Name: simultaneously capturing real-time images in two emission channels using a dual camera emission splitting system: applications to cell adhesion
Uploaded: 2013-09-04T12:00:00
Description: Watch this Scientific Journal Video about Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion at JoVE.com

作者要感谢道格拉斯博士戈茨（化学系和生物分子工程，俄亥俄大学）和费边Benencia博士（生物医学俄亥俄大学系）对有见地的讨论和审稿。我们也感谢克里斯托弗Huppenbauer博士的有益的技术讨论（W. Nuhsbaum公司）。这项工作是由美国国立卫生科学基金会（CBET-1106118）和国家研究院​​（1R15CA161830-01）资助。

1。软件安装<ol><li>购买StreamPix 5多摄像头软件SimulPix模块或能够收集并结合了黑白CCD相机拍摄的图像的其它图像软件。 </li><li>对于StreamPix 5最低要求包括：搭载Core 2 Duo处理器，2.4 GHz或更好地与2 GB RAM或更高的PC。受支持的IEEE数字或模拟相机和兼容图像采集卡。窗户XP/7/Vista 32位或64位。监视器支持分辨率1024×768或更高。 （建议OCU表达16倍或更高）的图形适配器具有良好的2D性能和7,200 RPM的硬盘驱动...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+diagram+for+cell+adhesion+under+flow:+leukocyte+rolling+and+firm+adhesion.">The state diagram for cell adhesion under flow: leukocyte rolling and firm adhesion.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (21), 11262-11267 (2000).</li><li>Lichtman, J. W., Conchello, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat. Methods. 2 (12), 910-919 (2005).</li><li>Kummitha, C. M., Shirure, V. S., Delgadillo, L. F., Deosarkar, S. P., Tees, D. F., Burdick, M. M., Goetz, D. 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Oncol. 2, 103 (2012).</li><li>Hoffman, G. E., Le, W. W., Sita, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Importance+of+Titrating+Antibodies+for+Immunocytochemical+Methods.">The Importance of Titrating Antibodies for Immunocytochemical Methods.</a> Current Protocols in Neuroscience. , (2001).</li><li>Weinberg, S., Lipke, E. A., Tung, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+electrophysiological+mapping+of+stem+cells.">In vitro electrophysiological mapping of stem cells.</a> Methods Mol. Biol. 660, 215-237 (2010).</li><li>Kong, K. V., Leong, W. K., Lim, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmium+carbonyl+clusters+containing+labile+ligands+hyperstabilize+microtubules.">Osmium carbonyl clusters containing labile ligands hyperstabilize microtubules.</a> Chem. Res. Toxicol. 22 (6), 1116-1122 (2009).</li><li>Vermot, J., Fraser, S. E., Liebling, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+fluorescence+microscopy+for+imaging+the+dynamics+of+embryonic+development.">Fast fluorescence microscopy for imaging the dynamics of embryonic development.</a> HFSP J. 2 (3), 143-155 (2008).</li><li>Bullen, A., Friedman, R. S., Krummel, M. F. <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+imaging+of+the+immune+system:+a+custom+technology+platform+for+high-speed,+multicolor+tissue+imaging+of+immune+responses.">Two-photon imaging of the immune system: a custom technology platform for high-speed, multicolor tissue imaging of immune responses.</a> Curr. Top. Microbiol. Immunol. 334, 1-29 (2009).</li><li>Worth, D. C., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+imaging+cell-matrix+adhesions.">Advances in imaging cell-matrix adhesions.</a> J. Cell Sci. 123, 3629-3638 (2010).</li><li>Xiao, Z., Visentin, G. P., Dayananda, K. 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双摄像头发射分光系统需要在应用中的细胞或分子的运动是快速捕捉高品质图像的空间，时间，以及光学分辨率。在产生代表性的结果，在双相机发射分光系统，包括软件的设置，相机设置的参数，进行优化以获得合并的图像序列，其中轧制和贴壁细胞，空间和时间上对齐。此优化步骤是关键的，因为未对准可导致图像不正确传达所研究的物理现象， 例如 ，观察到滚在两个绿色和红色通?...

