366 (2013).</li><li>Hovorka, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+analysis+of+doxorubicin+accumulation+and+the+indirect+quantification+of+its+DNA+intercalation.">Spectral analysis of doxorubicin accumulation and the indirect quantification of its DNA intercalation.</a> Eur. J. Pharm. Biopharm. 76 (3), 514-524 (2010).</li><li>Schickinger, S., Bruns, T., Wittig, R., Weber, P., Wagner, M., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosecond+ratio+imaging+of+redox+states+in+tumor+cell+spheroids+using+light+sheet-based+fluorescence+microscopy.">Nanosecond ratio imaging of redox states in tumor cell spheroids using light sheet-based fluorescence microscopy.</a> J. Biomed. Opt. 18 (12), 126007 (2013).</li><li>Lippert, H., Wald, M., Radt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+arrangement+for+the+production+of+a+light+sheet.">Optical arrangement for the production of a light sheet.</a> U.S. Patent. , (2010).</li><li>Lorenzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+division+dynamics+monitoring+in+3D+large+spheroid+tumor+models+using+light+sheet+microscopy.">Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy.</a> Cell Division. 6 (22), 1-8 (2011).</li><li>Bruns, T., Schneckenburger, H., Schickinger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+holder+for+rotation+of+three-dimensional+specimens+in+microscopy.">Sample holder for rotation of three-dimensional specimens in microscopy.</a> European Patent Application. , (2013).</li></ol>

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">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:243px;">Name of the Material/Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:124px;">&#160;Catalog Number</td>
			<td style="width:376px;">Comments/Description(optional)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">microtiter plate</td>
			<td>Orange Scientific</td>
			<td>4430100</td>
			<td>for cell spheroid growing</td>
		</tr>
		<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>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">MCF-7 cell line</td>
			<td>CLS Cell Lines Service GmbH</td>
			<td>300273</td>
			<td>cell line</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">U251-MG-L106 cell line</td>
			<td></td>
			<td></td>
			<td>cell line</td>
		</tr>
		<tr height="26">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">green cytotoxicity dye</td>
			<td>Promega GmbH</td>
			<td>G8742</td>
			<td>CellTox - fluorescent cytotoxicity dye</td>
		</tr>
		<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>
		</tr>
		<tr height="26">
			<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>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">capillary</td>
			<td>VitroCom&#160;</td>
			<td>8260-050</td>
			<td>sample preparation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">microscope</td>
			<td>Carl Zeiss Jena</td>
			<td>Axiovert 200M</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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 ...

Research

JoVE Journal

Engineering

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

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

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

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

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

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

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

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

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

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

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

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 ...