方法分子在细胞表面上，如免疫染色分析，采用被化学偶联至荧光基团，从而允许检测目标分子的探针。活细胞成像和流体动力流动为基础的细胞黏附测定法通常记录的黑白CCD相机设计在细胞和/或分子水平1，2捕获的生理过程。这些摄像机是高度敏感的，提供快速的帧速率（大于每秒30帧），和（由于快速的帧速率和较短的曝光时间），提供出色的时间分辨率。然而，黑白相机只能捕捉到一个单一的发射通道（检测单个荧光基团）来采集图像。单个相机发射分光系统可并入到捕获多个发射信道，但往往减少视场并需要相同的曝光时间进行摄像的所有通道。要抓紧细胞标记MULTIP的全彩色光谱勒荧光团，一个彩色照相机可以作为一种替代方法。然而，彩色相机通常不能够提供期望在某些应用中的活细胞成像的时间分辨率。另一个成像装置所需的应用中，有利的是，图象的活细胞在多个波长，同时保持高的时间分辨率。一个主要的实验应用是在平行平板流动腔粘附试验，其中细胞被灌注在生理学相关条件下在一个潜在的反应基片1，3。在流动细胞表达特定细胞表面分子可能粘附和轧辊的基体如表达粘附分子或表面吸附的细胞外基质蛋白4，5的细胞单层上。轧制细胞可以在几分之一秒进行旋转和平移运动。上的滚动和粘附的细胞，如簇的细胞表面分子的分子功能，还具有电位人经过积极的重组在细胞表面上。因此，成像系统必须提供一个特殊的时间分辨率（每秒或更高30帧和“近零”的曝光时间），以生成示出的细胞轧制6,7的一步一步的进展的图像序列。双摄像头发射分光系统能满足这些要求的成像单元带有多种荧光团。 双摄像发射分光系统拆分和滤波器荧光通道分成两个相似的摄像机同时捕获两个空间上相同，但荧光团特定的图象，同时保留的全视场。该技术使实时在每个通道中捕获的图像进行直接比较，并允许用户快速照相机模型之间具有不同的成像能力切换。此功能可用于制作在一个摄像头，更好地让系统捕捉调整图像捕捉设置有用荧光基团具有不同强度，寿命和消光系数8。再加上成像软件，双摄像头发射分光系统允许在多个波长活细胞成像分析的实时记录，并可以提高在体外实验使用的荧光来研究细胞行为。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>BT-20 cells</td>
 <td>ATCC</td>
 <td>HTB-19</td>
 <td></td>
 </tr>
 <tr>
 <td>CHO-E cells</td>
 <td>Gift from Dr. R. Sackstein (Brigham and Women's Hospital, Harvard Medical School)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>MEM </td>
 <td>Thermo Scientific</td>
 <td>SH30024.01</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Thermo Scientific</td>
 <td>SH30396.03</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Thermo Scientific</td>
 <td>SV30010</td>
 <td></td>
 </tr>
 <tr>
 <td>0.25% Trypsin / 0.1% EDTA</td>
 <td>Thermo Scientific</td>
 <td>SV30031.01</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Thermo Scientific </td>
 <td>SH30028.02</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS+</td>
 <td>Life Technologies</td>
 <td>14080-055</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A9647</td>
 <td></td>
 </tr>
 <tr>
 <td>HECA-452 monoclonal antibody</td>
 <td>BD Biosciences</td>
 <td>555946</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-human CD24 monoclonal antibody</td>
 <td>BD Biosciences</td>
 <td>555426</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-rat IgM AlexaFluor 488</td>
 <td>Invitrogen</td>
 <td>A21212</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse IgG AlexaFluor 568</td>
 <td>Invitrogen</td>
 <td>A11004</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>EXi Blue Fluorescence Microscopy Digital CCD Camera</td>
 <td>Q Imaging</td>
 <td>EXI-BLU-R-F-M-14-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Retiga EXi FAST 1394 Digital CCD Camera</td>
 <td>Q Imaging</td>
 <td>RET-EXi-F-M-12-C</td>
 <td></td>
 </tr>
 <tr>
 <td>DC2 Emission Splitter</td>
 <td>Photometrics</td>
 <td>DC2 </td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted Fluorescence Microscope</td>
 <td>Leica</td>
 <td> DMI6000 B</td>
 <td></td>
 </tr>
 <tr>
 <td>Streampix 5 software</td>
 <td>Norpix</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

simultaneously capturing real-time images in two emission channels using a dual camera emission splitting system: applications to cell adhesion

多色免疫荧光显微镜来检测特定的分子在细胞膜上可以加上平行平板流动腔实验来调查管动态流动条件下的细胞粘附机制。例如，标有多种荧光团的癌细胞可灌注了一个潜在的反应底物来建模癌转移的机制。然而，多通道单相机系统和彩色摄像机表现出的缺点在图像采集的实时活细胞分析。为了克服这些局限性，我们采用了双摄像头发射分光系统同时捕获荧光标记细胞的实时图像序列中的流室。双摄像头发射分光系统过滤器中定义的波长范围分为两个黑白CCD摄像机，从而同时捕获两个空间相同，但荧光的特定图像。随后，psuedocolored 1通道的图像被组合成一个单一的实时合并序列，可以揭示多个目标分子对细胞运动迅速在一个地区的利益。

平行板流动腔粘连实验检测证明，同时捕获实时图像序列的两个发射信道的双摄像头发射分光系统（ 图1）。双相机发射分光系统检测出BT-20细胞，进行了荧光标记的抗人CD24和HECA-452（检测sialofucosylated抗原）的单克隆抗体和合适的第二抗体（ 图2）。一些细胞滚动显示红色信号（CD24）还检测不到绿色信号（HECA-452），其他显示绿色信号和检测不到的红色信号，并且还在其?...

Watch this Scientific Journal Video about Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion at JoVE.com

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统：应用到细胞粘附

双摄像头发射分光系统，双色荧光显微镜生成具有出色的光学分辨率和时间分辨率，一定的活细胞检测的要求，包括平行板流动腔粘连检测的实时图像序列。当软件被用来从合并收购的同时发射通道的图像，伪彩色图像序列产生。

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Study of Cell Migration in Microfabricated Channels

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

手持式实时非侵入性小动物成像的临床光声成像系统

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

A Protocol for Real-time 3D Single Particle Tracking

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

在原位生物过程单细胞形态实时测定的显微镜

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...